Reference sequence mapping horserace

ipyrad is capable of incorporating a reference sequence to aid in the assembly. There are actually 4 different assembly methods, 3 of which use reference sequence in some way. Here we test reference assisted assembly for ipyrad and stacks, as ddocent and aftrrad do not allow for . Though aftrRAD performs nicely on empirical data and does allow for reference assisted assembly, we consider runtimes to be prohibitive and so exclude it from analysis here.

Ideas for datasets (all have data in SRA):

Selection and sex-biased dispersal in a coastal shark: the influence of philopatry on adaptive variation
- 134 individuals (paper assembled w/ ddocent)

Genome-wide data reveal cryptic diversity and genetic introgression in an Oriental cynopterine fruit bat radiation
- < 45 samples, 2 reference genomes in the same family

Beyond the Coral Triangle: high genetic diversity and near panmixia in Singapore's populations of the broadcast spawning sea star Protoreaster nodosus
- 77 samples, it's a passerine, so there must be something reasonably close

PSMC (pairwise sequentially Markovian coalescent) analysis of RAD (restriction site associated DNA) sequencing data
- 17 sticklebacks, they used stacks

For this analysis we chose the paired-end ddRAD dataset from Lah et al 2016 (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162792#sec002): Spatially Explicit Analysis of Genome-Wide SNPs Detects Subtle Population Structure in a Mobile Marine Mammal, the Harbor Porpoise

- 49 samples from 3 populations of European Harbor Porpoise
In [28]:
import subprocess
import ipyrad as ip
import shutil
import glob
import sys
import os

## Set the default directories for exec and data. 
WORK_DIR="/home/iovercast/manuscript-analysis/"
REFMAP_EMPIRICAL_DIR=os.path.join(WORK_DIR, "Phocoena_empirical/")
REFMAP_FASTQS=os.path.join(REFMAP_EMPIRICAL_DIR, "Final_Files_forDryad/Bbif_ddRADseq/fastq/")
IPYRAD_DIR=os.path.join(WORK_DIR, "ipyrad/")
STACKS_DIR=os.path.join(WORK_DIR, "stacks/")

for dir in [WORK_DIR, REFMAP_EMPIRICAL_DIR, IPYRAD_DIR, STACKS_DIR]:
    if not os.path.exists(dir):
        os.makedirs(dir)

Simulated reference sequence mapping

To get some idea of how ipyrad and stacks perform with reference sequence mapping we'll first assemble a simulated dataset.

Right now i'm just grabbing the simulated data from the ipyrad ipsimdata directory cuz it's quick and dirty but if you want to get crazy you can simulate new seqs by ripping code from here: https://github.com/dereneaton/ipyrad/blob/master/tests/cookbook-making-sim-data.ipynb

Make directories and fetch the simulated data

The toy simulated data that lives in the ipyrad git repo consists of 1000 simulated loci, 500 of which are present in the simulated reference sequence.

In [66]:
REFMAP_SIM_DIR = os.path.join(WORK_DIR, "REFMAP_SIM/")
REFMAP_DAT_DIR = os.path.join(REFMAP_SIM_DIR, "ipsimdata/")
IPYRAD_SIM_DIR = os.path.join(REFMAP_SIM_DIR, "ipyrad/")
STACKS_SIM_DIR = os.path.join(REFMAP_SIM_DIR, "stacks/")
DDOCENT_SIM_DIR = os.path.join(REFMAP_SIM_DIR, "ddocent/")
## REFMAP_DAT_DIR will be created when we untar ipsimdata.tar.gz
for d in [REFMAP_SIM_DIR, IPYRAD_SIM_DIR, STACKS_SIM_DIR, DDOCENT_SIM_DIR]:
    if not os.path.exists(d):
        os.makedirs(d)
os.chdir(REFMAP_SIM_DIR)

!wget https://github.com/dereneaton/ipyrad/raw/master/tests/ipsimdata.tar.gz
!tar -xzf ipsimdata.tar.gz
--2016-12-31 14:12:22--  https://github.com/dereneaton/ipyrad/raw/master/tests/ipsimdata.tar.gz
Resolving github.com (github.com)... 192.30.253.113, 192.30.253.112
Connecting to github.com (github.com)|192.30.253.113|:443... connected.
HTTP request sent, awaiting response... 302 Found
Location: https://raw.githubusercontent.com/dereneaton/ipyrad/master/tests/ipsimdata.tar.gz [following]
--2016-12-31 14:12:23--  https://raw.githubusercontent.com/dereneaton/ipyrad/master/tests/ipsimdata.tar.gz
Resolving raw.githubusercontent.com (raw.githubusercontent.com)... 151.101.20.133
Connecting to raw.githubusercontent.com (raw.githubusercontent.com)|151.101.20.133|:443... connected.
HTTP request sent, awaiting response... 200 OK
Length: 12433112 (12M) [application/octet-stream]
Saving to: ‘ipsimdata.tar.gz.1’

100%[======================================>] 12,433,112  17.8MB/s   in 0.7s   

2016-12-31 14:12:24 (17.8 MB/s) - ‘ipsimdata.tar.gz.1’ saved [12433112/12433112]

Do ipyrad simulated reference mapping

For the first analysis we'll use the reference assembly method which will just toss out all reads that don't map the the reference sequence. We should expect to recover 500 reads per sample.

The toy data runs in ~2 minutes.

In [95]:
os.chdir(IPYRAD_SIM_DIR)

## Make a new assembly and set some assembly parameters
data = ip.Assembly("refmap-sim")
data.set_params("raw_fastq_path", REFMAP_DAT_DIR + "pairddrad_wmerge_example_R*_.fastq.gz")
data.set_params("barcodes_path", REFMAP_DAT_DIR + "pairddrad_wmerge_example_barcodes.txt")
data.set_params("project_dir", "reference-assembly")
data.set_params("assembly_method", "reference")
data.set_params("reference_sequence", REFMAP_DAT_DIR + "pairddrad_wmerge_example_genome.fa")
data.set_params("datatype", "pairddrad")
data.set_params("restriction_overhang", ("TGCAG", "CGG"))

data.write_params(force=True)

cmd = "ipyrad -p params-refmap-sim.txt -s 1234567 -c 40".format(dir)
print(cmd)
!time $cmd
  New Assembly: refmap-sim
ipyrad -p params-refmap-sim.txt -s 1234567 -c 40

 -------------------------------------------------------------
  ipyrad [v.0.5.15]
  Interactive assembly and analysis of RAD-seq data
 -------------------------------------------------------------
  loading Assembly: refmap-sim
  from saved path: ~/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim.json
  local compute node: [40 cores] on node001

  Step 1: Demultiplexing fastq data to Samples
    Skipping: 12 Samples already found in Assembly refmap-sim.
    (can overwrite with force argument)    

  Step 2: Filtering reads 
    Skipping: All 12 selected Samples already edited.
    (can overwrite with force argument)    

  Step 3: Clustering/Mapping reads
    Skipping: All 12 selected Samples already clustered.
    (can overwrite with force argument)    

  Step 4: Joint estimation of error rate and heterozygosity
    Skipping: All 12 selected Samples already joint estimated
    (can overwrite with force argument)    

  Step 5: Consensus base calling 
    Skipping: All 12 selected Samples already consensus called
    (can overwrite with force argument)    

  Step 6: Clustering at 0.85 similarity across 12 samples
    Skipping: All 12 selected Samples already clustered.
    (can overwrite with force argument)    

  Step 7: Filter and write output files for 12 Samples
ERROR:ipyrad.core.assembly:IPyradWarningExit:     Output files already created for this Assembly in:
    /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_outfiles
    To overwrite, rerun using the force argument.
    

  Encountered an error, see ./ipyrad_log.txt. 
      Output files already created for this Assembly in:
    /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_outfiles
    To overwrite, rerun using the force argument.
    


real	0m18.623s
user	0m1.899s
sys	0m0.552s

Do ipyrad denovo+reference

Create a new branch and set the assembly method to denovo+reference. Now we will expect to recover 1000 loci per sample (500 from the reference mapping and 500 from de novo).

Again, the toy data runs in slighly less than 3 minutes.

In [97]:
data2 = data.branch("denovo_plus_reference-sim")
data2.set_params("assembly_method", "denovo+reference")

data2.write_params(force=True)

cmd = "ipyrad -p params-denovo_plus_reference-sim.txt -s 1234567 -c 40".format(dir)
print(cmd)
!time $cmd
ipyrad -p params-denovo_plus_reference-sim.txt -s 1234567 -c 40

 -------------------------------------------------------------
  ipyrad [v.0.5.15]
  Interactive assembly and analysis of RAD-seq data
 -------------------------------------------------------------
  loading Assembly: denovo_plus_reference-sim
  from saved path: ~/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/denovo_plus_reference-sim.json
  New Assembly: denovo_plus_reference-sim
  local compute node: [40 cores] on node001

  Step 1: Demultiplexing fastq data to Samples
  [####################] 100%  chunking large files  | 0:00:00  
  [####################] 100%  sorting reads         | 0:00:09  
  [####################] 100%  writing/compressing   | 0:00:01  

  Step 2: Filtering reads 
  [####################] 100%  processing reads      | 0:00:02  

  Step 3: Clustering/Mapping reads
  [####################] 100%  dereplicating         | 0:00:00  
  [####################] 100%  mapping               | 0:00:00  
  [####################] 100%  clustering            | 0:00:01  
  [####################] 100%  building clusters     | 0:00:00  
  [####################] 100%  finalize mapping      | 0:01:07  
  [####################] 100%  chunking              | 0:00:00  
  [####################] 100%  aligning              | 0:00:14  
  [####################] 100%  concatenating         | 0:00:00  

  Step 4: Joint estimation of error rate and heterozygosity
  [####################] 100%  inferring [H, E]      | 0:00:15  

  Step 5: Consensus base calling 
  Mean error  [0.00074 sd=0.00001]
  Mean hetero [0.00195 sd=0.00015]
  [####################] 100%  calculating depths    | 0:00:00  
  [####################] 100%  chunking clusters     | 0:00:00  
  [####################] 100%  consens calling       | 0:00:12  

  Step 6: Clustering at 0.85 similarity across 12 samples
  [####################] 100%  concat/shuffle input  | 0:00:00  
  [####################] 100%  clustering across     | 0:00:01  
  [####################] 100%  building clusters     | 0:00:00  
  [####################] 100%  aligning clusters     | 0:00:02  
  [####################] 100%  database indels       | 0:00:00  
  [####################] 100%  indexing clusters     | 0:00:02  
  [####################] 100%  building database     | 0:00:00  

  Step 7: Filter and write output files for 12 Samples
  [####################] 100%  filtering loci        | 0:00:06  
  [####################] 100%  building loci/stats   | 0:00:00  
  [####################] 100%  building vcf file     | 0:00:01  
  [####################] 100%  writing vcf file      | 0:00:00  
  [####################] 100%  building arrays       | 0:00:02  
  [####################] 100%  writing outfiles      | 0:00:01  
  Outfiles written to: ~/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/denovo_plus_reference-sim_outfiles


real	2m50.283s
user	0m22.885s
sys	0m1.653s

Do ipyrad denovo-reference

Create a new branch and set the assembly method to denovo+reference. Now we will expect to recover 1000 loci per sample (500 from the reference mapping and 500 from de novo).

Again, the toy data runs in slighly less than 2 minutes.

In [98]:
data2 = data.branch("denovo_minus_reference-sim")
data2.set_params("assembly_method", "denovo-reference")

data2.write_params(force=True)

cmd = "ipyrad -p params-denovo_minus_reference-sim.txt -s 1234567 -c 40".format(dir)
print(cmd)
!time $cmd
ipyrad -p params-denovo_minus_reference-sim.txt -s 1234567 -c 40

 -------------------------------------------------------------
  ipyrad [v.0.5.15]
  Interactive assembly and analysis of RAD-seq data
 -------------------------------------------------------------
  loading Assembly: denovo_minus_reference-sim
  from saved path: ~/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/denovo_minus_reference-sim.json
  New Assembly: denovo_minus_reference-sim
  local compute node: [40 cores] on node001

  Step 1: Demultiplexing fastq data to Samples
  [####################] 100%  chunking large files  | 0:00:00  
  [####################] 100%  sorting reads         | 0:00:07  
  [####################] 100%  writing/compressing   | 0:00:01  

  Step 2: Filtering reads 
  [####################] 100%  processing reads      | 0:00:02  

  Step 3: Clustering/Mapping reads
  [####################] 100%  dereplicating         | 0:00:00  
  [####################] 100%  mapping               | 0:00:01  
  [####################] 100%  clustering            | 0:00:00  
  [####################] 100%  building clusters     | 0:00:01  
  [####################] 100%  chunking              | 0:00:00  
  [####################] 100%  aligning              | 0:00:07  
  [####################] 100%  concatenating         | 0:00:00  

  Step 4: Joint estimation of error rate and heterozygosity
  [####################] 100%  inferring [H, E]      | 0:00:12  

  Step 5: Consensus base calling 
  Mean error  [0.00074 sd=0.00002]
  Mean hetero [0.00192 sd=0.00023]
  [####################] 100%  calculating depths    | 0:00:00  
  [####################] 100%  chunking clusters     | 0:00:00  
  [####################] 100%  consens calling       | 0:00:05  

  Step 6: Clustering at 0.85 similarity across 12 samples
  [####################] 100%  concat/shuffle input  | 0:00:00  
  [####################] 100%  clustering across     | 0:00:01  
  [####################] 100%  building clusters     | 0:00:00  
  [####################] 100%  aligning clusters     | 0:00:01  
  [####################] 100%  database indels       | 0:00:00  
  [####################] 100%  indexing clusters     | 0:00:01  
  [####################] 100%  building database     | 0:00:00  

  Step 7: Filter and write output files for 12 Samples
  [####################] 100%  filtering loci        | 0:00:06  
  [####################] 100%  building loci/stats   | 0:00:00  
  [####################] 100%  building vcf file     | 0:00:01  
  [####################] 100%  writing vcf file      | 0:00:00  
  [####################] 100%  building arrays       | 0:00:02  
  [####################] 100%  writing outfiles      | 0:00:00  
  Outfiles written to: ~/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/denovo_minus_reference-sim_outfiles


real	1m17.382s
user	0m14.547s
sys	0m1.512s

Stacks simulated reference sequence assembly

Stacks needs reference mapped sequences in .bam or .sam format. Since we already did the mapping in ipyrad we'll just pluck the *-mapped-sorted.bam files out of the ipyrad _refmapping directory. Nope! That would certainly be nice, but we dereplicate reads before we map. This is obviously important information (which ipyrad records and uses downstream), but stacks doesn't know about this, so you get bad quality mapping. I learned this the hard way. We need to map each sample by hand from the original ipyrad _edits files.

On the toy simulated data stacks runs in an impressive 5 seconds.

bwa mapping

We are going to poach bwa and samtools from the dDocent install, since we already installed them there. ugh.

This proceeds in 3 steps. First bwa maps to the reference (-t is # of cores, -v shuts down verbosity). Then samtools pulls out mapped and properly paired reads (-b outputs .bam format, -F 0x804 filters on mapping/pairing). Then it sorts the bam file, and cleans up the dangling sam file. Mapping is quick for the simulated data (<2 minutes).

In [14]:
IPYRAD_SIMEDITS_DIR = IPYRAD_SIM_DIR + "reference-assembly/refmap-sim_edits/"
REF_SEQ = REFMAP_DAT_DIR + "pairddrad_wmerge_example_genome.fa"

## Sim sample names
pop1 = ["1A_0", "1B_0", "1C_0", "1D_0"]
pop2 = ["2E_0", "2F_0", "2G_0", "2H_0"]
pop3 = ["3I_0", "3J_0", "3K_0", "3L_0"]
sim_sample_names = pop1 + pop2 + pop3

for samp in sim_sample_names:
    R1 = IPYRAD_SIMEDITS_DIR + samp + ".trimmed_R1_.fastq.gz"
    R2 = IPYRAD_SIMEDITS_DIR + samp + ".trimmed_R2_.fastq.gz"
    samout = STACKS_SIM_DIR + samp + ".sam"
    bamout = STACKS_SIM_DIR + samp + ".bam"
    export_cmd = "export PATH=~/manuscript-analysis/dDocent:$PATH"
    bwa_cmd = "bwa mem -t 40 -v 1 " + REF_SEQ\
                + " " + R1\
                + " " + R2\
                + " > " + samout
    samtools_cmd = "samtools view -b -F 0x804 " + samout\
                    + " | samtools sort -T /tmp/{}.sam -O bam -o {}".format(samp, bamout)
    cleanup_cmd = "rm {}".format(samout)
    cmd = ";".join([export_cmd, bwa_cmd, samtools_cmd, cleanup_cmd])
    !$cmd
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40080 sequences (3827640 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10025, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.20, 14.47)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40080 reads in 2.988 CPU sec, 0.112 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1A_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1A_0.trimmed_R2_.fastq.gz
[main] Real time: 0.434 sec; CPU: 3.219 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 39964 sequences (3816562 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10017, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.28, 14.44)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 39964 reads in 2.968 CPU sec, 0.108 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1B_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1B_0.trimmed_R2_.fastq.gz
[main] Real time: 0.262 sec; CPU: 3.031 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40210 sequences (3840055 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10125, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.14, 14.41)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40210 reads in 3.001 CPU sec, 0.112 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1C_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1C_0.trimmed_R2_.fastq.gz
[main] Real time: 0.271 sec; CPU: 3.068 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40344 sequences (3852852 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10084, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.31, 14.37)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40344 reads in 3.024 CPU sec, 0.113 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1D_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/1D_0.trimmed_R2_.fastq.gz
[main] Real time: 0.273 sec; CPU: 3.092 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40164 sequences (3835662 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10021, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 476)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (404, 524)
[M::mem_pestat] mean and std.dev: (464.12, 14.42)
[M::mem_pestat] low and high boundaries for proper pairs: (380, 548)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40164 reads in 2.835 CPU sec, 0.114 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2E_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2E_0.trimmed_R2_.fastq.gz
[main] Real time: 0.269 sec; CPU: 2.900 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40164 sequences (3835662 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10003, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.13, 14.39)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40164 reads in 2.821 CPU sec, 0.111 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2F_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2F_0.trimmed_R2_.fastq.gz
[main] Real time: 0.265 sec; CPU: 2.884 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40190 sequences (3838145 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10068, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.24, 14.37)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40190 reads in 2.563 CPU sec, 0.106 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2G_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2G_0.trimmed_R2_.fastq.gz
[main] Real time: 0.260 sec; CPU: 2.626 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40010 sequences (3820955 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10056, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.25, 14.46)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40010 reads in 2.893 CPU sec, 0.109 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2H_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/2H_0.trimmed_R2_.fastq.gz
[main] Real time: 0.264 sec; CPU: 2.958 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 39648 sequences (3786384 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10000, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.29, 14.39)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 39648 reads in 3.018 CPU sec, 0.108 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3I_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3I_0.trimmed_R2_.fastq.gz
[main] Real time: 0.264 sec; CPU: 3.084 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40200 sequences (3839100 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10060, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.19, 14.41)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40200 reads in 3.027 CPU sec, 0.133 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3J_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3J_0.trimmed_R2_.fastq.gz
[main] Real time: 0.287 sec; CPU: 3.090 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 40152 sequences (3834516 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 10123, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.27, 14.41)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 40152 reads in 3.084 CPU sec, 0.120 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3K_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3K_0.trimmed_R2_.fastq.gz
[main] Real time: 0.282 sec; CPU: 3.155 sec
[M::bwa_idx_load_from_disk] read 0 ALT contigs
[M::process] read 39864 sequences (3807012 bp)...
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 9964, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (452, 464, 477)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (402, 527)
[M::mem_pestat] mean and std.dev: (464.23, 14.40)
[M::mem_pestat] low and high boundaries for proper pairs: (377, 552)
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_process_seqs] Processed 39864 reads in 3.026 CPU sec, 0.122 real sec
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 /home/iovercast/manuscript-analysis/REFMAP_SIM/ipsimdata/pairddrad_wmerge_example_genome.fa /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3L_0.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/refmap-sim_edits/3L_0.trimmed_R2_.fastq.gz
[main] Real time: 0.286 sec; CPU: 3.100 sec
In [22]:
## This is how we'd do it since we weren't using a popmap file
infiles = ["-s "+ff+" " for ff in glob.glob(STACKS_SIM_DIR + "*.bam")]

## Toggle the dryrun flag for testing
DRYRUN=""
DRYRUN="-d"

## Options
## -T    The number of threads to use
## -O    The popmap file specifying individuals and populations
## -S    Disable database business
## -o    Output directory. Just write to the empirical stacks directory
## -X    Tell populations to create the output formats specified
## -X    and use `-m 6` which sets min depth per locus
OUTPUT_FORMATS = "--vcf --genepop --structure --phylip "
cmd = "ref_map.pl -T 40 -b 1 -S " + DRYRUN\
        + " -X \'populations:" + OUTPUT_FORMATS + "\'"\
        + " -X \'populations:-m 6\'"\
        + " -o " + STACKS_SIM_DIR + " "\
        + " ".join(infiles)
print("\nCommand to run - {}".format(cmd))
Command to run - ref_map.pl -T 40 -b 1 -S -d -X 'populations:--vcf --genepop --structure --phylip ' -X 'populations:-m 6' -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/ -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1A_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1B_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1C_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1D_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2E_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2F_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2G_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2H_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3I_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3J_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3K_0.bam  -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3L_0.bam 
In [21]:
%%bash -s "$WORK_DIR" "$STACKS_SIM_DIR" "$cmd"
export PATH="$1/miniconda/bin:$PATH"; export "STACKS_SIM_DIR=$2"; export "cmd=$3"

## We have to play a little cat and mouse game here because of quoting in some of the args
## and how weird bash is we have to write the cmd to a file and then exec it.
## If you try to just run $cmd it truncates the command at the first single tic. Hassle.
cd $STACKS_SIM_DIR
echo $cmd > stacks.sh; chmod 777 stacks.sh
time ./stacks.sh
Identifying unique stacks; file   1 of  12 [1A_0]
Identifying unique stacks; file   2 of  12 [1B_0]
Identifying unique stacks; file   3 of  12 [1C_0]
Identifying unique stacks; file   4 of  12 [1D_0]
Identifying unique stacks; file   5 of  12 [2E_0]
Identifying unique stacks; file   6 of  12 [2F_0]
Identifying unique stacks; file   7 of  12 [2G_0]
Identifying unique stacks; file   8 of  12 [2H_0]
Identifying unique stacks; file   9 of  12 [3I_0]
Identifying unique stacks; file  10 of  12 [3J_0]
Identifying unique stacks; file  11 of  12 [3K_0]
Identifying unique stacks; file  12 of  12 [3L_0]
Found 12 sample file(s).
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1A_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 1  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1B_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 2  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1C_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 3  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1D_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 4  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2E_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 5  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2F_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 6  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2G_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 7  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2H_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 8  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3I_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 9  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3J_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 10  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3K_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 11  -p 40  -m 1 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3L_0.bam -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -i 12  -p 40  -m 1 2>&1

Depths of Coverage for Processed Samples:

Generating catalog...
  /home/iovercast/manuscript-analysis/miniconda/bin/cstacks -g -b 1 -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1A_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1B_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1C_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1D_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2E_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2F_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2G_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2H_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3I_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3J_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3K_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3L_0  -p 40 2>&1
Matching samples to the catalog...
  /home/iovercast/manuscript-analysis/miniconda/bin/sstacks -g -b 1 -c /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/batch_1 -o /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1A_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1B_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1C_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/1D_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2E_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2F_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2G_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/2H_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3I_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3J_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3K_0 -s /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks/3L_0  -p 40 2>&1
Calculating population-level summary statistics
/home/iovercast/manuscript-analysis/miniconda/bin/populations -b 1 -P /home/iovercast/manuscript-analysis/REFMAP_SIM/stacks -s -t 40 --vcf --genepop --structure --phylip  -m 6 2>&1

real	0m0.040s
user	0m0.030s
sys	0m0.007s

dDocent simulated reference assembly

dDocent reference sequence mapping is not clearly documented. It turns out you can skip the initial assembly and go right to mapping/snp calling, but it requires you to copy the reference sequence to the dDocent workind directory as "reference.fasta". This uses the trimmed and QC'd fastq files from the ipyrad simulated run, so it assumes you have already done that and the fq files are still in place.

In [92]:
IPYRAD_SIMEDITS_DIR = IPYRAD_SIM_DIR + "reference-assembly/refmap-sim_edits/"
REF_SEQ = REFMAP_DAT_DIR + "pairddrad_wmerge_example_genome.fa"
DDOCENT_DIR = "/home/iovercast/manuscript-analysis/dDocent/"
os.chdir(DDOCENT_SIM_DIR)

## Create a simlink to the reference sequence in the current directory
cmd = "ln -sf {} reference.fasta".format(REF_SEQ)
!$cmd

## Sim sample names
pop1 = ["1A_0", "1B_0", "1C_0", "1D_0"]
pop2 = ["2E_0", "2F_0", "2G_0", "2H_0"]
pop3 = ["3I_0", "3J_0", "3K_0", "3L_0"]
sim_sample_names = pop1 + pop2 + pop3
sim_mapping_dict = {}

for pop_num, samps in enumerate([pop1, pop2, pop3]):
    for samp_num, samp_name in enumerate(samps):
        sim_mapping_dict[samp_name] = "Pop{}_{:03d}".format(pop_num+1, samp_num+1)

## Now we have to rename all the files in the way dDocent expects them:
## 1A_0_R1_.fastq.gz -> Pop1_001.F.fq.gz
for k, v in sim_mapping_dict.items():
    ## Symlink R1 and R2
    for i in ["1", "2"]:
        source = os.path.join(IPYRAD_SIMEDITS_DIR, k + ".trimmed_R{}_.fastq.gz".format(i))
        ## This is the way the current documentation says to name imported trimmed
        ## files, but it doesn't work.
        ## dest = os.path.join(DDOCENT_SIM_DIR, v + ".R{}.fq.gz".format(i))
        if i == "1":
            dest = os.path.join(DDOCENT_SIM_DIR, v + ".F.fq.gz".format(i))
        else:
            dest = os.path.join(DDOCENT_SIM_DIR, v + ".R.fq.gz".format(i))
        cmd = "ln -sf {} {}".format(source, dest)
        !$cmd

config_file = "{}/sim-config.txt".format(DDOCENT_SIM_DIR)
with open(config_file, 'w') as outfile:
    outfile.write('Number of Processors\n40\nMaximum Memory\n0\nTrimming\nno\nAssembly?\nno\nType_of_Assembly\nPE\nClustering_Similarity%\n0.85\nMapping_Reads?\nyes\nMapping_Match_Value\n1\nMapping_MisMatch_Value\n3\nMapping_GapOpen_Penalty\n5\nCalling_SNPs?\nyes\nEmail\n[email protected]\n')

cmd = "export LD_LIBRARY_PATH={}/freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; ".format(DDOCENT_DIR)
cmd += "export PATH={}:$PATH; time dDocent {}".format(DDOCENT_DIR, config_file)
print(cmd)
with open("ddocent.sh", 'w') as outfile:
    outfile.write("#!/bin/bash\n")
    outfile.write(cmd)
!chmod 777 ddocent.sh
export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH; time dDocent /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent//sim-config.txt
In [93]:
## You have to post-process the vcf files to decompose complex genotypes and remove indels
os.chdir(DDOCENT_SIM_DIR)
exports = "export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH"

fullvcf = os.path.join(DDOCENT_SIM_DIR, "TotalRawSNPs.vcf")
filtvcf = os.path.join(DDOCENT_SIM_DIR, "Final.recode.vcf")
for f in [fullvcf, filtvcf]:
    print("Finalizing - {}".format(f))
    
    ## Rename the samples to make them agree with the ipyrad/stacks names so
    ## the results analysis will work.
    vcffile = os.path.join(DDOCENT_SIM_DIR, f)
    infile = open(vcffile,'r')
    filedata = infile.readlines()
    infile.close()
    
    outfile = open(vcffile,'w')
    for line in filedata:
        if "CHROM" in line:
            for ipname, ddname in sim_mapping_dict.items():
                line = line.replace(ddname, ipname)
        outfile.write(line)
    outfile.close()
    
    ## Naming the new outfiles as <curname>.snps.vcf
    ## Decompose complex genotypes and remove indels
    outfile = os.path.join(DDOCENT_SIM_DIR, f.split("/")[-1].split(".vcf")[0] + ".snps.vcf")
    cmd = "{}; vcfallelicprimitives {} > ddoc-tmp.vcf".format(exports, f)
    print(cmd)
    !$cmd
    cmd = "{}; vcftools --vcf ddoc-tmp.vcf --remove-indels --recode --recode-INFO-all --out {}".format(exports, outfile)
    print(cmd)
    !$cmd
    !rm ddoc-tmp.vcf
Finalizing - /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/TotalRawSNPs.vcf
export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH; vcfallelicprimitives /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/TotalRawSNPs.vcf > ddoc-tmp.vcf
export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH; vcftools --vcf ddoc-tmp.vcf --remove-indels --recode --recode-INFO-all --out /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/TotalRawSNPs.snps.vcf

VCFtools - v0.1.11
(C) Adam Auton 2009

Parameters as interpreted:
	--vcf ddoc-tmp.vcf
	--recode-INFO-all
	--out /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/TotalRawSNPs.snps.vcf
	--recode
	--remove-indels

Index file is older than variant file. Will regenerate.
Building new index file.
	Scanning Chromosome: MT
Writing Index file.
File contains 4842 entries and 12 individuals.
Applying Required Filters.
Filtering sites by allele type
After filtering, kept 12 out of 12 Individuals
After filtering, kept 4840 out of a possible 4842 Sites
Outputting VCF file... Done
Run Time = 0.00 seconds
Finalizing - /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/Final.recode.vcf
export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH; vcfallelicprimitives /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/Final.recode.vcf > ddoc-tmp.vcf
export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH; vcftools --vcf ddoc-tmp.vcf --remove-indels --recode --recode-INFO-all --out /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/Final.recode.snps.vcf

VCFtools - v0.1.11
(C) Adam Auton 2009

Parameters as interpreted:
	--vcf ddoc-tmp.vcf
	--recode-INFO-all
	--out /home/iovercast/manuscript-analysis/REFMAP_SIM/ddocent/Final.recode.snps.vcf
	--recode
	--remove-indels

Index file is older than variant file. Will regenerate.
Building new index file.
	Scanning Chromosome: MT
Writing Index file.
File contains 4723 entries and 12 individuals.
Applying Required Filters.
Filtering sites by allele type
After filtering, kept 12 out of 12 Individuals
After filtering, kept 4722 out of a possible 4723 Sites
Outputting VCF file... Done
Run Time = 1.00 seconds

Empirical reference sequence mapping

Fetch the Phocoena raw sequence data

We will use the sra-toolkit command fastq-dump to pull the PE reads out of SRA. This maybe isn't the best way, or the quickest, but it'll get the job done. Takes ~30 minutes and requires ~70GB of space. After I downloaded the fq I looked at a couple random samples in FastQC to get an idea where to trim in step2.

In [ ]:
os.chdir(REFMAP_EMPIRICAL_DIR)
!mkdir raws
!cd raws
## Grab the sra-toolkit pre-built binaries to download from SRA
## This works, but commented for now so it doesn't keep redownloading
!wget http://ftp-trace.ncbi.nlm.nih.gov/sra/sdk/2.8.0/sratoolkit.2.8.0-ubuntu64.tar.gz
!tar -xvzf sratoolkit*
FQ_DUMP = os.path.join(REFMAP_EMPIRICAL_DIR, "sratoolkit.2.8.0-ubuntu64/bin/fastq-dump")
res = subprocess.check_output(FQ_DUMP + " -version", shell=True)

## The SRR numbers for the samples from this bioproject range from SRR4291662 to SRR4291705
## so go fetch them one by one
for samp in range(662, 706):
    print("Doing {}\t".format(samp)),
    res = subprocess.check_output(FQ_DUMP + " --split-files SRR4291" + str(samp), shell=True)
Doing 662	Doing 663	Doing 664	Doing 665	Doing 666	Doing 667	Doing 668	Doing 669	Doing 670	Doing 671	Doing 672	Doing 673	Doing 674	Doing 675	Doing 676	Doing 677	Doing 678	Doing 679	Doing 680	Doing 681	Doing 682	Doing 683	Doing 684	Doing 685	Doing 686	Doing 687	Doing 688	Doing 689	Doing 690	Doing 691	Doing 692	Doing 693	Doing 694	Doing 695	Doing 696	Doing 697	Doing 698	Doing 699	Doing 700	Doing 701	Doing 702	
In [48]:
## The SRA download files have wonky names, like SRR1234_R1.fastq.gz, but ipyrad expects SRR1234_R1_.fastq.gz,
## so we have to fix the filenames. Filename hax...
import glob
for f in glob.glob(REFMAP_EMPIRICAL_DIR + "raws/*.fastq.gz"):
    splits = f.split("/")[-1].split("_")
    newf = REFMAP_EMPIRICAL_DIR + "raws/" + splits[0] + "_R" + splits[1].split(".")[0] + "_.fastq.gz"
    os.rename(f, newf)

Fetch the bottlenose dolphin genome

Tursiops truncatus reference genome. Divergence time between dolphin and porpoise is approximately 15Mya, which is on the order of divergence between humans and orang. There is also a genome for the Minke whale, which is much more deeply diverged (~30Mya), could be interesting to try both to see how it works.

Minke whale - http://www.nature.com/ng/journal/v46/n1/full/ng.2835.html#accessions

SRA Data table for converting fq files to sample name as used in the paper: file:///home/chronos/u-b20882bda0f801c7265d4462f127d0cb4376d46d/Downloads/Tune/SraRunTable.txt

In [30]:
os.chdir(REFMAP_EMPIRICAL_DIR)
!mkdir TurtrunRef
!cd TurtrunRef
!wget ftp://ftp.ensembl.org/pub/release-87/fasta/tursiops_truncatus/dna/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa.gz

## Ensembl distributes gzip'd reference sequence files, but samtools really wants it to be bgzipped or uncompressed
!gunzip Tursiops_truncatus.turTru1.dna_rm.toplevel.fa.gz
--2016-12-18 10:29:57--  ftp://ftp.ensembl.org/pub/release-87/fasta/tursiops_truncatus/dna/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa.gz
           => ‘Tursiops_truncatus.turTru1.dna_rm.toplevel.fa.gz’
Resolving ftp.ensembl.org (ftp.ensembl.org)... 193.62.203.85
Connecting to ftp.ensembl.org (ftp.ensembl.org)|193.62.203.85|:21... connected.
Logging in as anonymous ... Logged in!
==> SYST ... done.    ==> PWD ... done.
==> TYPE I ... done.  ==> CWD (1) /pub/release-87/fasta/tursiops_truncatus/dna ... done.
==> SIZE Tursiops_truncatus.turTru1.dna_rm.toplevel.fa.gz ... 453168562
==> PASV ... done.    ==> RETR Tursiops_truncatus.turTru1.dna_rm.toplevel.fa.gz ... done.
Length: 453168562 (432M) (unauthoritative)

100%[======================================>] 453,168,562 2.88MB/s   in 3m 33s 

2016-12-18 10:33:32 (2.03 MB/s) - ‘Tursiops_truncatus.turTru1.dna_rm.toplevel.fa.gz’ saved [453168562]

Trim reads w/ cutadapt

To reduce any potential bias introduced by differences in trimming and filtering methods we will trim reads w/ cutadapt, and QC w/ ipyrad and use this dataset as the starting point for assembly. If you are wondering, you can run fastqc from the command line, like this

fastqc -o fastqc_out/ SRR4291662_1.fastq SRR4291662_2.fastq

We'll trim R1 and R2 to 85bp following Lah et al 2016. The -l flag for cutadapt specifies the length to which each read will be trimmed.

In [54]:
%%bash -s "$REFMAP_EMPIRICAL_DIR"
cd $1
mkdir trimmed
for i in `ls raws`; do echo $i; cutadapt -l 85 raws/$i | gzip > trimmed/$i; done
## Housekeeping
rm -rf raws
mv trimmed raws
SRR4291662_R1_.fastq.gz
SRR4291662_R2_.fastq.gz
SRR4291663_R1_.fastq.gz
SRR4291663_R2_.fastq.gz
SRR4291664_R1_.fastq.gz
SRR4291664_R2_.fastq.gz
SRR4291665_R1_.fastq.gz
SRR4291665_R2_.fastq.gz
SRR4291666_R1_.fastq.gz
SRR4291666_R2_.fastq.gz
SRR4291667_R1_.fastq.gz
SRR4291667_R2_.fastq.gz
SRR4291668_R1_.fastq.gz
SRR4291668_R2_.fastq.gz
SRR4291669_R1_.fastq.gz
SRR4291669_R2_.fastq.gz
SRR4291670_R1_.fastq.gz
SRR4291670_R2_.fastq.gz
SRR4291671_R1_.fastq.gz
SRR4291671_R2_.fastq.gz
SRR4291672_R1_.fastq.gz
SRR4291672_R2_.fastq.gz
SRR4291673_R1_.fastq.gz
SRR4291673_R2_.fastq.gz
SRR4291674_R1_.fastq.gz
SRR4291674_R2_.fastq.gz
SRR4291675_R1_.fastq.gz
SRR4291675_R2_.fastq.gz
SRR4291676_R1_.fastq.gz
SRR4291676_R2_.fastq.gz
SRR4291677_R1_.fastq.gz
SRR4291677_R2_.fastq.gz
SRR4291678_R1_.fastq.gz
SRR4291678_R2_.fastq.gz
SRR4291679_R1_.fastq.gz
SRR4291679_R2_.fastq.gz
SRR4291680_R1_.fastq.gz
SRR4291680_R2_.fastq.gz
SRR4291681_R1_.fastq.gz
SRR4291681_R2_.fastq.gz
SRR4291682_R1_.fastq.gz
SRR4291682_R2_.fastq.gz
SRR4291683_R1_.fastq.gz
SRR4291683_R2_.fastq.gz
SRR4291684_R1_.fastq.gz
SRR4291684_R2_.fastq.gz
SRR4291685_R1_.fastq.gz
SRR4291685_R2_.fastq.gz
SRR4291686_R1_.fastq.gz
SRR4291686_R2_.fastq.gz
SRR4291687_R1_.fastq.gz
SRR4291687_R2_.fastq.gz
SRR4291688_R1_.fastq.gz
SRR4291688_R2_.fastq.gz
SRR4291689_R1_.fastq.gz
SRR4291689_R2_.fastq.gz
SRR4291690_R1_.fastq.gz
SRR4291690_R2_.fastq.gz
SRR4291691_R1_.fastq.gz
SRR4291691_R2_.fastq.gz
SRR4291692_R1_.fastq.gz
SRR4291692_R2_.fastq.gz
SRR4291693_R1_.fastq.gz
SRR4291693_R2_.fastq.gz
SRR4291694_R1_.fastq.gz
SRR4291694_R2_.fastq.gz
SRR4291695_R1_.fastq.gz
SRR4291695_R2_.fastq.gz
SRR4291696_R1_.fastq.gz
SRR4291696_R2_.fastq.gz
SRR4291697_R1_.fastq.gz
SRR4291697_R2_.fastq.gz
SRR4291698_R1_.fastq.gz
SRR4291698_R2_.fastq.gz
SRR4291699_R1_.fastq.gz
SRR4291699_R2_.fastq.gz
SRR4291700_R1_.fastq.gz
SRR4291700_R2_.fastq.gz
SRR4291701_R1_.fastq.gz
SRR4291701_R2_.fastq.gz
SRR4291702_R1_.fastq.gz
SRR4291702_R2_.fastq.gz
SRR4291703_R1_.fastq.gz
SRR4291703_R2_.fastq.gz
SRR4291704_R1_.fastq.gz
SRR4291704_R2_.fastq.gz
SRR4291705_R1_.fastq.gz
SRR4291705_R2_.fastq.gz
mkdir: cannot create directory ‘trimmed’: File exists
This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291662_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.66 s (6 us/read; 10.37 M reads/minute).

=== Summary ===

Total reads processed:               3,225,657
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,225,657 (100.0%)

Total basepairs processed:   302,535,710 bp
Total written (filtered):    273,671,316 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291662_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.43 s (6 us/read; 10.50 M reads/minute).

=== Summary ===

Total reads processed:               3,225,657
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,225,657 (100.0%)

Total basepairs processed:   321,680,483 bp
Total written (filtered):    273,633,566 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291663_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 14.78 s (6 us/read; 10.56 M reads/minute).

=== Summary ===

Total reads processed:               2,602,278
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,602,278 (100.0%)

Total basepairs processed:   241,637,058 bp
Total written (filtered):    220,902,242 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291663_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 14.96 s (6 us/read; 10.44 M reads/minute).

=== Summary ===

Total reads processed:               2,602,278
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,602,278 (100.0%)

Total basepairs processed:   259,728,172 bp
Total written (filtered):    220,886,902 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291664_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 7.47 s (6 us/read; 10.51 M reads/minute).

=== Summary ===

Total reads processed:               1,308,140
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,308,140 (100.0%)

Total basepairs processed:   124,071,961 bp
Total written (filtered):    111,051,112 bp (89.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291664_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 7.39 s (6 us/read; 10.62 M reads/minute).

=== Summary ===

Total reads processed:               1,308,140
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,308,140 (100.0%)

Total basepairs processed:   130,566,117 bp
Total written (filtered):    111,041,864 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291665_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 8.12 s (6 us/read; 10.51 M reads/minute).

=== Summary ===

Total reads processed:               1,422,513
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,422,513 (100.0%)

Total basepairs processed:   132,119,445 bp
Total written (filtered):    120,780,161 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291665_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 8.14 s (6 us/read; 10.49 M reads/minute).

=== Summary ===

Total reads processed:               1,422,513
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,422,513 (100.0%)

Total basepairs processed:   142,019,450 bp
Total written (filtered):    120,774,460 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291666_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.56 s (6 us/read; 10.47 M reads/minute).

=== Summary ===

Total reads processed:               3,237,438
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,237,438 (100.0%)

Total basepairs processed:   300,710,625 bp
Total written (filtered):    274,900,243 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291666_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.72 s (6 us/read; 10.38 M reads/minute).

=== Summary ===

Total reads processed:               3,237,438
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,237,438 (100.0%)

Total basepairs processed:   323,231,561 bp
Total written (filtered):    274,880,218 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291667_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 23.73 s (6 us/read; 10.54 M reads/minute).

=== Summary ===

Total reads processed:               4,170,153
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     4,170,153 (100.0%)

Total basepairs processed:   390,976,823 bp
Total written (filtered):    353,700,081 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291667_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 23.96 s (6 us/read; 10.44 M reads/minute).

=== Summary ===

Total reads processed:               4,170,153
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     4,170,153 (100.0%)

Total basepairs processed:   415,710,634 bp
Total written (filtered):    353,654,174 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291668_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 20.04 s (6 us/read; 10.33 M reads/minute).

=== Summary ===

Total reads processed:               3,450,882
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,450,882 (100.0%)

Total basepairs processed:   310,304,790 bp
Total written (filtered):    293,093,219 bp (94.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291668_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.70 s (6 us/read; 10.51 M reads/minute).

=== Summary ===

Total reads processed:               3,450,882
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,450,882 (100.0%)

Total basepairs processed:   344,647,193 bp
Total written (filtered):    293,069,094 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291669_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 21.57 s (6 us/read; 10.18 M reads/minute).

=== Summary ===

Total reads processed:               3,658,457
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,658,457 (100.0%)

Total basepairs processed:   332,569,307 bp
Total written (filtered):    310,678,470 bp (93.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291669_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 21.07 s (6 us/read; 10.42 M reads/minute).

=== Summary ===

Total reads processed:               3,658,457
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,658,457 (100.0%)

Total basepairs processed:   365,337,470 bp
Total written (filtered):    310,656,388 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291670_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 8.09 s (6 us/read; 10.59 M reads/minute).

=== Summary ===

Total reads processed:               1,428,040
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,428,040 (100.0%)

Total basepairs processed:   135,277,065 bp
Total written (filtered):    121,110,383 bp (89.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291670_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 8.11 s (6 us/read; 10.57 M reads/minute).

=== Summary ===

Total reads processed:               1,428,040
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,428,040 (100.0%)

Total basepairs processed:   142,339,159 bp
Total written (filtered):    121,090,656 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291671_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 21.11 s (6 us/read; 10.59 M reads/minute).

=== Summary ===

Total reads processed:               3,726,102
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,726,102 (100.0%)

Total basepairs processed:   346,070,011 bp
Total written (filtered):    316,369,233 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291671_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 20.70 s (6 us/read; 10.80 M reads/minute).

=== Summary ===

Total reads processed:               3,726,102
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,726,102 (100.0%)

Total basepairs processed:   371,995,572 bp
Total written (filtered):    316,342,060 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291672_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 26.01 s (8 us/read; 7.51 M reads/minute).

=== Summary ===

Total reads processed:               3,256,943
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,256,943 (100.0%)

Total basepairs processed:   305,656,861 bp
Total written (filtered):    276,475,866 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291672_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 20.99 s (6 us/read; 9.31 M reads/minute).

=== Summary ===

Total reads processed:               3,256,943
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,256,943 (100.0%)

Total basepairs processed:   325,037,311 bp
Total written (filtered):    276,448,443 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291673_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 12.23 s (6 us/read; 10.44 M reads/minute).

=== Summary ===

Total reads processed:               2,128,431
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,128,431 (100.0%)

Total basepairs processed:   199,673,694 bp
Total written (filtered):    180,616,031 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291673_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 11.72 s (6 us/read; 10.90 M reads/minute).

=== Summary ===

Total reads processed:               2,128,431
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,128,431 (100.0%)

Total basepairs processed:   212,332,918 bp
Total written (filtered):    180,599,472 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291674_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 14.54 s (6 us/read; 10.76 M reads/minute).

=== Summary ===

Total reads processed:               2,606,704
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,606,704 (100.0%)

Total basepairs processed:   234,287,729 bp
Total written (filtered):    221,303,963 bp (94.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291674_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 14.52 s (6 us/read; 10.77 M reads/minute).

=== Summary ===

Total reads processed:               2,606,704
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,606,704 (100.0%)

Total basepairs processed:   260,154,896 bp
Total written (filtered):    221,272,346 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291675_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.16 s (6 us/read; 10.71 M reads/minute).

=== Summary ===

Total reads processed:               3,242,962
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,242,962 (100.0%)

Total basepairs processed:   297,982,996 bp
Total written (filtered):    275,355,483 bp (92.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291675_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.60 s (6 us/read; 10.46 M reads/minute).

=== Summary ===

Total reads processed:               3,242,962
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,242,962 (100.0%)

Total basepairs processed:   323,769,609 bp
Total written (filtered):    275,327,109 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291676_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 14.46 s (6 us/read; 10.23 M reads/minute).

=== Summary ===

Total reads processed:               2,466,111
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,466,111 (100.0%)

Total basepairs processed:   221,799,201 bp
Total written (filtered):    209,492,582 bp (94.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291676_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 14.33 s (6 us/read; 10.33 M reads/minute).

=== Summary ===

Total reads processed:               2,466,111
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,466,111 (100.0%)

Total basepairs processed:   246,375,382 bp
Total written (filtered):    209,484,079 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291677_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.64 s (6 us/read; 10.38 M reads/minute).

=== Summary ===

Total reads processed:               3,397,721
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,397,721 (100.0%)

Total basepairs processed:   308,581,562 bp
Total written (filtered):    288,301,569 bp (93.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291677_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.30 s (6 us/read; 10.56 M reads/minute).

=== Summary ===

Total reads processed:               3,397,721
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,397,721 (100.0%)

Total basepairs processed:   338,887,177 bp
Total written (filtered):    288,267,890 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291678_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.95 s (6 us/read; 9.65 M reads/minute).

=== Summary ===

Total reads processed:               3,048,039
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,048,039 (100.0%)

Total basepairs processed:   276,868,465 bp
Total written (filtered):    258,666,672 bp (93.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291678_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 17.05 s (6 us/read; 10.73 M reads/minute).

=== Summary ===

Total reads processed:               3,048,039
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,048,039 (100.0%)

Total basepairs processed:   304,094,709 bp
Total written (filtered):    258,647,431 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291679_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 23.03 s (6 us/read; 10.75 M reads/minute).

=== Summary ===

Total reads processed:               4,127,787
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     4,127,787 (100.0%)

Total basepairs processed:   378,825,733 bp
Total written (filtered):    350,115,785 bp (92.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291679_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 23.27 s (6 us/read; 10.64 M reads/minute).

=== Summary ===

Total reads processed:               4,127,787
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     4,127,787 (100.0%)

Total basepairs processed:   411,474,061 bp
Total written (filtered):    350,061,395 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291680_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 16.20 s (6 us/read; 10.81 M reads/minute).

=== Summary ===

Total reads processed:               2,917,555
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,917,555 (100.0%)

Total basepairs processed:   265,159,848 bp
Total written (filtered):    247,711,645 bp (93.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291680_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 21.15 s (7 us/read; 8.28 M reads/minute).

=== Summary ===

Total reads processed:               2,917,555
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,917,555 (100.0%)

Total basepairs processed:   291,272,774 bp
Total written (filtered):    247,692,864 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291681_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.07 s (6 us/read; 10.91 M reads/minute).

=== Summary ===

Total reads processed:               3,466,670
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,466,670 (100.0%)

Total basepairs processed:   325,229,207 bp
Total written (filtered):    294,186,403 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291681_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.76 s (6 us/read; 10.53 M reads/minute).

=== Summary ===

Total reads processed:               3,466,670
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,466,670 (100.0%)

Total basepairs processed:   345,853,871 bp
Total written (filtered):    294,161,220 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291682_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 20.14 s (6 us/read; 10.49 M reads/minute).

=== Summary ===

Total reads processed:               3,519,888
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,519,888 (100.0%)

Total basepairs processed:   326,570,566 bp
Total written (filtered):    298,588,290 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291682_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.88 s (6 us/read; 10.62 M reads/minute).

=== Summary ===

Total reads processed:               3,519,888
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,519,888 (100.0%)

Total basepairs processed:   350,929,362 bp
Total written (filtered):    298,543,164 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291683_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 11.83 s (6 us/read; 10.47 M reads/minute).

=== Summary ===

Total reads processed:               2,064,559
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,064,559 (100.0%)

Total basepairs processed:   187,046,314 bp
Total written (filtered):    174,797,570 bp (93.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291683_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 11.70 s (6 us/read; 10.59 M reads/minute).

=== Summary ===

Total reads processed:               2,064,559
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,064,559 (100.0%)

Total basepairs processed:   205,296,143 bp
Total written (filtered):    174,759,321 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291684_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 17.51 s (6 us/read; 10.58 M reads/minute).

=== Summary ===

Total reads processed:               3,086,468
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,086,468 (100.0%)

Total basepairs processed:   289,461,086 bp
Total written (filtered):    261,848,374 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291684_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 17.64 s (6 us/read; 10.50 M reads/minute).

=== Summary ===

Total reads processed:               3,086,468
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,086,468 (100.0%)

Total basepairs processed:   307,783,378 bp
Total written (filtered):    261,819,338 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291685_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 13.52 s (6 us/read; 10.86 M reads/minute).

=== Summary ===

Total reads processed:               2,446,083
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,446,083 (100.0%)

Total basepairs processed:   229,320,135 bp
Total written (filtered):    207,457,048 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291685_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 13.74 s (6 us/read; 10.68 M reads/minute).

=== Summary ===

Total reads processed:               2,446,083
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,446,083 (100.0%)

Total basepairs processed:   243,821,853 bp
Total written (filtered):    207,429,219 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291686_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.07 s (6 us/read; 10.63 M reads/minute).

=== Summary ===

Total reads processed:               3,201,430
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,201,430 (100.0%)

Total basepairs processed:   287,489,343 bp
Total written (filtered):    271,572,152 bp (94.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291686_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 24.01 s (7 us/read; 8.00 M reads/minute).

=== Summary ===

Total reads processed:               3,201,430
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,201,430 (100.0%)

Total basepairs processed:   319,198,511 bp
Total written (filtered):    271,529,648 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291687_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.34 s (6 us/read; 10.62 M reads/minute).

=== Summary ===

Total reads processed:               3,245,019
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,245,019 (100.0%)

Total basepairs processed:   291,561,672 bp
Total written (filtered):    275,408,840 bp (94.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291687_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 17.94 s (6 us/read; 10.85 M reads/minute).

=== Summary ===

Total reads processed:               3,245,019
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,245,019 (100.0%)

Total basepairs processed:   323,754,288 bp
Total written (filtered):    275,370,853 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291688_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 8.72 s (6 us/read; 10.76 M reads/minute).

=== Summary ===

Total reads processed:               1,564,117
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,564,117 (100.0%)

Total basepairs processed:   146,438,492 bp
Total written (filtered):    132,507,950 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291688_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 8.69 s (6 us/read; 10.80 M reads/minute).

=== Summary ===

Total reads processed:               1,564,117
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,564,117 (100.0%)

Total basepairs processed:   155,657,876 bp
Total written (filtered):    132,487,616 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291689_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.13 s (6 us/read; 10.53 M reads/minute).

=== Summary ===

Total reads processed:               3,181,938
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,181,938 (100.0%)

Total basepairs processed:   298,646,806 bp
Total written (filtered):    270,124,604 bp (90.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291689_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 17.88 s (6 us/read; 10.68 M reads/minute).

=== Summary ===

Total reads processed:               3,181,938
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,181,938 (100.0%)

Total basepairs processed:   317,599,989 bp
Total written (filtered):    270,100,955 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291690_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 15.60 s (6 us/read; 10.29 M reads/minute).

=== Summary ===

Total reads processed:               2,674,772
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,674,772 (100.0%)

Total basepairs processed:   253,734,600 bp
Total written (filtered):    227,093,686 bp (89.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291690_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 15.38 s (6 us/read; 10.43 M reads/minute).

=== Summary ===

Total reads processed:               2,674,772
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,674,772 (100.0%)

Total basepairs processed:   267,026,097 bp
Total written (filtered):    227,081,425 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291691_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 35.33 s (6 us/read; 10.02 M reads/minute).

=== Summary ===

Total reads processed:               5,903,027
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     5,903,027 (100.0%)

Total basepairs processed:   548,419,385 bp
Total written (filtered):    501,329,616 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291691_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 34.24 s (6 us/read; 10.34 M reads/minute).

=== Summary ===

Total reads processed:               5,903,027
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     5,903,027 (100.0%)

Total basepairs processed:   589,507,289 bp
Total written (filtered):    501,290,530 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291692_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 6.55 s (6 us/read; 10.74 M reads/minute).

=== Summary ===

Total reads processed:               1,172,868
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,172,868 (100.0%)

Total basepairs processed:   108,479,568 bp
Total written (filtered):     99,222,769 bp (91.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291692_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 6.43 s (5 us/read; 10.94 M reads/minute).

=== Summary ===

Total reads processed:               1,172,868
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,172,868 (100.0%)

Total basepairs processed:   116,504,462 bp
Total written (filtered):     99,193,009 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291693_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 12.27 s (6 us/read; 10.63 M reads/minute).

=== Summary ===

Total reads processed:               2,174,717
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,174,717 (100.0%)

Total basepairs processed:   199,675,649 bp
Total written (filtered):    184,528,700 bp (92.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291693_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 12.04 s (6 us/read; 10.84 M reads/minute).

=== Summary ===

Total reads processed:               2,174,717
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,174,717 (100.0%)

Total basepairs processed:   216,926,920 bp
Total written (filtered):    184,508,611 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291694_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 20.30 s (6 us/read; 10.74 M reads/minute).

=== Summary ===

Total reads processed:               3,632,242
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,632,242 (100.0%)

Total basepairs processed:   340,683,039 bp
Total written (filtered):    308,182,877 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291694_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 20.48 s (6 us/read; 10.64 M reads/minute).

=== Summary ===

Total reads processed:               3,632,242
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,632,242 (100.0%)

Total basepairs processed:   362,256,218 bp
Total written (filtered):    308,150,487 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291695_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 10.68 s (6 us/read; 10.43 M reads/minute).

=== Summary ===

Total reads processed:               1,856,718
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,856,718 (100.0%)

Total basepairs processed:   173,941,660 bp
Total written (filtered):    157,377,022 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291695_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 10.78 s (6 us/read; 10.33 M reads/minute).

=== Summary ===

Total reads processed:               1,856,718
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,856,718 (100.0%)

Total basepairs processed:   184,905,679 bp
Total written (filtered):    157,341,499 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291696_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.31 s (6 us/read; 10.39 M reads/minute).

=== Summary ===

Total reads processed:               3,345,439
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,345,439 (100.0%)

Total basepairs processed:   307,309,204 bp
Total written (filtered):    283,984,631 bp (92.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291696_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.43 s (6 us/read; 10.89 M reads/minute).

=== Summary ===

Total reads processed:               3,345,439
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,345,439 (100.0%)

Total basepairs processed:   333,884,503 bp
Total written (filtered):    283,956,832 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291697_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.52 s (6 us/read; 10.40 M reads/minute).

=== Summary ===

Total reads processed:               3,210,823
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,210,823 (100.0%)

Total basepairs processed:   294,875,650 bp
Total written (filtered):    272,500,412 bp (92.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291697_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.37 s (6 us/read; 10.49 M reads/minute).

=== Summary ===

Total reads processed:               3,210,823
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,210,823 (100.0%)

Total basepairs processed:   320,361,719 bp
Total written (filtered):    272,471,463 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291698_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 10.47 s (6 us/read; 10.26 M reads/minute).

=== Summary ===

Total reads processed:               1,790,978
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,790,978 (100.0%)

Total basepairs processed:   167,594,570 bp
Total written (filtered):    151,658,671 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291698_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 10.25 s (6 us/read; 10.48 M reads/minute).

=== Summary ===

Total reads processed:               1,790,978
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     1,790,978 (100.0%)

Total basepairs processed:   178,125,550 bp
Total written (filtered):    151,625,435 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291699_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 15.90 s (6 us/read; 10.63 M reads/minute).

=== Summary ===

Total reads processed:               2,816,420
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,816,420 (100.0%)

Total basepairs processed:   261,543,984 bp
Total written (filtered):    239,104,344 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291699_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 16.04 s (6 us/read; 10.54 M reads/minute).

=== Summary ===

Total reads processed:               2,816,420
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,816,420 (100.0%)

Total basepairs processed:   281,129,007 bp
Total written (filtered):    239,083,585 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291700_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 15.41 s (6 us/read; 10.39 M reads/minute).

=== Summary ===

Total reads processed:               2,668,256
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,668,256 (100.0%)

Total basepairs processed:   249,952,844 bp
Total written (filtered):    226,146,252 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291700_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 15.27 s (6 us/read; 10.48 M reads/minute).

=== Summary ===

Total reads processed:               2,668,256
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     2,668,256 (100.0%)

Total basepairs processed:   265,721,099 bp
Total written (filtered):    226,097,178 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291701_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 20.10 s (6 us/read; 10.37 M reads/minute).

=== Summary ===

Total reads processed:               3,474,962
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,474,962 (100.0%)

Total basepairs processed:   315,839,017 bp
Total written (filtered):    295,059,339 bp (93.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291701_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.76 s (6 us/read; 10.55 M reads/minute).

=== Summary ===

Total reads processed:               3,474,962
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,474,962 (100.0%)

Total basepairs processed:   346,913,613 bp
Total written (filtered):    295,029,825 bp (85.0%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291702_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 28.11 s (6 us/read; 10.50 M reads/minute).

=== Summary ===

Total reads processed:               4,918,786
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     4,918,786 (100.0%)

Total basepairs processed:   460,455,562 bp
Total written (filtered):    416,655,206 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291702_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 28.02 s (6 us/read; 10.53 M reads/minute).

=== Summary ===

Total reads processed:               4,918,786
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     4,918,786 (100.0%)

Total basepairs processed:   489,428,555 bp
Total written (filtered):    416,577,729 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291703_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.27 s (6 us/read; 10.46 M reads/minute).

=== Summary ===

Total reads processed:               3,186,044
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,186,044 (100.0%)

Total basepairs processed:   295,683,146 bp
Total written (filtered):    270,343,584 bp (91.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291703_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.17 s (6 us/read; 10.52 M reads/minute).

=== Summary ===

Total reads processed:               3,186,044
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,186,044 (100.0%)

Total basepairs processed:   317,754,200 bp
Total written (filtered):    270,312,770 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291704_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.37 s (6 us/read; 10.44 M reads/minute).

=== Summary ===

Total reads processed:               3,197,026
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,197,026 (100.0%)

Total basepairs processed:   299,931,145 bp
Total written (filtered):    271,300,448 bp (90.5%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291704_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 18.23 s (6 us/read; 10.52 M reads/minute).

=== Summary ===

Total reads processed:               3,197,026
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,197,026 (100.0%)

Total basepairs processed:   318,941,014 bp
Total written (filtered):    271,271,223 bp (85.1%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291705_R1_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.39 s (6 us/read; 10.60 M reads/minute).

=== Summary ===

Total reads processed:               3,424,403
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,424,403 (100.0%)

Total basepairs processed:   314,439,868 bp
Total written (filtered):    290,585,202 bp (92.4%)

This is cutadapt 1.12 with Python 2.7.12
Command line parameters: -l 85 raws/SRR4291705_R2_.fastq.gz
Trimming 0 adapters with at most 10.0% errors in single-end mode ...
Finished in 19.99 s (6 us/read; 10.28 M reads/minute).

=== Summary ===

Total reads processed:               3,424,403
Reads with adapters:                         0 (0.0%)
Reads written (passing filters):     3,424,403 (100.0%)

Total basepairs processed:   341,609,530 bp
Total written (filtered):    290,553,612 bp (85.1%)

Import reads into ipyrad and do QC

Now take the trimmed reads and filter for adapters, minimum sequence length, and max low quality bases.

In [29]:
IPYRAD_REFMAP_DIR = os.path.join(REFMAP_EMPIRICAL_DIR, "ipyrad/")
if not os.path.exists(IPYRAD_REFMAP_DIR):
    os.makedirs(IPYRAD_REFMAP_DIR)
os.chdir(IPYRAD_REFMAP_DIR)
In [57]:
## Make a new assembly and set some assembly parameters
data = ip.Assembly("refmap-empirical")
data.set_params("sorted_fastq_path", REFMAP_EMPIRICAL_DIR + "raws/*.fastq.gz")
data.set_params("project_dir", "reference-assembly")
data.set_params("assembly_method", "reference")
data.set_params("reference_sequence", REFMAP_EMPIRICAL_DIR + "TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa")
data.set_params("datatype", "pairddrad")
data.set_params("restriction_overhang", ("TGCAG", "CGG"))
data.set_params('max_low_qual_bases', 5)
data.set_params('filter_adapters', 2)

data.write_params(force=True)

cmd = "ipyrad -p params-refmap-empirical.txt -s 1 --force".format(dir)
print(cmd)
!time $cmd
cmd = "ipyrad -p params-refmap-empirical.txt -s 2 --force".format(dir)
print(cmd)
!time $cmd
  New Assembly: refmap-empirical
ipyrad -p params-refmap-empirical.txt -s 1 --force

 -------------------------------------------------------------
  ipyrad [v.0.5.15]
  Interactive assembly and analysis of RAD-seq data
 -------------------------------------------------------------
  New Assembly: refmap-empirical
  local compute node: [40 cores] on node001

  Step 1: Loading sorted fastq data to Samples
  [####################] 100%  loading reads         | 0:00:21  
  88 fastq files loaded to 44 Samples.


real	0m42.604s
user	0m2.510s
sys	0m0.791s
ipyrad -p params-refmap-empirical.txt -s 2 --force

 -------------------------------------------------------------
  ipyrad [v.0.5.15]
  Interactive assembly and analysis of RAD-seq data
 -------------------------------------------------------------
  loading Assembly: refmap-empirical
  from saved path: ~/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical.json
  local compute node: [40 cores] on node001

  Step 2: Filtering reads 
  [####################] 100%  processing reads      | 0:13:44  


real	13m57.667s
user	0m10.699s
sys	0m0.653s

Do ipyrad refmap empirical

Here we will use the ipyrad 'reference' assembly method which will only use reads that successfully map to the reference sequence. This is similar and should be comparable to the way stacks incorporates reference sequences.

Steps 1 & 2 run in ~15 minutes.

In [58]:
## Oops. If you run some other cell while this is running it steals stdout, so you lose track of progress.
cmd = "ipyrad -p params-refmap-empirical.txt -s 34567".format(dir)
print(cmd)
!time $cmd
ipyrad -p params-refmap-empirical.txt -s 3

 -------------------------------------------------------------
  ipyrad [v.0.5.15]
  Interactive assembly and analysis of RAD-seq data
 -------------------------------------------------------------
  loading Assembly: refmap-empirical
  from saved path: ~/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical.json
  local compute node: [40 cores] on node001

  Step 3: Clustering/Mapping reads
  [####################] 100%  dereplicating         | 0:10:16  
  [#########           ]  45%  mapping               | 0:24:11  

Do ipyrad denovo+reference

Create a new branch and set the assembly method to denovo+reference.

In [174]:
data2 = data.branch("denovo_ref-empirical")
data2.set_params("assembly_method", "denovo+reference")

data2.write_params(force=True)

cmd = "ipyrad -p params-denovo_ref-empirical.txt -s 34567 -c 40".format(dir)
print(cmd)
!time $cmd
ipyrad -p params-denovo_ref-sim.txt -s 1234567 -c 40

 -------------------------------------------------------------
  ipyrad [v.0.5.15]
  Interactive assembly and analysis of RAD-seq data
 -------------------------------------------------------------
  loading Assembly: denovo_ref-sim
  from saved path: ~/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/denovo_ref-sim.json
  New Assembly: denovo_ref-sim
  local compute node: [40 cores] on node001

  Step 1: Demultiplexing fastq data to Samples
  [####################] 100%  chunking large files  | 0:00:00  
  [####################] 100%  sorting reads         | 0:00:07  
  [####################] 100%  writing/compressing   | 0:00:01  

  Step 2: Filtering reads 
  [####################] 100%  processing reads      | 0:00:02  

  Step 3: Clustering/Mapping reads
  [####################] 100%  dereplicating         | 0:00:00  
  [####################] 100%  mapping               | 0:00:00  
  [####################] 100%  clustering            | 0:00:00  
  [####################] 100%  building clusters     | 0:00:01  
  [####################] 100%  finalize mapping      | 0:00:29  
  [####################] 100%  chunking              | 0:00:00  
  [####################] 100%  aligning              | 0:00:15  
  [####################] 100%  concatenating         | 0:00:00  

  Step 4: Joint estimation of error rate and heterozygosity
  [####################] 100%  inferring [H, E]      | 0:00:15  

  Step 5: Consensus base calling 
  Mean error  [0.00074 sd=0.00001]
  Mean hetero [0.00195 sd=0.00015]
  [####################] 100%  calculating depths    | 0:00:00  
  [####################] 100%  chunking clusters     | 0:00:00  
  [####################] 100%  consens calling       | 0:00:11  

  Step 6: Clustering at 0.85 similarity across 12 samples
  [####################] 100%  concat/shuffle input  | 0:00:00  
  [####################] 100%  clustering across     | 0:00:01  
  [####################] 100%  building clusters     | 0:00:00  
  [####################] 100%  aligning clusters     | 0:00:03  
  [####################] 100%  database indels       | 0:00:00  
  [####################] 100%  indexing clusters     | 0:00:01  
  [####################] 100%  building database     | 0:00:00  

  Step 7: Filter and write output files for 12 Samples
  [####################] 100%  filtering loci        | 0:00:06  
  [####################] 100%  building loci/stats   | 0:00:00  
  [####################] 100%  building vcf file     | 0:00:01  
  [####################] 100%  writing vcf file      | 0:00:00  
  [####################] 100%  building arrays       | 0:00:02  
  [####################] 100%  writing outfiles      | 0:00:01  
  Outfiles written to: ~/manuscript-analysis/REFMAP_SIM/ipyrad/reference-assembly/denovo_ref-sim_outfiles


real	2m12.816s
user	0m23.420s
sys	0m1.675s

Do Stacks refmap empirical

Lah et al trim R1 and R2 to 85bp and then concatenate them for stacks analysis. I thought this was weird, but the stacks manual says "For double-digest RAD data that has been paired-end sequenced, Stacks supports this type of data by treating the loci built from the single-end and paired-end as two independent loci", which I guess is effectively the same thing as concatenating.

For reference sequence assembly stacks doesn't actually handle the mapping, you need to Do It Yourself and then pull in .sam or .bam files. Since we already do the bwa mapping in ipyrad lets just use those files. The question is do we use the full .sam file or the *-mapped.bam (which excludes unmapped and unpaired reads). We'll use the *-mapped.bam files because the stacks manual says the mapped reads should be clean and tidy, so no junk.

NB: Stacks makes the strong assumption that the "left" edge of all reads within a stack line up. It looks like it just uses the StartPos to define a stack (assuming the cut site is the same). We will clean up soft-clipped sequences with ngsutils (https://github.com/ngsutils/ngsutils.git).

NB: Stacks treats R1 and R2 as independent.

NB: The other consequence of being forced to remove soft clipped sequence is that you are bound to be losing some real variation.

For the empirical data stacks runs in ~2 hours (~8 if you include the time it takes to map the sequences).

In [38]:
## Set directories and make the popmap file
STACKS_REFMAP_DIR = os.path.join(REFMAP_EMPIRICAL_DIR, "stacks/")
if not os.path.exists(STACKS_REFMAP_DIR):
    os.makedirs(STACKS_REFMAP_DIR)
os.chdir(STACKS_REFMAP_DIR)

make_stacks_popmap(STACKS_REFMAP_DIR)
Writing popmap file to /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/popmap.txt

Map ipyrad trimmed reads to the reference sequence

Based on the same logic as above with the simulated data, we need to remap the raw reads from the empirical ipyrad _edits directory to make stacks happy. This step consumes a ton of ram and takes a non-negligible amount of time. See docs for sim mapping above for the meaning of all the bwa/samtools flags.

Mapping the real data will take several hours (5-6 hours if you're lucky).

In [ ]:
IPYRAD_EDITS_DIR = os.path.join(IPYRAD_REFMAP_DIR, "reference-assembly/refmap-empirical_edits/")
REF_SEQ = REFMAP_EMPIRICAL_DIR + "TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa"

## Just get the 
sample_names = glob.glob(IPYRAD_EDITS_DIR + "*.trimmed_R1_.fastq.gz")
sample_names = [x.split(".")[0].split("/")[-1] for x in sample_names]

for samp in sample_names:
    R1 = IPYRAD_EDITS_DIR + samp + ".trimmed_R1_.fastq.gz"
    R2 = IPYRAD_EDITS_DIR + samp + ".trimmed_R2_.fastq.gz"
    samout = STACKS_REFMAP_DIR + samp + ".sam"
    bamout = STACKS_REFMAP_DIR + samp + ".bam"
    export_cmd = "export PATH=~/manuscript-analysis/dDocent:$PATH"
    bwa_cmd = "bwa mem -t 40 -v 0 " + REF_SEQ\
                + " " + R1\
                + " " + R2\
                + " > " + samout
    samtools_cmd = "samtools view -b -F 0x804 " + samout\
                    + " | samtools sort -T /tmp/{}.sam -O bam -o {}".format(samp, bamout)
    cleanup_cmd = "rm {}".format(samout)
    cmd = "; ".join([export_cmd, bwa_cmd, samtools_cmd, cleanup_cmd])
    !time $cmd
real	0m0.000s
user	0m0.000s
sys	0m0.000s

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (604, 1784, 2185)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5347)
[M::mem_pestat] mean and std.dev: (1788.75, 1421.66)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7475)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (193, 229, 272)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (35, 430)
[M::mem_pestat] mean and std.dev: (234.03, 57.92)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 509)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1516, 2019, 6187)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15529)
[M::mem_pestat] mean and std.dev: (3734.25, 2720.10)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20200)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (685, 2931, 5098)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 13924)
[M::mem_pestat] mean and std.dev: (2765.14, 2259.57)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 18337)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (740, 1784, 2182)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5066)
[M::mem_pestat] mean and std.dev: (1764.10, 1151.54)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 6508)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (194, 230, 273)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (36, 431)
[M::mem_pestat] mean and std.dev: (234.42, 57.91)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 510)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1536, 4455, 6187)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15489)
[M::mem_pestat] mean and std.dev: (4025.73, 2707.42)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20140)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291701.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291701.trimmed_R2_.fastq.gz
[main] Real time: 303.408 sec; CPU: 4020.255 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (604, 1850, 2472)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 6208)
[M::mem_pestat] mean and std.dev: (1776.17, 1180.59)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 8076)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (203, 242, 287)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (35, 455)
[M::mem_pestat] mean and std.dev: (245.55, 60.92)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 539)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1531, 2530, 6183)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15487)
[M::mem_pestat] mean and std.dev: (3645.37, 2595.32)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20139)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (685, 2931, 5098)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 13924)
[M::mem_pestat] mean and std.dev: (3050.90, 1983.19)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 18337)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (740, 2182, 2472)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5936)
[M::mem_pestat] mean and std.dev: (2053.10, 1324.06)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7668)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (203, 242, 287)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (35, 455)
[M::mem_pestat] mean and std.dev: (245.70, 60.87)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 539)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1556, 4515, 6183)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15437)
[M::mem_pestat] mean and std.dev: (4229.70, 2491.04)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20064)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291681.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291681.trimmed_R2_.fastq.gz
[main] Real time: 280.003 sec; CPU: 3767.582 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (604, 1850, 2418)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 6046)
[M::mem_pestat] mean and std.dev: (1658.53, 1230.99)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7860)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (190, 224, 265)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (40, 415)
[M::mem_pestat] mean and std.dev: (228.78, 56.99)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 490)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1531, 2530, 6183)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15487)
[M::mem_pestat] mean and std.dev: (3686.63, 2574.13)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20139)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (685, 1465, 4752)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 12886)
[M::mem_pestat] mean and std.dev: (2641.26, 2199.88)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 16953)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (740, 1850, 2472)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5936)
[M::mem_pestat] mean and std.dev: (1866.46, 1192.82)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7668)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (190, 224, 266)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (38, 418)
[M::mem_pestat] mean and std.dev: (229.05, 56.99)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 494)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1516, 2581, 6176)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15496)
[M::mem_pestat] mean and std.dev: (3641.20, 2518.20)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20156)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291682.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291682.trimmed_R2_.fastq.gz
[main] Real time: 278.177 sec; CPU: 3841.945 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (391, 1784, 2182)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5764)
[M::mem_pestat] mean and std.dev: (1495.00, 1179.37)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7555)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (192, 228, 270)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (36, 426)
[M::mem_pestat] mean and std.dev: (232.69, 58.10)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 504)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1516, 2581, 6183)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15517)
[M::mem_pestat] mean and std.dev: (3892.85, 2693.16)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20184)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (685, 4153, 4219)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 11287)
[M::mem_pestat] mean and std.dev: (2734.53, 1833.27)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 14821)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (191, 226, 269)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (35, 425)
[M::mem_pestat] mean and std.dev: (231.42, 57.61)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 503)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1708, 4468, 6247)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15325)
[M::mem_pestat] mean and std.dev: (4684.27, 2680.30)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 19864)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation RF
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291678.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291678.trimmed_R2_.fastq.gz
[main] Real time: 278.955 sec; CPU: 3769.486 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (740, 1850, 2472)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5936)
[M::mem_pestat] mean and std.dev: (1827.28, 1175.99)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7668)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (195, 232, 276)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (33, 438)
[M::mem_pestat] mean and std.dev: (236.03, 59.48)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 519)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1514, 2678, 6183)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15521)
[M::mem_pestat] mean and std.dev: (3759.94, 2525.84)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20190)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (966, 2318, 4219)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 10725)
[M::mem_pestat] mean and std.dev: (2703.32, 1997.38)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 13978)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (969, 2182, 2182)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 4608)
[M::mem_pestat] mean and std.dev: (2038.79, 1330.03)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7359)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (193, 231, 274)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (31, 436)
[M::mem_pestat] mean and std.dev: (234.46, 59.58)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 517)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1516, 4468, 6187)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15529)
[M::mem_pestat] mean and std.dev: (4027.00, 2526.71)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20200)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291686.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291686.trimmed_R2_.fastq.gz
[main] Real time: 263.595 sec; CPU: 3605.401 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (670, 1850, 2418)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5914)
[M::mem_pestat] mean and std.dev: (1741.52, 1201.23)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7662)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (207, 250, 299)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (23, 483)
[M::mem_pestat] mean and std.dev: (253.12, 63.72)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 575)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1513, 1777, 6173)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15493)
[M::mem_pestat] mean and std.dev: (3434.44, 2553.81)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20153)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (685, 2931, 4219)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 11287)
[M::mem_pestat] mean and std.dev: (3135.72, 2399.62)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 14821)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (785, 1850, 2418)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5684)
[M::mem_pestat] mean and std.dev: (1647.73, 822.81)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7317)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (205, 247, 296)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (23, 478)
[M::mem_pestat] mean and std.dev: (250.71, 63.42)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 569)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1536, 1777, 6219)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15585)
[M::mem_pestat] mean and std.dev: (3547.52, 2557.66)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20268)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291689.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291689.trimmed_R2_.fastq.gz
[main] Real time: 270.172 sec; CPU: 3508.493 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (604, 1850, 2418)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 6046)
[M::mem_pestat] mean and std.dev: (1720.91, 1142.69)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7860)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (190, 228, 272)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (26, 436)
[M::mem_pestat] mean and std.dev: (231.34, 61.11)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 518)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1539, 2581, 6176)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15450)
[M::mem_pestat] mean and std.dev: (3768.03, 2522.60)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20087)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (685, 2931, 4436)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 11938)
[M::mem_pestat] mean and std.dev: (3005.27, 2207.49)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 15689)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291683.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291683.trimmed_R2_.fastq.gz
[main] Real time: 199.643 sec; CPU: 2349.067 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (604, 1784, 2297)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5683)
[M::mem_pestat] mean and std.dev: (1676.03, 1147.20)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7376)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (192, 227, 270)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (36, 426)
[M::mem_pestat] mean and std.dev: (232.32, 57.67)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 504)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1531, 2530, 6183)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15487)
[M::mem_pestat] mean and std.dev: (3638.20, 2588.46)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20139)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (685, 2931, 5098)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 13924)
[M::mem_pestat] mean and std.dev: (2756.81, 2182.51)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 18337)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (357, 969, 2182)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 5832)
[M::mem_pestat] mean and std.dev: (1471.56, 1306.32)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7657)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (192, 227, 270)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (36, 426)
[M::mem_pestat] mean and std.dev: (232.40, 57.61)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 504)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1495, 2581, 6173)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15529)
[M::mem_pestat] mean and std.dev: (3508.49, 2429.33)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20207)
[M::mem_pestat] analyzing insert size distribution for orientation RR...
[M::mem_pestat] (25, 50, 75) percentile: (4219, 5098, 5098)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (2461, 6856)
[M::mem_pestat] mean and std.dev: (4801.57, 460.97)
[M::mem_pestat] low and high boundaries for proper pairs: (1582, 7735)
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[M::mem_pestat] skip orientation RR
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291705.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291705.trimmed_R2_.fastq.gz
[main] Real time: 268.112 sec; CPU: 3609.621 sec
[bam_sort_core] merging from 2 files...

real	0m0.000s
user	0m0.000s
sys	0m0.000s
[M::mem_pestat] analyzing insert size distribution for orientation FF...
[M::mem_pestat] (25, 50, 75) percentile: (604, 1784, 2418)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 6046)
[M::mem_pestat] mean and std.dev: (1640.13, 1243.11)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 7860)
[M::mem_pestat] analyzing insert size distribution for orientation FR...
[M::mem_pestat] (25, 50, 75) percentile: (181, 211, 247)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (49, 379)
[M::mem_pestat] mean and std.dev: (214.95, 53.03)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 445)
[M::mem_pestat] analyzing insert size distribution for orientation RF...
[M::mem_pestat] (25, 50, 75) percentile: (1556, 4410, 6183)
[M::mem_pestat] low and high boundaries for computing mean and std.dev: (1, 15437)
[M::mem_pestat] mean and std.dev: (3951.14, 2572.36)
[M::mem_pestat] low and high boundaries for proper pairs: (1, 20064)
[M::mem_pestat] skip orientation RR as there are not enough pairs
[M::mem_pestat] skip orientation FF
[M::mem_pestat] skip orientation RF
[main] Version: 0.7.15-r1142-dirty
[main] CMD: bwa mem -t 40 -v 1 /home/iovercast/manuscript-analysis/Phocoena_empirical/TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291688.trimmed_R1_.fastq.gz /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/reference-assembly/refmap-empirical_edits/SRR4291688.trimmed_R2_.fastq.gz
[main] Real time: 142.756 sec; CPU: 1975.479 sec

real	0m0.000s
user	0m0.000s
sys	0m0.000s

Stacks prereqs - install ngsutils

Install ngsutils and use it to remove soft-clipped sequences from each read. We could do this by rerunning bwa with the -H flag, but that would be slightly more annoying. This is not guaranteed to 'work' because i had a hell of a time getting ngsutils to install on my system.

In [ ]:
%%bash -s "$REFMAP_EMPIRICAL_DIR"
cd $1/stacks
git clone https://github.com/ngsutils/ngsutils.git
cd ngsutils
make

Now do the filtering

This will apply the removeclipping method of bamutils to each mapped bam file in the stacks refmap directory. Then it deletes the old bam file and moves the new bam file to have the name of the old one. On a nice system this takes ~2 minutes per sample (so ~2 hours total).

In [56]:
infiles = glob.glob(STACKS_REFMAP_DIR + "SRR*.bam")

for f in infiles:
    outfile = f + ".tmp"
    print(f, outfile)
    subprocess.call("bamutils removeclipping {} {}".format(f, outfile), shell=True)
    subprocess.call("rm {}".format(f), shell=True)
    subprocess.call("mv {} {}".format(outfile, f), shell=True)
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291679.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291679.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291702.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291702.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291701.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291701.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291681.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291681.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291682.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291682.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291678.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291678.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291686.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291686.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291705.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291705.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291688.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291688.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291674.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291674.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291673.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291673.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291684.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291684.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291677.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291677.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291675.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291675.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291703.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291703.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291672.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291672.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291687.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291687.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291696.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291696.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291699.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291699.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291668.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291668.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291698.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291698.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291690.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291690.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291692.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291692.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291666.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291666.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291664.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291664.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291689.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291689.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291683.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291683.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291680.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291680.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291685.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291685.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291700.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291700.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291671.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291671.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291693.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291693.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291704.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291704.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291676.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291676.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291695.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291695.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291697.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291697.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291691.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291691.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291662.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291662.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291663.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291663.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291669.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291669.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291670.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291670.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291694.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291694.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291667.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291667.bam.tmp')
('/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291665.bam', '/home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291665.bam.tmp')

Now run stacks ref_map pipeline

We build the stacks command w/ python because then we call the command inside a bash cell because denovo_map expects all the submodules to be in the PATH. The default command line created will include the -d flag to do the dry run, so the bash cell won't actually do anything real. But it does create a stacks.sh file in the stacks directory that includes the command to run.

In [57]:
## This is how we'd do it if we weren't using a popmap file
#infiles = ["-s "+ff+" " for ff in glob.glob(IPYRAD_REFMAP_DIR+"*-mapped-sorted.bam")]

## Toggle the dryrun flag for testing
DRYRUN=""
DRYRUN="-d"

## Options
## -T    The number of threads to use
## -O    The popmap file specifying individuals and populations
## -S    Disable database business
## -o    Output directory. Just write to the empirical stacks directory
## -X    The first -X tells populations to create the output formats sepcified
## -X    The second one passes `-m 6` which sets min depth per locus
OUTPUT_FORMATS = "--vcf --genepop --structure --phylip "
cmd = "ref_map.pl -T 40 -b 1 -S " + DRYRUN\
        + " -O {}/popmap.txt".format(STACKS_REFMAP_DIR)\
        + " --samples {}".format(STACKS_REFMAP_DIR)\
        + " -X \'populations:" + OUTPUT_FORMATS + "\'"\
        + " -X \'populations:-m 6\'"\
        + " -o " + STACKS_REFMAP_DIR
print("\nCommand to run - {}".format(cmd))
Command to run - ref_map.pl -T 40 -b 1 -S -d -O /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks//popmap.txt --samples /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/ -X 'populations:--vcf --genepop --structure --phylip ' -X 'populations:-m 6' -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/
In [58]:
%%bash -s "$WORK_DIR" "$STACKS_REFMAP_DIR" "$cmd"
export PATH="$1/miniconda/bin:$PATH"; export "STACKS_REFMAP_DIR=$2"; export "cmd=$3"

## We have to play a little cat and mouse game here because of quoting in some of the args
## and how weird bash is we have to write the cmd to a file and then exec it.
## If you try to just run $cmd it truncates the command at the first single tic. Hassle.
cd $STACKS_REFMAP_DIR
echo $cmd > stacks.sh; chmod 777 stacks.sh
time ./stacks.sh
Identifying unique stacks; file   1 of  44 [SRR4291704]
Identifying unique stacks; file   2 of  44 [SRR4291705]
Identifying unique stacks; file   3 of  44 [SRR4291700]
Identifying unique stacks; file   4 of  44 [SRR4291701]
Identifying unique stacks; file   5 of  44 [SRR4291702]
Identifying unique stacks; file   6 of  44 [SRR4291703]
Identifying unique stacks; file   7 of  44 [SRR4291685]
Identifying unique stacks; file   8 of  44 [SRR4291684]
Identifying unique stacks; file   9 of  44 [SRR4291687]
Identifying unique stacks; file  10 of  44 [SRR4291686]
Identifying unique stacks; file  11 of  44 [SRR4291681]
Identifying unique stacks; file  12 of  44 [SRR4291680]
Identifying unique stacks; file  13 of  44 [SRR4291683]
Identifying unique stacks; file  14 of  44 [SRR4291682]
Identifying unique stacks; file  15 of  44 [SRR4291689]
Identifying unique stacks; file  16 of  44 [SRR4291688]
Identifying unique stacks; file  17 of  44 [SRR4291674]
Identifying unique stacks; file  18 of  44 [SRR4291675]
Identifying unique stacks; file  19 of  44 [SRR4291676]
Identifying unique stacks; file  20 of  44 [SRR4291677]
Identifying unique stacks; file  21 of  44 [SRR4291670]
Identifying unique stacks; file  22 of  44 [SRR4291671]
Identifying unique stacks; file  23 of  44 [SRR4291672]
Identifying unique stacks; file  24 of  44 [SRR4291673]
Identifying unique stacks; file  25 of  44 [SRR4291678]
Identifying unique stacks; file  26 of  44 [SRR4291679]
Identifying unique stacks; file  27 of  44 [SRR4291696]
Identifying unique stacks; file  28 of  44 [SRR4291697]
Identifying unique stacks; file  29 of  44 [SRR4291694]
Identifying unique stacks; file  30 of  44 [SRR4291695]
Identifying unique stacks; file  31 of  44 [SRR4291692]
Identifying unique stacks; file  32 of  44 [SRR4291693]
Identifying unique stacks; file  33 of  44 [SRR4291690]
Identifying unique stacks; file  34 of  44 [SRR4291691]
Identifying unique stacks; file  35 of  44 [SRR4291698]
Identifying unique stacks; file  36 of  44 [SRR4291699]
Identifying unique stacks; file  37 of  44 [SRR4291669]
Identifying unique stacks; file  38 of  44 [SRR4291668]
Identifying unique stacks; file  39 of  44 [SRR4291663]
Identifying unique stacks; file  40 of  44 [SRR4291662]
Identifying unique stacks; file  41 of  44 [SRR4291667]
Identifying unique stacks; file  42 of  44 [SRR4291666]
Identifying unique stacks; file  43 of  44 [SRR4291665]
Identifying unique stacks; file  44 of  44 [SRR4291664]
Parsed population map: 44 files in 7 populations and 1 group.
Found 44 sample file(s).
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291704.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 1  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291705.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 2  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291700.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 3  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291701.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 4  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291702.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 5  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291703.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 6  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291685.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 7  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291684.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 8  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291687.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 9  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291686.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 10  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291681.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 11  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291680.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 12  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291683.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 13  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291682.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 14  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291689.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 15  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291688.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 16  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291674.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 17  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291675.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 18  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291676.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 19  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291677.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 20  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291670.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 21  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291671.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 22  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291672.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 23  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291673.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 24  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291678.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 25  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291679.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 26  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291696.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 27  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291697.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 28  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291694.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 29  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291695.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 30  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291692.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 31  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291693.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 32  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291690.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 33  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291691.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 34  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291698.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 35  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291699.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 36  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291669.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 37  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291668.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 38  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291663.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 39  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291662.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 40  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291667.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 41  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291666.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 42  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291665.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 43  -p 40 2>&1
  /home/iovercast/manuscript-analysis/miniconda/bin/pstacks -t bam -f /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291664.bam -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -i 44  -p 40 2>&1

Depths of Coverage for Processed Samples:

Generating catalog...
  /home/iovercast/manuscript-analysis/miniconda/bin/cstacks -g -b 1 -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291704 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291705 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291700 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291701 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291702 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291703 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291685 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291684 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291687 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291686 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291681 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291680 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291683 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291682 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291689 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291688 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291674 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291675 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291676 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291677 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291670 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291671 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291672 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291673 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291678 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291679 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291696 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291697 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291694 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291695 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291692 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291693 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291690 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291691 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291698 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291699 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291669 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291668 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291663 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291662 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291667 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291666 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291665 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291664  -p 40 2>&1
Matching samples to the catalog...
  /home/iovercast/manuscript-analysis/miniconda/bin/sstacks -g -b 1 -c /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/batch_1 -o /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291704 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291705 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291700 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291701 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291702 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291703 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291685 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291684 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291687 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291686 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291681 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291680 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291683 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291682 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291689 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291688 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291674 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291675 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291676 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291677 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291670 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291671 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291672 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291673 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291678 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291679 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291696 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291697 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291694 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291695 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291692 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291693 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291690 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291691 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291698 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291699 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291669 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291668 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291663 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291662 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291667 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291666 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291665 -s /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks/SRR4291664  -p 40 2>&1
Calculating population-level summary statistics
/home/iovercast/manuscript-analysis/miniconda/bin/populations -b 1 -P /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks -s -t 40 -M /home/iovercast/manuscript-analysis/Phocoena_empirical/stacks//popmap.txt --vcf --genepop --structure --phylip  -m 6 2>&1

real	0m0.385s
user	0m0.031s
sys	0m0.012s

Do dDocent refmap empirical

dDocent requires a lot of pre-flight housekeeping. You can't use an arbitrary directory for raw files, you have to puth the raw reads in a specific directory. You also can't use arbitrary file names, you have to use specific filenames that dDocent requires. zomg. I wonder if we can trick it with symlinks.

This next cell will do all the housekeeping and then print the nicely formatted command to run. You have to copy this command and run it by hand in a terminal on the machine you want to run it on bcz it doesn't like running inside the notebook.

~1 hour to munge reads and index the reference sequence. ~3hrs to map the reads and build the vcf. Several hours getting the .fq.gz files to have a naming format that dDocent agreed with.

In [59]:
## A housekeeping function for getting a dictionary to map SRR* filenames in the ipyrad edits directory
## to ddocent style.
##
## Gotcha: Nice 1-based indexing for the dDocent format.
##
## For raw reads the format (for R1) is pop1_sample1.F.fq.gz format a la:
## 1A_0_R1_.fastq.gz -> Pop1_Sample1.F.fq.gz
##
## For trimmed reads the format is pop1_001.R1.fq.gz a la:
## 1A_0_R1_.fastq.gz -> Pop1_001.R1.fq.gz
## So annoying because we have to translate across a bunch of different mappings. ugh.
def get_ddocent_filename_mapping():
    mapping_dict = {}
    
    ## Maps sample name to population
    pop_dict = get_popdict()
    pops = set(pop_dict.values())

    ## For each population go through and add items to the dict per sample
    ## So we have to map the sample name to the SRR and then make an entry
    ## mapping SRR file name to ddocent format
    for i, pop in enumerate(pops):
        ## Get a list of all the samples in this population. This is probably a stupid way but it works.
        samps = [item[0] for item in pop_dict.items() if item[1] == pop]
        for j, samp in enumerate(samps):
            mapping_dict[samp] = "Pop{}_{:03d}".format(i+1, j+1)
            ## For the untrimmed format, if you want dDocent to do the trimming
            ## mapping_dict[samp] = "Pop{}_Sample{}".format(i, j)

    return mapping_dict
print(get_ddocent_filename_mapping())
{'SRR4291704': 'Pop7_001', 'SRR4291705': 'Pop7_002', 'SRR4291700': 'Pop6_001', 'SRR4291701': 'Pop4_001', 'SRR4291702': 'Pop4_002', 'SRR4291703': 'Pop7_003', 'SRR4291685': 'Pop3_001', 'SRR4291684': 'Pop5_001', 'SRR4291687': 'Pop5_002', 'SRR4291686': 'Pop5_003', 'SRR4291681': 'Pop5_004', 'SRR4291680': 'Pop5_005', 'SRR4291683': 'Pop5_006', 'SRR4291682': 'Pop5_007', 'SRR4291689': 'Pop5_008', 'SRR4291688': 'Pop5_009', 'SRR4291674': 'Pop3_002', 'SRR4291675': 'Pop1_001', 'SRR4291676': 'Pop1_002', 'SRR4291677': 'Pop1_003', 'SRR4291670': 'Pop2_001', 'SRR4291671': 'Pop2_002', 'SRR4291672': 'Pop1_004', 'SRR4291673': 'Pop1_005', 'SRR4291678': 'Pop1_006', 'SRR4291679': 'Pop5_010', 'SRR4291696': 'Pop4_003', 'SRR4291697': 'Pop6_002', 'SRR4291694': 'Pop6_003', 'SRR4291695': 'Pop6_004', 'SRR4291692': 'Pop6_005', 'SRR4291693': 'Pop6_006', 'SRR4291690': 'Pop6_007', 'SRR4291691': 'Pop6_008', 'SRR4291698': 'Pop6_009', 'SRR4291699': 'Pop6_010', 'SRR4291669': 'Pop2_003', 'SRR4291668': 'Pop2_004', 'SRR4291663': 'Pop3_003', 'SRR4291662': 'Pop3_004', 'SRR4291667': 'Pop2_005', 'SRR4291666': 'Pop7_004', 'SRR4291665': 'Pop7_005', 'SRR4291664': 'Pop7_006'}
In [87]:
## Set up directory structures. change the force flag if you want to
## blow everything away and restart
# force = True
force = ""

DDOCENT_DIR = "/home/iovercast/manuscript-analysis/dDocent/"
DDOCENT_REFMAP_DIR = os.path.join(REFMAP_EMPIRICAL_DIR, "ddocent/")
if force and os.path.exists(DDOCENT_REFMAP_DIR):
    shutil.rmtree(DDOCENT_REFMAP_DIR)
if not os.path.exists(DDOCENT_REFMAP_DIR):
    os.makedirs(DDOCENT_REFMAP_DIR)
os.chdir(DDOCENT_REFMAP_DIR)

## Create a simlink to the reference sequence in the current directory
REF_SEQ = REFMAP_EMPIRICAL_DIR + "TurtrunRef/Tursiops_truncatus.turTru1.dna_rm.toplevel.fa"
cmd = "ln -s {} reference.fasta".format(REF_SEQ)
!$cmd

## Now we have to rename all the files in the way dDocent expects them:
## 1A_0_R1_.fastq.gz -> Pop1_Sample1.F.fq.gz
## Make symlinks to the trimmed data files in the ipyrad directory. It _should_ work.
## Trimmed reads in the ipyrad directory are of the format: SRR4291681.trimmed_R1_.fastq.gz
IPYRAD_EDITS_DIR = os.path.join(IPYRAD_REFMAP_DIR, "reference-assembly/refmap-empirical_edits/")

name_mapping = get_ddocent_filename_mapping()

for k,v in name_mapping.items():
    ## Symlink R1 and R2
    for i in ["1", "2"]:
        source = os.path.join(IPYRAD_EDITS_DIR, k + ".trimmed_R{}_.fastq.gz".format(i))
        ##dest = os.path.join(DDOCENT_REFMAP_DIR, v + ".R{}.fq.gz".format(i))
        if i == "1":
            dest = os.path.join(DDOCENT_REFMAP_DIR, v + ".R1.fq.gz".format(i))
        else:
            dest = os.path.join(DDOCENT_REFMAP_DIR, v + ".R2.fq.gz".format(i))
        cmd = "ln -sf {} {}".format(source, dest)
        !$cmd

## Write out the config file for this run.
## Compacted the config file into one long line here to make it not take up so much room
## Trimming           = no because we trimmed in ipyrad
## Assembly           = no because we are providing a reverence sequence
## Type of Assembly   = PE for paired-end
config_file = "{}/empirical-config.txt".format(DDOCENT_REFMAP_DIR)
with open(config_file, 'w') as outfile:
    outfile.write('Number of Processors\n40\nMaximum Memory\n0\nTrimming\nno\nAssembly?\nno\nType_of_Assembly\nPE\nClustering_Similarity%\n0.85\nMapping_Reads?\nyes\nMapping_Match_Value\n1\nMapping_MisMatch_Value\n3\nMapping_GapOpen_Penalty\n5\nCalling_SNPs?\nyes\nEmail\n[email protected]\n')

cmd = "export LD_LIBRARY_PATH={}/freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; ".format(DDOCENT_DIR)
cmd += "export PATH={}:$PATH; time dDocent {}".format(DDOCENT_DIR, config_file)
print(cmd)
with open("ddocent.sh", 'w') as outfile:
    outfile.write("#!/bin/bash\n")
    outfile.write(cmd)
!chmod 777 ddocent.sh
## Have to run the printed command by hand from the ddocent REALDATA dir bcz it doesn't like running in the notebook
#!$cmd

## NB: Must rename all the samples in the output vcf and then use vcf-shuffle-cols
## perl script in the vcf/perl directory to reorder the vcf file to match
## the output of stacks and ipyrad for pca/heatmaps to work.
ln: failed to create symbolic link ‘reference.fasta’: File exists
export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH; time dDocent /home/iovercast/manuscript-analysis/Phocoena_empirical/ddocent//empirical-config.txt
In [ ]:
## You have to post-process the vcf files to decompose complex genotypes and remove indels
os.chdir(DDOCENT_REFMAP_DIR)
exports = "export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH"

fullvcf = os.path.join(DDOCENT_REFMAP_DIR, "TotalRawSNPs.vcf")
filtvcf = os.path.join(DDOCENT_REFMAP_DIR, "Final.recode.vcf")
for f in [fullvcf, filtvcf]:
    print("Finalizing - {}".format(f))
    
    ## Rename the samples to make them agree with the ipyrad/stacks names so
    ## the results analysis will work.
    vcffile = f
    infile = open(vcffile,'r')
    filedata = infile.readlines()
    infile.close()
    
    outfile = open(vcffile,'w')
    for line in filedata:
        if "CHROM" in line:
            for ipname, ddname in name_mapping.items():
                line = line.replace(ddname, ipname)
        outfile.write(line)
    outfile.close()
    
    ## Rename columns to match ipyrad and then resort columns to be in same order
    IPYRAD_VCF = os.path.join(IPYRAD_REFMAP_DIR, "refmap-empirical_outfiles/refmap-empirical.vcf")
    os.chdir(os.path.join(DDOCENT_DIR, "vcftools_0.1.11/perl"))
    tmpvcf = os.path.join(DDOCENT_REFMAP_DIR, "ddocent-tmp.vcf")
    cmd = "perl vcf-shuffle-cols -t {} {} > {}".format(IPYRAD_VCF, vcffile, tmpvcf)
    print(cmd)
    #!$cmd

    os.chdir(DDOCENT_REFMAP_DIR)
    ## Naming the new outfiles as <curname>.snps.vcf
    ## Decompose complex genotypes and remove indels
    outfile = os.path.join(DDOCENT_REFMAP_DIR, f.split("/")[-1].split(".vcf")[0] + ".snps.vcf")
    cmd = "{}; vcfallelicprimitives {} > ddoc-tmp.vcf".format(exports, f)
    print(cmd)
    !$cmd
    cmd = "{}; vcftools --vcf ddoc-tmp.vcf --remove-indels --recode --recode-INFO-all --out {}".format(exports, outfile)
    print(cmd)
    !$cmd
    !rm ddoc-tmp.vcf
Finalizing - /home/iovercast/manuscript-analysis/Phocoena_empirical/ddocent/TotalRawSNPs.vcf
perl vcf-shuffle-cols -t /home/iovercast/manuscript-analysis/Phocoena_empirical/ipyrad/refmap-empirical_outfiles/refmap-empirical.vcf /home/iovercast/manuscript-analysis/Phocoena_empirical/ddocent/TotalRawSNPs.vcf > /home/iovercast/manuscript-analysis/Phocoena_empirical/ddocent/ddocent-tmp.vcf
export LD_LIBRARY_PATH=/home/iovercast/manuscript-analysis/dDocent//freebayes-src/vcflib/tabixpp/htslib/:$LD_LIBRARY_PATH; export PATH=/home/iovercast/manuscript-analysis/dDocent/:$PATH; vcfallelicprimitives /home/iovercast/manuscript-analysis/Phocoena_empirical/ddocent/TotalRawSNPs.vcf > ddoc-tmp.vcf

Housekeeping

This should be at the top, but i'm leaving it here so it's out of the way. Just some housekeeping for translating between SRA sequence file name and sample name from the paper. This is only for the benefit of stacks populations script.

Fetch info from SRA for mapping between SRR accession numbers and sample names

The files that come down from SRA are not helpfully named. We have to create a mapping between sample names from the paper and SRR numbers.

Get the RunInfo table from here: https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=SRP090334

In [23]:
def get_sampsdict():
    info_header = "BioSample_s	Experiment_s	Library_Name_s	MBases_l	MBytes_l	Run_s	SRA_Sample_s	Sample_Name_s	dev_stage_s	ecotype_s	lat_lon_s	sex_s	tissue_s	Assay_Type_s	AssemblyName_s	BioProject_s	BioSampleModel_s	Center_Name_s	Consent_s	InsertSize_l	LibraryLayout_s	LibrarySelection_s	LibrarySource_s	LoadDate_s	Organism_s	Platform_s	ReleaseDate_s	SRA_Study_s	g1k_analysis_group_s	g1k_pop_code_s	source_s"
    info = """SAMN05806468	SRX2187156	Pp01	595	395	SRR4291662	SRS1709994	Pp01	<not provided>	relicta	44.09 N 29.81 E	female	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806469	SRX2187157	Pp02	478	318	SRR4291663	SRS1709995	Pp02	<not provided>	relicta	41.42 N 28.92 E	female	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806478	SRX2187158	Pp11	242	162	SRR4291664	SRS1709996	Pp11	adult	phocoena	54.96 N 8.32 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806479	SRX2187159	Pp12	261	174	SRR4291665	SRS1709997	Pp12	adult	phocoena	54.95 N 8.32 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806480	SRX2187160	Pp13	595	397	SRR4291666	SRS1709998	Pp13	juvenile	phocoena	54.16 N 8.82 E	male	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806481	SRX2187161	Pp14	769	511	SRR4291667	SRS1709999	Pp14	<not provided>	phocoena	57.00 N 11.00 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806482	SRX2187162	Pp15	624	414	SRR4291668	SRS1710000	Pp15	<not provided>	phocoena	56.89 N 12.50 E	male	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806483	SRX2187163	Pp16	665	446	SRR4291669	SRS1710001	Pp16	<not provided>	phocoena	57.37 N 9.68 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806484	SRX2187164	Pp17	264	177	SRR4291670	SRS1710002	Pp17	<not provided>	phocoena	57.59 N 10.10 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806485	SRX2187165	Pp18	684	453	SRR4291671	SRS1710003	Pp18	<not provided>	phocoena	58.93 N 11.15 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806486	SRX2187166	Pp19	601	398	SRR4291672	SRS1710004	Pp19	<not provided>	phocoena	55.43 N 107.0 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806487	SRX2187167	Pp20	392	261	SRR4291673	SRS1710005	Pp20	<not provided>	phocoena	55.97 N 11.18 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806470	SRX2187168	Pp03	471	316	SRR4291674	SRS1710006	Pp03	<not provided>	relicta	41.48 N 28.31 E	female	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806488	SRX2187169	Pp21	592	397	SRR4291675	SRS1710007	Pp21	<not provided>	phocoena	55.43 N 10.70 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806489	SRX2187170	Pp22	446	300	SRR4291676	SRS1710008	Pp22	<not provided>	phocoena	56.25 N 12.82 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806490	SRX2187171	Pp23	617	409	SRR4291677	SRS1710009	Pp23	<not provided>	phocoena	56.65 N 12.85 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806491	SRX2187172	Pp24	554	367	SRR4291678	SRS1710010	Pp24	<not provided>	phocoena	56.00 N 12.00 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806492	SRX2187173	Pp25	753	500	SRR4291679	SRS1710011	Pp25	juvenile	phocoena	55.00 N 10.23 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806493	SRX2187174	Pp26	530	353	SRR4291680	SRS1710012	Pp26	<not provided>	phocoena	54.38 N 10.99 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806494	SRX2187175	Pp27	639	426	SRR4291681	SRS1710013	Pp27	juvenile	phocoena	54.83 N 9.62 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806495	SRX2187176	Pp28	646	430	SRR4291682	SRS1710014	Pp28	juvenile	phocoena	54.59 N 10.03 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806496	SRX2187177	Pp29	374	247	SRR4291683	SRS1710015	Pp29	juvenile	phocoena	54.42 N 11.55 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806497	SRX2187178	Pp30	569	376	SRR4291684	SRS1710016	Pp30	juvenile	phocoena	54.53 N 11.12 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806471	SRX2187179	Pp04	451	303	SRR4291685	SRS1710017	Pp04	<not provided>	relicta	41.65 N 28.27 E	female	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806498	SRX2187180	Pp31	578	384	SRR4291686	SRS1710018	Pp31	adult	phocoena	54.53 N 11.11 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806499	SRX2187181	Pp32	586	392	SRR4291687	SRS1710019	Pp32	juvenile	phocoena	54.32 N 13.09 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806500	SRX2187182	Pp33	288	189	SRR4291688	SRS1710020	Pp33	juvenile	phocoena	54.46 N 12.54 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806501	SRX2187183	Pp34	587	389	SRR4291689	SRS1710021	Pp34	<not provided>	phocoena	54.32 N 13.09 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806502	SRX2187184	Pp35	496	330	SRR4291690	SRS1710022	Pp35	<not provided>	phocoena	55.00 N 14.00 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806503	SRX2187185	Pp36	1085	720	SRR4291691	SRS1710023	Pp36	juvenile	phocoena	56.00 N 15.00 E	male	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806504	SRX2187186	Pp37	214	141	SRR4291692	SRS1710024	Pp37	<not provided>	phocoena	55.56 N 17.63 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806505	SRX2187187	Pp38	397	263	SRR4291693	SRS1710025	Pp38	<not provided>	phocoena	55.50 N 17.00 E	female	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806506	SRX2187188	Pp39	670	447	SRR4291694	SRS1710026	Pp39	juvenile	phocoena	56.00 N 16.00 E	male	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806507	SRX2187189	Pp40	342	226	SRR4291695	SRS1710027	Pp40	<not provided>	phocoena	54.73 N 18.58 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806472	SRX2187190	Pp05	611	406	SRR4291696	SRS1710028	Pp05	<not provided>	phocoena	64.78 N 13.22 E	male	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806508	SRX2187191	Pp41	586	389	SRR4291697	SRS1710029	Pp41	<not provided>	phocoena	54.80 N 18.44 E	female	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806509	SRX2187192	Pp42	329	219	SRR4291698	SRS1710030	Pp42	<not provided>	phocoena	54.67 N 18.59 E	male	muscle	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806510	SRX2187193	Pp43	517	343	SRR4291699	SRS1710031	Pp43	juvenile	phocoena	57.00 N 20.00 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806511	SRX2187194	Pp44	491	326	SRR4291700	SRS1710032	Pp44	adult	phocoena	57.01 N 20.00 E	male	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806473	SRX2187195	Pp06	632	423	SRR4291701	SRS1710033	Pp06	<not provided>	phocoena	64.58 N 13.58 E	male	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806474	SRX2187196	Pp07	905	602	SRR4291702	SRS1710034	Pp07	<not provided>	phocoena	64.31 N 14.00 E	male	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806475	SRX2187197	Pp08	585	390	SRR4291703	SRS1710035	Pp08	adult	phocoena	54.70 N 8.33 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806476	SRX2187198	Pp09	590	392	SRR4291704	SRS1710036	Pp09	<not provided>	phocoena	54.30 N 8.93 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>
    SAMN05806477	SRX2187199	Pp10	625	414	SRR4291705	SRS1710037	Pp10	adult	phocoena	55.47 N 8.38 E	female	skin	OTHER	<not provided>	PRJNA343959	Model organism or animal	<not provided>	public	0	PAIRED	Restriction Digest	GENOMIC	2016-09-22	Phocoena phocoena	ILLUMINA	2016-09-27	SRP090334	<not provided>	<not provided>	<not provided>""".split("\n")
    samps_dict = {}
    for i in info:
        line = i.split("\t")
        samps_dict[line[2]] = line[5]
    return(samps_dict)

Create a population map

Supplementary table S1 (http://journals.plos.org/plosone/article/file?type=supplementary&id=info:doi/10.1371/journal.pone.0162792.s006) contains detailed sample information. We can just extract the sample names and populations they belong to.

In [24]:
def get_popdict():
    samps_dict = get_sampsdict()
    popmap = \
"""01	WBS
02	WBS
03	WBS
04	WBS
05	IS
06	IS
07	IS
08	NOS
09	NOS
10	NOS
11	NOS
12	NOS
13	NOS
14	SK1
15	SK1
16	SK1
17	SK1
18	SK1
19	KB1
20	KB1
21	KB1
22	KB1
23	KB1
24	KB1
25	BES2
26	BES2
27	BES2
28	BES2
29	BES2
30	BES2
31	BES2
32	BES2
33	BES2
34	BES2
35	IBS
36	IBS
37	IBS
38	IBS
39	IBS
40	IBS
41	IBS
42	IBS
43	IBS
44	IBS""".split("\n")
    pop_dict = {}
    for i in popmap:
        line = i.split("\t")
        pop_dict[samps_dict["Pp"+line[0]]] = line[1]
    return(pop_dict)
In [25]:
## Adding "-mapped-sorted" to each individual name to avoid having to rename the .bam files created by ipyrad
def make_stacks_popmap(OUTDIR):
    pop_dict = get_popdict()
    out = os.path.join(OUTDIR, "popmap.txt")
    print("Writing popmap file to {}".format(out))
    with open(out, 'w') as outfile:
        for k,v in pop_dict.items():
            outfile.write(k + "\t" + v + "\n")

Ignore all below here

Oh man. I thought dDocent could use a reference sequence for assembly, but it totally can't. I did all this work to get the housekeeping in order, but then finally figured out at the last minute while setting the config that it just can't use an external reference sequence. zomg. Keeping this here cuz it might be useful some day in the event that dDocent makes this possible. Nvm, it can do reference sequence mapping, it's just not well documented.

In [ ]: