Source of the materials: Biopython cookbook (adapted) Status: Draft
This chapter is about Multiple Sequence Alignments, by which we mean a
collection of multiple sequences which have been aligned together –
usually with the insertion of gap characters, and addition of leading or
trailing gaps – such that all the sequence strings are the same length.
Such an alignment can be regarded as a matrix of letters, where each row
is held as a SeqRecord
object internally.
We will introduce the MultipleSeqAlignment
object which holds this
kind of data, and the Bio.AlignIO
module for reading and writing them
as various file formats (following the design of the Bio.SeqIO
module
from the previous chapter). Note that both Bio.SeqIO
and Bio.AlignIO
can read and write sequence alignment files. The appropriate choice will
depend largely on what you want to do with the data.
The final part of this chapter is about our command line wrappers for common multiple sequence alignment tools like ClustalW and MUSCLE.
We have two functions for reading in sequence alignments,
Bio.AlignIO.read()
and Bio.AlignIO.parse()
which following the
convention introduced in Bio.SeqIO
are for files containing one or
multiple alignments respectively.
Using Bio.AlignIO.parse()
will return an iterator which
gives MultipleSeqAlignment
objects. Iterators are typically used in a
for loop. Examples of situations where you will have multiple different
alignments include resampled alignments from the PHYLIP tool seqboot
,
or multiple pairwise alignments from the EMBOSS tools water
or
needle
, or Bill Pearson’s FASTA tools.
However, in many situations you will be dealing with files which contain
only a single alignment. In this case, you should use the
Bio.AlignIO.read()
function which returns a single
MultipleSeqAlignment
object.
Both functions expect two mandatory arguments:
The first argument is a handle to read the data from, typically an open file (see Section [sec:appendix-handles]), or a filename.
The second argument is a lower case string specifying the
alignment format. As in Bio.SeqIO
we don’t try and guess the file
format for you! See http://biopython.org/wiki/AlignIO for a full
listing of supported formats.
There is also an optional seq_count
argument which is discussed in
Section [sec:AlignIO-count-argument] below for dealing with ambiguous
file formats which may contain more than one alignment.
A further optional alphabet
argument allowing you to specify the
expected alphabet. This can be useful as many alignment file formats do
not explicitly label the sequences as RNA, DNA or protein – which means
Bio.AlignIO
will default to using a generic alphabet.
As an example, consider the following annotation rich protein alignment in the PFAM or Stockholm file format:
# STOCKHOLM 1.0
#=GS COATB_BPIKE/30-81 AC P03620.1
#=GS COATB_BPIKE/30-81 DR PDB; 1ifl ; 1-52;
#=GS Q9T0Q8_BPIKE/1-52 AC Q9T0Q8.1
#=GS COATB_BPI22/32-83 AC P15416.1
#=GS COATB_BPM13/24-72 AC P69541.1
#=GS COATB_BPM13/24-72 DR PDB; 2cpb ; 1-49;
#=GS COATB_BPM13/24-72 DR PDB; 2cps ; 1-49;
#=GS COATB_BPZJ2/1-49 AC P03618.1
#=GS Q9T0Q9_BPFD/1-49 AC Q9T0Q9.1
#=GS Q9T0Q9_BPFD/1-49 DR PDB; 1nh4 A; 1-49;
#=GS COATB_BPIF1/22-73 AC P03619.2
#=GS COATB_BPIF1/22-73 DR PDB; 1ifk ; 1-50;
COATB_BPIKE/30-81 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA
#=GR COATB_BPIKE/30-81 SS -HHHHHHHHHHHHHH--HHHHHHHH--HHHHHHHHHHHHHHHHHHHHH----
Q9T0Q8_BPIKE/1-52 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA
COATB_BPI22/32-83 DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA
COATB_BPM13/24-72 AEGDDP...AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
#=GR COATB_BPM13/24-72 SS ---S-T...CHCHHHHCCCCTCCCTTCHHHHHHHHHHHHHHHHHHHHCTT--
COATB_BPZJ2/1-49 AEGDDP...AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA
Q9T0Q9_BPFD/1-49 AEGDDP...AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
#=GR Q9T0Q9_BPFD/1-49 SS ------...-HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH--
COATB_BPIF1/22-73 FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA
#=GR COATB_BPIF1/22-73 SS XX-HHHH--HHHHHH--HHHHHHH--HHHHHHHHHHHHHHHHHHHHHHH---
#=GC SS_cons XHHHHHHHHHHHHHHHCHHHHHHHHCHHHHHHHHHHHHHHHHHHHHHHHC--
#=GC seq_cons AEssss...AptAhDSLpspAT-hIu.sWshVsslVsAsluIKLFKKFsSKA
//
This is the seed alignment for the Phage_Coat_Gp8 (PF05371) PFAM entry, downloaded from a now out of date release of PFAM from http://pfam.sanger.ac.uk/. We can load this file as follows (assuming it has been saved to disk as “PF05371_seed.sth” in the current working directory):
from Bio import AlignIO
alignment = AlignIO.read("data/PF05371_seed.sth", "stockholm")
This code will print out a summary of the alignment:
print(alignment)
SingleLetterAlphabet() alignment with 7 rows and 52 columns AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRL...SKA COATB_BPIKE/30-81 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKL...SRA Q9T0Q8_BPIKE/1-52 DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRL...SKA COATB_BPI22/32-83 AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPM13/24-72 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPZJ2/1-49 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA Q9T0Q9_BPFD/1-49 FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKL...SRA COATB_BPIF1/22-73
You’ll notice in the above output the sequences have been truncated. We
could instead write our own code to format this as we please by
iterating over the rows as SeqRecord
objects:
from Bio import AlignIO
alignment = AlignIO.read("data/PF05371_seed.sth", "stockholm")
print("Alignment length %i" % alignment.get_alignment_length())
Alignment length 52
for record in alignment:
print("%s - %s" % (record.seq, record.id))
AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA - COATB_BPIKE/30-81 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA - Q9T0Q8_BPIKE/1-52 DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA - COATB_BPI22/32-83 AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - COATB_BPM13/24-72 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA - COATB_BPZJ2/1-49 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - Q9T0Q9_BPFD/1-49 FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA - COATB_BPIF1/22-73
You could also use the alignment object’s format
method to show it in
a particular file format – see Section [sec:alignment-format-method]
for details.
Did you notice in the raw file above that several of the sequences include database cross-references to the PDB and the associated known secondary structure? Try this:
for record in alignment:
if record.dbxrefs:
print("%s %s" % (record.id, record.dbxrefs))
COATB_BPIKE/30-81 ['PDB; 1ifl ; 1-52;'] COATB_BPM13/24-72 ['PDB; 2cpb ; 1-49;', 'PDB; 2cps ; 1-49;'] Q9T0Q9_BPFD/1-49 ['PDB; 1nh4 A; 1-49;'] COATB_BPIF1/22-73 ['PDB; 1ifk ; 1-50;']
To have a look at all the sequence annotation, try this:
for record in alignment:
print(record)
ID: COATB_BPIKE/30-81 Name: COATB_BPIKE Description: COATB_BPIKE/30-81 Database cross-references: PDB; 1ifl ; 1-52; Number of features: 0 /start=30 /accession=P03620.1 /end=81 Per letter annotation for: secondary_structure Seq('AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA', SingleLetterAlphabet()) ID: Q9T0Q8_BPIKE/1-52 Name: Q9T0Q8_BPIKE Description: Q9T0Q8_BPIKE/1-52 Number of features: 0 /start=1 /accession=Q9T0Q8.1 /end=52 Seq('AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA', SingleLetterAlphabet()) ID: COATB_BPI22/32-83 Name: COATB_BPI22 Description: COATB_BPI22/32-83 Number of features: 0 /start=32 /accession=P15416.1 /end=83 Seq('DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA', SingleLetterAlphabet()) ID: COATB_BPM13/24-72 Name: COATB_BPM13 Description: COATB_BPM13/24-72 Database cross-references: PDB; 2cpb ; 1-49;, PDB; 2cps ; 1-49; Number of features: 0 /start=24 /accession=P69541.1 /end=72 Per letter annotation for: secondary_structure Seq('AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA', SingleLetterAlphabet()) ID: COATB_BPZJ2/1-49 Name: COATB_BPZJ2 Description: COATB_BPZJ2/1-49 Number of features: 0 /start=1 /accession=P03618.1 /end=49 Seq('AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA', SingleLetterAlphabet()) ID: Q9T0Q9_BPFD/1-49 Name: Q9T0Q9_BPFD Description: Q9T0Q9_BPFD/1-49 Database cross-references: PDB; 1nh4 A; 1-49; Number of features: 0 /start=1 /accession=Q9T0Q9.1 /end=49 Per letter annotation for: secondary_structure Seq('AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA', SingleLetterAlphabet()) ID: COATB_BPIF1/22-73 Name: COATB_BPIF1 Description: COATB_BPIF1/22-73 Database cross-references: PDB; 1ifk ; 1-50; Number of features: 0 /start=22 /accession=P03619.2 /end=73 Per letter annotation for: secondary_structure Seq('FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA', SingleLetterAlphabet())
Sanger provide a nice web interface at http://pfam.sanger.ac.uk/family?acc=PF05371 which will actually let you download this alignment in several other formats. This is what the file looks like in the FASTA file format:
>COATB_BPIKE/30-81
AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA
>Q9T0Q8_BPIKE/1-52
AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA
>COATB_BPI22/32-83
DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA
>COATB_BPM13/24-72
AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
>COATB_BPZJ2/1-49
AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA
>Q9T0Q9_BPFD/1-49
AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA
>COATB_BPIF1/22-73
FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA
Note the website should have an option about showing gaps as periods (dots) or dashes, we’ve shown dashes above. Assuming you download and save this as file “PF05371_seed.faa” then you can load it with almost exactly the same code:
from Bio import AlignIO
alignment = AlignIO.read("PF05371_seed.faa", "fasta")
print(alignment)
All that has changed in this code is the filename and the format string.
You’ll get the same output as before, the sequences and record
identifiers are the same. However, as you should expect, if you check
each SeqRecord
there is no annotation nor database cross-references
because these are not included in the FASTA file format.
Note that rather than using the Sanger website, you could have used
Bio.AlignIO
to convert the original Stockholm format file into a FASTA
file yourself (see below).
With any supported file format, you can load an alignment in exactly the same way just by changing the format string. For example, use “phylip” for PHYLIP files, “nexus” for NEXUS files or “emboss” for the alignments output by the EMBOSS tools. There is a full listing on the wiki page (http://biopython.org/wiki/AlignIO) and in the built in documentation (also online):
from Bio import AlignIO
help(AlignIO)
Help on package Bio.AlignIO in Bio: NAME Bio.AlignIO - Multiple sequence alignment input/output as alignment objects. DESCRIPTION The Bio.AlignIO interface is deliberately very similar to Bio.SeqIO, and in fact the two are connected internally. Both modules use the same set of file format names (lower case strings). From the user's perspective, you can read in a PHYLIP file containing one or more alignments using Bio.AlignIO, or you can read in the sequences within these alignmenta using Bio.SeqIO. Bio.AlignIO is also documented at http://biopython.org/wiki/AlignIO and by a whole chapter in our tutorial: * `HTML Tutorial`_ * `PDF Tutorial`_ .. _`HTML Tutorial`: http://biopython.org/DIST/docs/tutorial/Tutorial.html .. _`PDF Tutorial`: http://biopython.org/DIST/docs/tutorial/Tutorial.pdf Input ----- For the typical special case when your file or handle contains one and only one alignment, use the function Bio.AlignIO.read(). This takes an input file handle (or in recent versions of Biopython a filename as a string), format string and optional number of sequences per alignment. It will return a single MultipleSeqAlignment object (or raise an exception if there isn't just one alignment): >>> from Bio import AlignIO >>> align = AlignIO.read("Phylip/interlaced.phy", "phylip") >>> print(align) SingleLetterAlphabet() alignment with 3 rows and 384 columns -----MKVILLFVLAVFTVFVSS---------------RGIPPE...I-- CYS1_DICDI MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTL...VAA ALEU_HORVU ------MWATLPLLCAGAWLLGV--------PVCGAAELSVNSL...PLV CATH_HUMAN For the general case, when the handle could contain any number of alignments, use the function Bio.AlignIO.parse(...) which takes the same arguments, but returns an iterator giving MultipleSeqAlignment objects (typically used in a for loop). If you want random access to the alignments by number, turn this into a list: >>> from Bio import AlignIO >>> alignments = list(AlignIO.parse("Emboss/needle.txt", "emboss")) >>> print(alignments[2]) SingleLetterAlphabet() alignment with 2 rows and 120 columns -KILIVDDQYGIRILLNEVFNKEGYQTFQAANGLQALDIVTKER...--- ref_rec LHIVVVDDDPGTCVYIESVFAELGHTCKSFVRPEAAEEYILTHP...HKE gi|94967506|receiver Most alignment file formats can be concatenated so as to hold as many different multiple sequence alignments as possible. One common example is the output of the tool seqboot in the PHLYIP suite. Sometimes there can be a file header and footer, as seen in the EMBOSS alignment output. Output ------ Use the function Bio.AlignIO.write(...), which takes a complete set of Alignment objects (either as a list, or an iterator), an output file handle (or filename in recent versions of Biopython) and of course the file format:: from Bio import AlignIO alignments = ... count = SeqIO.write(alignments, "example.faa", "fasta") If using a handle make sure to close it to flush the data to the disk:: from Bio import AlignIO alignments = ... with open("example.faa", "w") as handle: count = SeqIO.write(alignments, handle, "fasta") In general, you are expected to call this function once (with all your alignments) and then close the file handle. However, for file formats like PHYLIP where multiple alignments are stored sequentially (with no file header and footer), then multiple calls to the write function should work as expected when using handles. If you are using a filename, the repeated calls to the write functions will overwrite the existing file each time. Conversion ---------- The Bio.AlignIO.convert(...) function allows an easy interface for simple alignnment file format conversions. Additionally, it may use file format specific optimisations so this should be the fastest way too. In general however, you can combine the Bio.AlignIO.parse(...) function with the Bio.AlignIO.write(...) function for sequence file conversion. Using generator expressions provides a memory efficient way to perform filtering or other extra operations as part of the process. File Formats ------------ When specifying the file format, use lowercase strings. The same format names are also used in Bio.SeqIO and include the following: - clustal - Output from Clustal W or X, see also the module Bio.Clustalw which can be used to run the command line tool from Biopython. - emboss - EMBOSS tools' "pairs" and "simple" alignment formats. - fasta - The generic sequence file format where each record starts with an identifer line starting with a ">" character, followed by lines of sequence. - fasta-m10 - For the pairswise alignments output by Bill Pearson's FASTA tools when used with the -m 10 command line option for machine readable output. - ig - The IntelliGenetics file format, apparently the same as the MASE alignment format. - nexus - Output from NEXUS, see also the module Bio.Nexus which can also read any phylogenetic trees in these files. - phylip - Interlaced PHYLIP, as used by the PHLIP tools. - phylip-sequential - Sequential PHYLIP. - phylip-relaxed - PHYLIP like format allowing longer names. - stockholm - A richly annotated alignment file format used by PFAM. Note that while Bio.AlignIO can read all the above file formats, it cannot write to all of them. You can also use any file format supported by Bio.SeqIO, such as "fasta" or "ig" (which are listed above), PROVIDED the sequences in your file are all the same length. PACKAGE CONTENTS ClustalIO EmbossIO FastaIO Interfaces NexusIO PhylipIO StockholmIO FUNCTIONS convert(in_file, in_format, out_file, out_format, alphabet=None) Convert between two alignment files, returns number of alignments. - in_file - an input handle or filename - in_format - input file format, lower case string - output - an output handle or filename - out_file - output file format, lower case string - alphabet - optional alphabet to assume **NOTE** - If you provide an output filename, it will be opened which will overwrite any existing file without warning. This may happen if even the conversion is aborted (e.g. an invalid out_format name is given). parse(handle, format, seq_count=None, alphabet=None) Iterate over an alignment file as MultipleSeqAlignment objects. Arguments: - handle - handle to the file, or the filename as a string (note older versions of Biopython only took a handle). - format - string describing the file format. - alphabet - optional Alphabet object, useful when the sequence type cannot be automatically inferred from the file itself (e.g. fasta, phylip, clustal) - seq_count - Optional integer, number of sequences expected in each alignment. Recommended for fasta format files. If you have the file name in a string 'filename', use: >>> from Bio import AlignIO >>> filename = "Emboss/needle.txt" >>> format = "emboss" >>> for alignment in AlignIO.parse(filename, format): ... print("Alignment of length %i" % alignment.get_alignment_length()) Alignment of length 124 Alignment of length 119 Alignment of length 120 Alignment of length 118 Alignment of length 125 If you have a string 'data' containing the file contents, use:: from Bio import AlignIO from StringIO import StringIO my_iterator = AlignIO.parse(StringIO(data), format) Use the Bio.AlignIO.read() function when you expect a single record only. read(handle, format, seq_count=None, alphabet=None) Turns an alignment file into a single MultipleSeqAlignment object. Arguments: - handle - handle to the file, or the filename as a string (note older versions of Biopython only took a handle). - format - string describing the file format. - alphabet - optional Alphabet object, useful when the sequence type cannot be automatically inferred from the file itself (e.g. fasta, phylip, clustal) - seq_count - Optional integer, number of sequences expected in each alignment. Recommended for fasta format files. If the handle contains no alignments, or more than one alignment, an exception is raised. For example, using a PFAM/Stockholm file containing one alignment: >>> from Bio import AlignIO >>> filename = "Clustalw/protein.aln" >>> format = "clustal" >>> alignment = AlignIO.read(filename, format) >>> print("Alignment of length %i" % alignment.get_alignment_length()) Alignment of length 411 If however you want the first alignment from a file containing multiple alignments this function would raise an exception. >>> from Bio import AlignIO >>> filename = "Emboss/needle.txt" >>> format = "emboss" >>> alignment = AlignIO.read(filename, format) Traceback (most recent call last): ... ValueError: More than one record found in handle Instead use: >>> from Bio import AlignIO >>> filename = "Emboss/needle.txt" >>> format = "emboss" >>> alignment = next(AlignIO.parse(filename, format)) >>> print("First alignment has length %i" % alignment.get_alignment_length()) First alignment has length 124 You must use the Bio.AlignIO.parse() function if you want to read multiple records from the handle. write(alignments, handle, format) Write complete set of alignments to a file. Arguments: - alignments - A list (or iterator) of Alignment objects (ideally the new MultipleSeqAlignment objects), or (if using Biopython 1.54 or later) a single alignment object. - handle - File handle object to write to, or filename as string (note older versions of Biopython only took a handle). - format - lower case string describing the file format to write. You should close the handle after calling this function. Returns the number of alignments written (as an integer). DATA __docformat__ = 'restructuredtext en' print_function = _Feature((2, 6, 0, 'alpha', 2), (3, 0, 0, 'alpha', 0)... FILE /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/AlignIO/__init__.py
The previous section focused on reading files containing a single
alignment. In general however, files can contain more than one
alignment, and to read these files we must use the Bio.AlignIO.parse()
function.
Suppose you have a small alignment in PHYLIP format:
5 6
Alpha AACAAC
Beta AACCCC
Gamma ACCAAC
Delta CCACCA
Epsilon CCAAAC
If you wanted to bootstrap a phylogenetic tree using the PHYLIP tools,
one of the steps would be to create a set of many resampled alignments
using the tool bootseq
. This would give output something like this,
which has been abbreviated for conciseness:
5 6
Alpha AAACCA
Beta AAACCC
Gamma ACCCCA
Delta CCCAAC
Epsilon CCCAAA
5 6
Alpha AAACAA
Beta AAACCC
Gamma ACCCAA
Delta CCCACC
Epsilon CCCAAA
5 6
Alpha AAAAAC
Beta AAACCC
Gamma AACAAC
Delta CCCCCA
Epsilon CCCAAC
...
5 6
Alpha AAAACC
Beta ACCCCC
Gamma AAAACC
Delta CCCCAA
Epsilon CAAACC
If you wanted to read this in using Bio.AlignIO
you could use:
from Bio import AlignIO
alignments = AlignIO.parse("data/resampled.phy", "phylip")
for alignment in alignments:
print(alignment)
print("")
--------------------------------------------------------------------------- FileNotFoundError Traceback (most recent call last) <ipython-input-8-13e6344aed5e> in <module>() 1 from Bio import AlignIO 2 alignments = AlignIO.parse("data/resampled.phy", "phylip") ----> 3 for alignment in alignments: 4 print(alignment) 5 print("") /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/AlignIO/__init__.py in parse(handle, format, seq_count, alphabet) 348 raise TypeError("Need integer for seq_count (sequences per alignment)") 349 --> 350 with as_handle(handle, 'rU') as fp: 351 # Map the file format to a sequence iterator: 352 if format in _FormatToIterator: /home/tiago_antao/miniconda/lib/python3.5/contextlib.py in __enter__(self) 57 def __enter__(self): 58 try: ---> 59 return next(self.gen) 60 except StopIteration: 61 raise RuntimeError("generator didn't yield") from None /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/File.py in as_handle(handleish, mode, **kwargs) 88 yield fp 89 else: ---> 90 with open(handleish, mode, **kwargs) as fp: 91 yield fp 92 else: FileNotFoundError: [Errno 2] No such file or directory: 'data/resampled.phy'
As with the function Bio.SeqIO.parse()
, using Bio.AlignIO.parse()
returns an iterator. If you want to keep all the alignments in memory at
once, which will allow you to access them in any order, then turn the
iterator into a list:
from Bio import AlignIO
alignments = list(AlignIO.parse("data/resampled.phy", "phylip"))
last_align = alignments[-1]
first_align = alignments[0]
--------------------------------------------------------------------------- FileNotFoundError Traceback (most recent call last) <ipython-input-10-0ef98bef9604> in <module>() 1 from Bio import AlignIO ----> 2 alignments = list(AlignIO.parse("data/resampled.phy", "phylip")) 3 last_align = alignments[-1] 4 first_align = alignments[0] /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/AlignIO/__init__.py in parse(handle, format, seq_count, alphabet) 348 raise TypeError("Need integer for seq_count (sequences per alignment)") 349 --> 350 with as_handle(handle, 'rU') as fp: 351 # Map the file format to a sequence iterator: 352 if format in _FormatToIterator: /home/tiago_antao/miniconda/lib/python3.5/contextlib.py in __enter__(self) 57 def __enter__(self): 58 try: ---> 59 return next(self.gen) 60 except StopIteration: 61 raise RuntimeError("generator didn't yield") from None /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/File.py in as_handle(handleish, mode, **kwargs) 88 yield fp 89 else: ---> 90 with open(handleish, mode, **kwargs) as fp: 91 yield fp 92 else: FileNotFoundError: [Errno 2] No such file or directory: 'data/resampled.phy'
Many alignment file formats can explicitly store more than one alignment, and the division between each alignment is clear. However, when a general sequence file format has been used there is no such block structure. The most common such situation is when alignments have been saved in the FASTA file format. For example consider the following:
>Alpha
ACTACGACTAGCTCAG--G
>Beta
ACTACCGCTAGCTCAGAAG
>Gamma
ACTACGGCTAGCACAGAAG
>Alpha
ACTACGACTAGCTCAGG--
>Beta
ACTACCGCTAGCTCAGAAG
>Gamma
ACTACGGCTAGCACAGAAG
This could be a single alignment containing six sequences (with repeated identifiers). Or, judging from the identifiers, this is probably two different alignments each with three sequences, which happen to all have the same length.
What about this next example?
>Alpha
ACTACGACTAGCTCAG--G
>Beta
ACTACCGCTAGCTCAGAAG
>Alpha
ACTACGACTAGCTCAGG--
>Gamma
ACTACGGCTAGCACAGAAG
>Alpha
ACTACGACTAGCTCAGG--
>Delta
ACTACGGCTAGCACAGAAG
Again, this could be a single alignment with six sequences. However this time based on the identifiers we might guess this is three pairwise alignments which by chance have all got the same lengths.
This final example is similar:
>Alpha
ACTACGACTAGCTCAG--G
>XXX
ACTACCGCTAGCTCAGAAG
>Alpha
ACTACGACTAGCTCAGG
>YYY
ACTACGGCAAGCACAGG
>Alpha
--ACTACGAC--TAGCTCAGG
>ZZZ
GGACTACGACAATAGCTCAGG
In this third example, because of the differing lengths, this cannot be treated as a single alignment containing all six records. However, it could be three pairwise alignments.
Clearly trying to store more than one alignment in a FASTA file is not
ideal. However, if you are forced to deal with these as input files
Bio.AlignIO
can cope with the most common situation where all the
alignments have the same number of records. One example of this is a
collection of pairwise alignments, which can be produced by the EMBOSS
tools needle
and water
– although in this situation, Bio.AlignIO
should be able to understand their native output using “emboss” as the
format string.
To interpret these FASTA examples as several separate alignments, we can
use Bio.AlignIO.parse()
with the optional seq_count
argument which
specifies how many sequences are expected in each alignment (in these
examples, 3, 2 and 2 respectively). For example, using the third example
as the input data:
for alignment in AlignIO.parse(handle, "fasta", seq_count=2):
print("Alignment length %i" % alignment.get_alignment_length())
for record in alignment:
print("%s - %s" % (record.seq, record.id))
print("")
--------------------------------------------------------------------------- NameError Traceback (most recent call last) <ipython-input-11-6e1f5efd5fb4> in <module>() ----> 1 for alignment in AlignIO.parse(handle, "fasta", seq_count=2): 2 print("Alignment length %i" % alignment.get_alignment_length()) 3 for record in alignment: 4 print("%s - %s" % (record.seq, record.id)) 5 print("") NameError: name 'handle' is not defined
Using Bio.AlignIO.read()
or Bio.AlignIO.parse()
without the
seq_count
argument would give a single alignment containing all six
records for the first two examples. For the third example, an exception
would be raised because the lengths differ preventing them being turned
into a single alignment.
If the file format itself has a block structure allowing Bio.AlignIO
to determine the number of sequences in each alignment directly, then
the seq_count
argument is not needed. If it is supplied, and doesn’t
agree with the file contents, an error is raised.
Note that this optional seq_count
argument assumes each alignment in
the file has the same number of sequences. Hypothetically you may come
across stranger situations, for example a FASTA file containing several
alignments each with a different number of sequences – although I would
love to hear of a real world example of this. Assuming you cannot get
the data in a nicer file format, there is no straight forward way to
deal with this using Bio.AlignIO
. In this case, you could consider
reading in the sequences themselves using Bio.SeqIO
and batching them
together to create the alignments as appropriate.
We’ve talked about using Bio.AlignIO.read()
and Bio.AlignIO.parse()
for alignment input (reading files), and now we’ll look at
Bio.AlignIO.write()
which is for alignment output (writing files).
This is a function taking three arguments: some MultipleSeqAlignment
objects (or for backwards compatibility the obsolete Alignment
objects), a handle or filename to write to, and a sequence format.
Here is an example, where we start by creating a few
MultipleSeqAlignment
objects the hard way (by hand, rather than by
loading them from a file). Note we create some SeqRecord
objects to
construct the alignment from.
from Bio.Alphabet import generic_dna
from Bio.Seq import Seq
from Bio.SeqRecord import SeqRecord
from Bio.Align import MultipleSeqAlignment
align1 = MultipleSeqAlignment([
SeqRecord(Seq("ACTGCTAGCTAG", generic_dna), id="Alpha"),
SeqRecord(Seq("ACT-CTAGCTAG", generic_dna), id="Beta"),
SeqRecord(Seq("ACTGCTAGDTAG", generic_dna), id="Gamma"),
])
align2 = MultipleSeqAlignment([
SeqRecord(Seq("GTCAGC-AG", generic_dna), id="Delta"),
SeqRecord(Seq("GACAGCTAG", generic_dna), id="Epsilon"),
SeqRecord(Seq("GTCAGCTAG", generic_dna), id="Zeta"),
])
align3 = MultipleSeqAlignment([
SeqRecord(Seq("ACTAGTACAGCTG", generic_dna), id="Eta"),
SeqRecord(Seq("ACTAGTACAGCT-", generic_dna), id="Theta"),
SeqRecord(Seq("-CTACTACAGGTG", generic_dna), id="Iota"),
])
my_alignments = [align1, align2, align3]
Now we have a list of Alignment
objects, we’ll write them to a PHYLIP
format file:
from Bio import AlignIO
AlignIO.write(my_alignments, "my_example.phy", "phylip")
3
And if you open this file in your favourite text editor it should look like this:
3 12
Alpha ACTGCTAGCT AG
Beta ACT-CTAGCT AG
Gamma ACTGCTAGDT AG
3 9
Delta GTCAGC-AG
Epislon GACAGCTAG
Zeta GTCAGCTAG
3 13
Eta ACTAGTACAG CTG
Theta ACTAGTACAG CT-
Iota -CTACTACAG GTG
Its more common to want to load an existing alignment, and save that, perhaps after some simple manipulation like removing certain rows or columns.
Suppose you wanted to know how many alignments the Bio.AlignIO.write()
function wrote to the handle? If your alignments were in a list like the
example above, you could just use len(my_alignments)
, however you
can’t do that when your records come from a generator/iterator.
Therefore the Bio.AlignIO.write()
function returns the number of
alignments written to the file.
Note - If you tell the Bio.AlignIO.write()
function to write to a
file that already exists, the old file will be overwritten without any
warning.
Converting between sequence alignment file formats with Bio.AlignIO
works in the same way as converting between sequence file formats with
Bio.SeqIO
(Section [sec:SeqIO-conversion]). We load generally the
alignment(s) using Bio.AlignIO.parse()
and then save them using the
Bio.AlignIO.write()
– or just use the Bio.AlignIO.convert()
helper
function.
For this example, we’ll load the PFAM/Stockholm format file used earlier and save it as a Clustal W format file:
from Bio import AlignIO
count = AlignIO.convert("data/PF05371_seed.sth", "stockholm", "PF05371_seed.aln", "clustal")
print("Converted %i alignments" % count)
Converted 1 alignments
Or, using Bio.AlignIO.parse()
and Bio.AlignIO.write()
:
from Bio import AlignIO
alignments = AlignIO.parse("data/PF05371_seed.sth", "stockholm")
count = AlignIO.write(alignments, "PF05371_seed.aln", "clustal")
print("Converted %i alignments" % count)
Converted 1 alignments
The Bio.AlignIO.write()
function expects to be given multiple
alignment objects. In the example above we gave it the alignment
iterator returned by Bio.AlignIO.parse()
.
In this case, we know there is only one alignment in the file so we
could have used Bio.AlignIO.read()
instead, but notice we have to pass
this alignment to Bio.AlignIO.write()
as a single element list:
from Bio import AlignIO
alignment = AlignIO.read("data/PF05371_seed.sth", "stockholm")
AlignIO.write([alignment], "PF05371_seed.aln", "clustal")
1
Either way, you should end up with the same new Clustal W format file “PF05371_seed.aln” with the following content:
CLUSTAL X (1.81) multiple sequence alignment
COATB_BPIKE/30-81 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSS
Q9T0Q8_BPIKE/1-52 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVS
COATB_BPI22/32-83 DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSS
COATB_BPM13/24-72 AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTS
COATB_BPZJ2/1-49 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFAS
Q9T0Q9_BPFD/1-49 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTS
COATB_BPIF1/22-73 FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVS
COATB_BPIKE/30-81 KA
Q9T0Q8_BPIKE/1-52 RA
COATB_BPI22/32-83 KA
COATB_BPM13/24-72 KA
COATB_BPZJ2/1-49 KA
Q9T0Q9_BPFD/1-49 KA
COATB_BPIF1/22-73 RA
Alternatively, you could make a PHYLIP format file which we’ll name “PF05371_seed.phy”:
from Bio import AlignIO
AlignIO.convert("data/PF05371_seed.sth", "stockholm", "PF05371_seed.phy", "phylip")
1
This time the output looks like this:
7 52
COATB_BPIK AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIRLFKKFSS
Q9T0Q8_BPI AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIKLFKKFVS
COATB_BPI2 DGTSTATSYA TEAMNSLKTQ ATDLIDQTWP VVTSVAVAGL AIRLFKKFSS
COATB_BPM1 AEGDDP---A KAAFNSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
COATB_BPZJ AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFAS
Q9T0Q9_BPF AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
COATB_BPIF FAADDATSQA KAAFDSLTAQ ATEMSGYAWA LVVLVVGATV GIKLFKKFVS
KA
RA
KA
KA
KA
KA
RA
One of the big handicaps of the original PHYLIP alignment file format is that the sequence identifiers are strictly truncated at ten characters. In this example, as you can see the resulting names are still unique - but they are not very readable. As a result, a more relaxed variant of the original PHYLIP format is now quite widely used:
from Bio import AlignIO
AlignIO.convert("data/PF05371_seed.sth", "stockholm", "PF05371_seed.phy", "phylip-relaxed")
1
This time the output looks like this, using a longer indentation to allow all the identifers to be given in full::
7 52
COATB_BPIKE/30-81 AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIRLFKKFSS
Q9T0Q8_BPIKE/1-52 AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIKLFKKFVS
COATB_BPI22/32-83 DGTSTATSYA TEAMNSLKTQ ATDLIDQTWP VVTSVAVAGL AIRLFKKFSS
COATB_BPM13/24-72 AEGDDP---A KAAFNSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
COATB_BPZJ2/1-49 AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFAS
Q9T0Q9_BPFD/1-49 AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
COATB_BPIF1/22-73 FAADDATSQA KAAFDSLTAQ ATEMSGYAWA LVVLVVGATV GIKLFKKFVS
KA
RA
KA
KA
KA
KA
RA
If you have to work with the original strict PHYLIP format, then you may need to compress the identifers somehow – or assign your own names or numbering system. This following bit of code manipulates the record identifiers before saving the output:
from Bio import AlignIO
alignment = AlignIO.read("data/PF05371_seed.sth", "stockholm")
name_mapping = {}
for i, record in enumerate(alignment):
name_mapping[i] = record.id
record.id = "seq%i" % i
print(name_mapping)
AlignIO.write([alignment], "PF05371_seed.phy", "phylip")
{0: 'COATB_BPIKE/30-81', 1: 'Q9T0Q8_BPIKE/1-52', 2: 'COATB_BPI22/32-83', 3: 'COATB_BPM13/24-72', 4: 'COATB_BPZJ2/1-49', 5: 'Q9T0Q9_BPFD/1-49', 6: 'COATB_BPIF1/22-73'}
1
Here is the new (strict) PHYLIP format output:
7 52
seq0 AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIRLFKKFSS
seq1 AEPNAATNYA TEAMDSLKTQ AIDLISQTWP VVTTVVVAGL VIKLFKKFVS
seq2 DGTSTATSYA TEAMNSLKTQ ATDLIDQTWP VVTSVAVAGL AIRLFKKFSS
seq3 AEGDDP---A KAAFNSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
seq4 AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFAS
seq5 AEGDDP---A KAAFDSLQAS ATEYIGYAWA MVVVIVGATI GIKLFKKFTS
seq6 FAADDATSQA KAAFDSLTAQ ATEMSGYAWA LVVLVVGATV GIKLFKKFVS
KA
RA
KA
KA
KA
KA
RA
In general, because of the identifier limitation, working with strict PHYLIP file formats shouldn’t be your first choice. Using the PFAM/Stockholm format on the other hand allows you to record a lot of additional annotation too.
The Bio.AlignIO
interface is based on handles, which means if you want
to get your alignment(s) into a string in a particular file format you
need to do a little bit more work (see below). However, you will
probably prefer to take advantage of the alignment object’s format()
method. This takes a single mandatory argument, a lower case string
which is supported by Bio.AlignIO
as an output format. For example:
from Bio import AlignIO
alignment = AlignIO.read("PF05371_seed.sth", "stockholm")
print(alignment.format("clustal"))
As described in Section [sec:SeqRecord-format], the SeqRecord
object
has a similar method using output formats supported by Bio.SeqIO
.
Internally the format()
method is using the StringIO
string based
handle and calling Bio.AlignIO.write()
. You can do this in your own
code if for example you are using an older version of Biopython:
from Bio import AlignIO
from StringIO import StringIO
alignments = AlignIO.parse("PF05371_seed.sth", "stockholm")
out_handle = StringIO()
AlignIO.write(alignments, out_handle, "clustal")
clustal_data = out_handle.getvalue()
print(clustal_data)
Now that we’ve covered loading and saving alignments, we’ll look at what else you can do with them.
First of all, in some senses the alignment objects act like a Python
list
of SeqRecord
objects (the rows). With this model in mind
hopefully the actions of len()
(the number of rows) and iteration
(each row as a SeqRecord
) make sense:
from Bio import AlignIO
alignment = AlignIO.read("data/PF05371_seed.sth", "stockholm")
print("Number of rows: %i" % len(alignment))
Number of rows: 7
for record in alignment:
print("%s - %s" % (record.seq, record.id))
AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA - COATB_BPIKE/30-81 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA - Q9T0Q8_BPIKE/1-52 DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA - COATB_BPI22/32-83 AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - COATB_BPM13/24-72 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA - COATB_BPZJ2/1-49 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA - Q9T0Q9_BPFD/1-49 FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA - COATB_BPIF1/22-73
You can also use the list-like append
and extend
methods to add more
rows to the alignment (as SeqRecord
objects). Keeping the list
metaphor in mind, simple slicing of the alignment should also make sense
print(alignment)
SingleLetterAlphabet() alignment with 7 rows and 52 columns AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRL...SKA COATB_BPIKE/30-81 AEPNAATNYATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKL...SRA Q9T0Q8_BPIKE/1-52 DGTSTATSYATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRL...SKA COATB_BPI22/32-83 AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPM13/24-72 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPZJ2/1-49 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA Q9T0Q9_BPFD/1-49 FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKL...SRA COATB_BPIF1/22-73
print(alignment[3:7])
SingleLetterAlphabet() alignment with 4 rows and 52 columns AEGDDP---AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPM13/24-72 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA COATB_BPZJ2/1-49 AEGDDP---AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKL...SKA Q9T0Q9_BPFD/1-49 FAADDATSQAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKL...SRA COATB_BPIF1/22-73
What if you wanted to select by column? Those of you who have used the NumPy matrix or array objects won’t be surprised at this - you use a double index.
print(alignment[2, 6])
T
Using two integer indices pulls out a single letter, short hand for this:
print(alignment[2].seq[6])
T
You can pull out a single column as a string like this:
print(alignment[:, 6])
TTT---T
You can also select a range of columns. For example, to pick out those same three rows we extracted earlier, but take just their first six columns:
print(alignment[3:6, :6])
SingleLetterAlphabet() alignment with 3 rows and 6 columns AEGDDP COATB_BPM13/24-72 AEGDDP COATB_BPZJ2/1-49 AEGDDP Q9T0Q9_BPFD/1-49
Leaving the first index as :
means take all the rows:
print(alignment[:, :6])
SingleLetterAlphabet() alignment with 7 rows and 6 columns AEPNAA COATB_BPIKE/30-81 AEPNAA Q9T0Q8_BPIKE/1-52 DGTSTA COATB_BPI22/32-83 AEGDDP COATB_BPM13/24-72 AEGDDP COATB_BPZJ2/1-49 AEGDDP Q9T0Q9_BPFD/1-49 FAADDA COATB_BPIF1/22-73
This brings us to a neat way to remove a section. Notice columns 7, 8 and 9 which are gaps in three of the seven sequences:
print(alignment[:, 6:9])
SingleLetterAlphabet() alignment with 7 rows and 3 columns TNY COATB_BPIKE/30-81 TNY Q9T0Q8_BPIKE/1-52 TSY COATB_BPI22/32-83 --- COATB_BPM13/24-72 --- COATB_BPZJ2/1-49 --- Q9T0Q9_BPFD/1-49 TSQ COATB_BPIF1/22-73
Again, you can slice to get everything after the ninth column:
print(alignment[:, 9:])
SingleLetterAlphabet() alignment with 7 rows and 43 columns ATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA COATB_BPIKE/30-81 ATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA Q9T0Q8_BPIKE/1-52 ATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA COATB_BPI22/32-83 AKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA COATB_BPM13/24-72 AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA COATB_BPZJ2/1-49 AKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA Q9T0Q9_BPFD/1-49 AKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA COATB_BPIF1/22-73
Now, the interesting thing is that addition of alignment objects works by column. This lets you do this as a way to remove a block of columns:
edited = alignment[:, :6] + alignment[:, 9:]
print(edited)
SingleLetterAlphabet() alignment with 7 rows and 49 columns AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA COATB_BPIKE/30-81 AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA Q9T0Q8_BPIKE/1-52 DGTSTAATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA COATB_BPI22/32-83 AEGDDPAKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA COATB_BPM13/24-72 AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA COATB_BPZJ2/1-49 AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA Q9T0Q9_BPFD/1-49 FAADDAAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA COATB_BPIF1/22-73
Another common use of alignment addition would be to combine alignments
for several different genes into a meta-alignment. Watch out though -
the identifiers need to match up (see Section [sec:SeqRecord-addition]
for how adding SeqRecord
objects works). You may find it helpful to
first sort the alignment rows alphabetically by id:
edited.sort()
print(edited)
SingleLetterAlphabet() alignment with 7 rows and 49 columns DGTSTAATEAMNSLKTQATDLIDQTWPVVTSVAVAGLAIRLFKKFSSKA COATB_BPI22/32-83 FAADDAAKAAFDSLTAQATEMSGYAWALVVLVVGATVGIKLFKKFVSRA COATB_BPIF1/22-73 AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIRLFKKFSSKA COATB_BPIKE/30-81 AEGDDPAKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA COATB_BPM13/24-72 AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFASKA COATB_BPZJ2/1-49 AEPNAAATEAMDSLKTQAIDLISQTWPVVTTVVVAGLVIKLFKKFVSRA Q9T0Q8_BPIKE/1-52 AEGDDPAKAAFDSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKA Q9T0Q9_BPFD/1-49
Note that you can only add two alignments together if they have the same number of rows.
Depending on what you are doing, it can be more useful to turn the alignment object into an array of letters – and you can do this with NumPy:
import numpy as np
from Bio import AlignIO
alignment = AlignIO.read("data/PF05371_seed.sth", "stockholm")
align_array = np.array([list(rec) for rec in alignment], np.character)
print("Array shape %i by %i" % align_array.shape)
Array shape 7 by 52
If you will be working heavily with the columns, you can tell NumPy to store the array by column (as in Fortran) rather then its default of by row (as in C):
align_array = np.array([list(rec) for rec in alignment], np.character, order="F")
Note that this leaves the original Biopython alignment object and the NumPy array in memory as separate objects - editing one will not update the other!
There are lots of algorithms out there for aligning sequences, both pairwise alignments and multiple sequence alignments. These calculations are relatively slow, and you generally wouldn’t want to write such an algorithm in Python. Instead, you can use Biopython to invoke a command line tool on your behalf. Normally you would:
Prepare an input file of your unaligned sequences, typically this
will be a FASTA file which you might create using Bio.SeqIO
(see Chapter [chapter:Bio.SeqIO]).
Call the command line tool to process this input file, typically via one of Biopython’s command line wrappers (which we’ll discuss here).
Read the output from the tool, i.e. your aligned sequences,
typically using Bio.AlignIO
(see earlier in this chapter).
All the command line wrappers we’re going to talk about in this chapter
follow the same style. You create a command line object specifying the
options (e.g. the input filename and the output filename), then invoke
this command line via a Python operating system call (e.g. using the
subprocess
module).
Most of these wrappers are defined in the Bio.Align.Applications
module:
import Bio.Align.Applications
dir(Bio.Align.Applications)
['ClustalOmegaCommandline', 'ClustalwCommandline', 'DialignCommandline', 'MSAProbsCommandline', 'MafftCommandline', 'MuscleCommandline', 'PrankCommandline', 'ProbconsCommandline', 'TCoffeeCommandline', '_ClustalOmega', '_Clustalw', '_Dialign', '_MSAProbs', '_Mafft', '_Muscle', '_Prank', '_Probcons', '_TCoffee', '__all__', '__builtins__', '__cached__', '__doc__', '__docformat__', '__file__', '__loader__', '__name__', '__package__', '__path__', '__spec__']
(Ignore the entries starting with an underscore – these have special
meaning in Python.) The module Bio.Emboss.Applications
has wrappers
for some of the EMBOSS suite,
including needle
and water
, which are described below in
Section [seq:emboss-needle-water], and wrappers for the EMBOSS
packaged versions of the PHYLIP tools (which EMBOSS refer to as one of
their EMBASSY packages - third party tools with an EMBOSS style
interface). We won’t explore all these alignment tools here in the
section, just a sample, but the same principles apply.
ClustalW is a popular command line tool for multiple sequence alignment
(there is also a graphical interface called ClustalX). Biopython’s
Bio.Align.Applications
module has a wrapper for this alignment tool
(and several others).
Before trying to use ClustalW from within Python, you should first try running the ClustalW tool yourself by hand at the command line, to familiarise yourself the other options. You’ll find the Biopython wrapper is very faithful to the actual command line API:
from Bio.Align.Applications import ClustalwCommandline
help(ClustalwCommandline)
Help on class ClustalwCommandline in module Bio.Align.Applications._Clustalw: class ClustalwCommandline(Bio.Application.AbstractCommandline) | Command line wrapper for clustalw (version one or two). | | http://www.clustal.org/ | | Example: | -------- | | >>> from Bio.Align.Applications import ClustalwCommandline | >>> in_file = "unaligned.fasta" | >>> clustalw_cline = ClustalwCommandline("clustalw2", infile=in_file) | >>> print(clustalw_cline) | clustalw2 -infile=unaligned.fasta | | You would typically run the command line with clustalw_cline() or via | the Python subprocess module, as described in the Biopython tutorial. | | Citation: | --------- | | Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, | McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, | Gibson TJ, Higgins DG. (2007). Clustal W and Clustal X version 2.0. | Bioinformatics, 23, 2947-2948. | | Last checked against versions: 1.83 and 2.1 | | Method resolution order: | ClustalwCommandline | Bio.Application.AbstractCommandline | builtins.object | | Methods defined here: | | __init__(self, cmd='clustalw', **kwargs) | Create a new instance of a command line wrapper object. | | ---------------------------------------------------------------------- | Methods inherited from Bio.Application.AbstractCommandline: | | __call__(self, stdin=None, stdout=True, stderr=True, cwd=None, env=None) | Executes the command, waits for it to finish, and returns output. | | Runs the command line tool and waits for it to finish. If it returns | a non-zero error level, an exception is raised. Otherwise two strings | are returned containing stdout and stderr. | | The optional stdin argument should be a string of data which will be | passed to the tool as standard input. | | The optional stdout and stderr argument may be filenames (string), | but otherwise are treated as a booleans, and control if the output | should be captured as strings (True, default), or ignored by sending | it to /dev/null to avoid wasting memory (False). If sent to a file | or ignored, then empty string(s) are returned. | | The optional cwd argument is a string giving the working directory | to run the command from. See Python's subprocess module documentation | for more details. | | The optional env argument is a dictionary setting the environment | variables to be used in the new process. By default the current | process' environment variables are used. See Python's subprocess | module documentation for more details. | | Default example usage:: | | from Bio.Emboss.Applications import WaterCommandline | water_cmd = WaterCommandline(gapopen=10, gapextend=0.5, | stdout=True, auto=True, | asequence="a.fasta", bsequence="b.fasta") | print("About to run: %s" % water_cmd) | std_output, err_output = water_cmd() | | This functionality is similar to subprocess.check_output() added in | Python 2.7. In general if you require more control over running the | command, use subprocess directly. | | As of Biopython 1.56, when the program called returns a non-zero error | level, a custom ApplicationError exception is raised. This includes | any stdout and stderr strings captured as attributes of the exception | object, since they may be useful for diagnosing what went wrong. | | __repr__(self) | Return a representation of the command line object for debugging. | | e.g. | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5) | >>> cline.asequence = "asis:ACCCGGGCGCGGT" | >>> cline.bsequence = "asis:ACCCGAGCGCGGT" | >>> cline.outfile = "temp_water.txt" | >>> print(cline) | water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5 | >>> cline | WaterCommandline(cmd='water', outfile='temp_water.txt', asequence='asis:ACCCGGGCGCGGT', bsequence='asis:ACCCGAGCGCGGT', gapopen=10, gapextend=0.5) | | __setattr__(self, name, value) | Set attribute name to value (PRIVATE). | | This code implements a workaround for a user interface issue. | Without this __setattr__ attribute-based assignment of parameters | will silently accept invalid parameters, leading to known instances | of the user assuming that parameters for the application are set, | when they are not. | | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5, stdout=True) | >>> cline.asequence = "a.fasta" | >>> cline.bsequence = "b.fasta" | >>> cline.csequence = "c.fasta" | Traceback (most recent call last): | ... | ValueError: Option name csequence was not found. | >>> print(cline) | water -stdout -asequence=a.fasta -bsequence=b.fasta -gapopen=10 -gapextend=0.5 | | This workaround uses a whitelist of object attributes, and sets the | object attribute list as normal, for these. Other attributes are | assumed to be parameters, and passed to the self.set_parameter method | for validation and assignment. | | __str__(self) | Make the commandline string with the currently set options. | | e.g. | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5) | >>> cline.asequence = "asis:ACCCGGGCGCGGT" | >>> cline.bsequence = "asis:ACCCGAGCGCGGT" | >>> cline.outfile = "temp_water.txt" | >>> print(cline) | water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5 | >>> str(cline) | 'water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5' | | set_parameter(self, name, value=None) | Set a commandline option for a program (OBSOLETE). | | Every parameter is available via a property and as a named | keyword when creating the instance. Using either of these is | preferred to this legacy set_parameter method which is now | OBSOLETE, and likely to be DEPRECATED and later REMOVED in | future releases. | | ---------------------------------------------------------------------- | Data descriptors inherited from Bio.Application.AbstractCommandline: | | __dict__ | dictionary for instance variables (if defined) | | __weakref__ | list of weak references to the object (if defined) | | ---------------------------------------------------------------------- | Data and other attributes inherited from Bio.Application.AbstractCommandline: | | parameters = None
For the most basic usage, all you need is to have a FASTA input file, such as opuntia.fasta (available online or in the Doc/examples subdirectory of the Biopython source code). This is a small FASTA file containing seven prickly-pear DNA sequences (from the cactus family Opuntia).
By default ClustalW will generate an alignment and guide tree file with
names based on the input FASTA file, in this case opuntia.aln
and
opuntia.dnd
, but you can override this or make it explicit:
from Bio.Align.Applications import ClustalwCommandline
cline = ClustalwCommandline("clustalw2", infile="data/opuntia.fasta")
print(cline)
clustalw2 -infile=data/opuntia.fasta
Notice here we have given the executable name as clustalw2
, indicating
we have version two installed, which has a different filename to version
one (clustalw
, the default). Fortunately both versions support the
same set of arguments at the command line (and indeed, should be
functionally identical).
You may find that even though you have ClustalW installed, the above command doesn’t work – you may get a message about “command not found” (especially on Windows). This indicated that the ClustalW executable is not on your PATH (an environment variable, a list of directories to be searched). You can either update your PATH setting to include the location of your copy of ClustalW tools (how you do this will depend on your OS), or simply type in the full path of the tool. For example:
import os
from Bio.Align.Applications import ClustalwCommandline
clustalw_exe = r"C:\Program Files\new clustal\clustalw2.exe"
clustalw_cline = ClustalwCommandline(clustalw_exe, infile="data/opuntia.fasta")
assert os.path.isfile(clustalw_exe), "Clustal W executable missing"
stdout, stderr = clustalw_cline()
--------------------------------------------------------------------------- AssertionError Traceback (most recent call last) <ipython-input-37-2132fefeb91f> in <module>() ----> 1 assert os.path.isfile(clustalw_exe), "Clustal W executable missing" 2 stdout, stderr = clustalw_cline() AssertionError: Clustal W executable missing
Remember, in Python strings \n
and \t
are by default interpreted as
a new line and a tab – which is why we’re put a letter “r” at the start
for a raw string that isn’t translated in this way. This is generally
good practice when specifying a Windows style file name.
Internally this uses the subprocess
module which is now the
recommended way to run another program in Python. This replaces older
options like the os.system()
and the os.popen*
functions.
Now, at this point it helps to know about how command line tools “work”. When you run a tool at the command line, it will often print text output directly to screen. This text can be captured or redirected, via two “pipes”, called standard output (the normal results) and standard error (for error messages and debug messages). There is also standard input, which is any text fed into the tool. These names get shortened to stdin, stdout and stderr. When the tool finishes, it has a return code (an integer), which by convention is zero for success.
When you run the command line tool like this via the Biopython wrapper, it will wait for it to finish, and check the return code. If this is non zero (indicating an error), an exception is raised. The wrapper then returns two strings, stdout and stderr.
In the case of ClustalW, when run at the command line all the important output is written directly to the output files. Everything normally printed to screen while you wait (via stdout or stderr) is boring and can be ignored (assuming it worked).
What we care about are the two output files, the alignment and the guide
tree. We didn’t tell ClustalW what filenames to use, but it defaults to
picking names based on the input file. In this case the output should be
in the file opuntia.aln
. You should be able to work out how to read in
the alignment using Bio.AlignIO
by now:
from Bio import AlignIO
align = AlignIO.read("data/opuntia.aln", "clustal")
print(align)
SingleLetterAlphabet() alignment with 7 rows and 906 columns TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273285|gb|AF191659.1|AF191 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273284|gb|AF191658.1|AF191 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273287|gb|AF191661.1|AF191 TATACATAAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273286|gb|AF191660.1|AF191 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273290|gb|AF191664.1|AF191 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273289|gb|AF191663.1|AF191 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273291|gb|AF191665.1|AF191
In case you are interested (and this is an aside from the main thrust of
this chapter), the opuntia.dnd
file ClustalW creates is just a
standard Newick tree file, and Bio.Phylo
can parse these:
from Bio import Phylo
tree = Phylo.read("data/opuntia.dnd", "newick")
Phylo.draw_ascii(tree)
_______________ gi|6273291|gb|AF191665.1|AF191665 __________________________| | | ______ gi|6273290|gb|AF191664.1|AF191664 | |__| | |_____ gi|6273289|gb|AF191663.1|AF191663 | _|_________________ gi|6273287|gb|AF191661.1|AF191661 | |__________ gi|6273286|gb|AF191660.1|AF191660 | | __ gi|6273285|gb|AF191659.1|AF191659 |___| | gi|6273284|gb|AF191658.1|AF191658
Chapter [sec:Phylo] covers Biopython’s support for phylogenetic trees in more depth.
MUSCLE is a more recent multiple sequence alignment tool than ClustalW,
and Biopython also has a wrapper for it under the
Bio.Align.Applications
module. As before, we recommend you try using
MUSCLE from the command line before trying it from within Python, as the
Biopython wrapper is very faithful to the actual command line API:
from Bio.Align.Applications import MuscleCommandline
help(MuscleCommandline)
Help on class MuscleCommandline in module Bio.Align.Applications._Muscle: class MuscleCommandline(Bio.Application.AbstractCommandline) | Command line wrapper for the multiple alignment program MUSCLE. | | http://www.drive5.com/muscle/ | | Example: | -------- | | >>> from Bio.Align.Applications import MuscleCommandline | >>> muscle_exe = r"C:\Program Files\Aligments\muscle3.8.31_i86win32.exe" | >>> in_file = r"C:\My Documents\unaligned.fasta" | >>> out_file = r"C:\My Documents\aligned.fasta" | >>> muscle_cline = MuscleCommandline(muscle_exe, input=in_file, out=out_file) | >>> print(muscle_cline) | "C:\Program Files\Aligments\muscle3.8.31_i86win32.exe" -in "C:\My Documents\unaligned.fasta" -out "C:\My Documents\aligned.fasta" | | You would typically run the command line with muscle_cline() or via | the Python subprocess module, as described in the Biopython tutorial. | | Citations: | ---------- | | Edgar, Robert C. (2004), MUSCLE: multiple sequence alignment with high | accuracy and high throughput, Nucleic Acids Research 32(5), 1792-97. | | Edgar, R.C. (2004) MUSCLE: a multiple sequence alignment method with | reduced time and space complexity. BMC Bioinformatics 5(1): 113. | | Last checked against version: 3.7, briefly against 3.8 | | Method resolution order: | MuscleCommandline | Bio.Application.AbstractCommandline | builtins.object | | Methods defined here: | | __init__(self, cmd='muscle', **kwargs) | Create a new instance of a command line wrapper object. | | ---------------------------------------------------------------------- | Methods inherited from Bio.Application.AbstractCommandline: | | __call__(self, stdin=None, stdout=True, stderr=True, cwd=None, env=None) | Executes the command, waits for it to finish, and returns output. | | Runs the command line tool and waits for it to finish. If it returns | a non-zero error level, an exception is raised. Otherwise two strings | are returned containing stdout and stderr. | | The optional stdin argument should be a string of data which will be | passed to the tool as standard input. | | The optional stdout and stderr argument may be filenames (string), | but otherwise are treated as a booleans, and control if the output | should be captured as strings (True, default), or ignored by sending | it to /dev/null to avoid wasting memory (False). If sent to a file | or ignored, then empty string(s) are returned. | | The optional cwd argument is a string giving the working directory | to run the command from. See Python's subprocess module documentation | for more details. | | The optional env argument is a dictionary setting the environment | variables to be used in the new process. By default the current | process' environment variables are used. See Python's subprocess | module documentation for more details. | | Default example usage:: | | from Bio.Emboss.Applications import WaterCommandline | water_cmd = WaterCommandline(gapopen=10, gapextend=0.5, | stdout=True, auto=True, | asequence="a.fasta", bsequence="b.fasta") | print("About to run: %s" % water_cmd) | std_output, err_output = water_cmd() | | This functionality is similar to subprocess.check_output() added in | Python 2.7. In general if you require more control over running the | command, use subprocess directly. | | As of Biopython 1.56, when the program called returns a non-zero error | level, a custom ApplicationError exception is raised. This includes | any stdout and stderr strings captured as attributes of the exception | object, since they may be useful for diagnosing what went wrong. | | __repr__(self) | Return a representation of the command line object for debugging. | | e.g. | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5) | >>> cline.asequence = "asis:ACCCGGGCGCGGT" | >>> cline.bsequence = "asis:ACCCGAGCGCGGT" | >>> cline.outfile = "temp_water.txt" | >>> print(cline) | water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5 | >>> cline | WaterCommandline(cmd='water', outfile='temp_water.txt', asequence='asis:ACCCGGGCGCGGT', bsequence='asis:ACCCGAGCGCGGT', gapopen=10, gapextend=0.5) | | __setattr__(self, name, value) | Set attribute name to value (PRIVATE). | | This code implements a workaround for a user interface issue. | Without this __setattr__ attribute-based assignment of parameters | will silently accept invalid parameters, leading to known instances | of the user assuming that parameters for the application are set, | when they are not. | | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5, stdout=True) | >>> cline.asequence = "a.fasta" | >>> cline.bsequence = "b.fasta" | >>> cline.csequence = "c.fasta" | Traceback (most recent call last): | ... | ValueError: Option name csequence was not found. | >>> print(cline) | water -stdout -asequence=a.fasta -bsequence=b.fasta -gapopen=10 -gapextend=0.5 | | This workaround uses a whitelist of object attributes, and sets the | object attribute list as normal, for these. Other attributes are | assumed to be parameters, and passed to the self.set_parameter method | for validation and assignment. | | __str__(self) | Make the commandline string with the currently set options. | | e.g. | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5) | >>> cline.asequence = "asis:ACCCGGGCGCGGT" | >>> cline.bsequence = "asis:ACCCGAGCGCGGT" | >>> cline.outfile = "temp_water.txt" | >>> print(cline) | water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5 | >>> str(cline) | 'water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5' | | set_parameter(self, name, value=None) | Set a commandline option for a program (OBSOLETE). | | Every parameter is available via a property and as a named | keyword when creating the instance. Using either of these is | preferred to this legacy set_parameter method which is now | OBSOLETE, and likely to be DEPRECATED and later REMOVED in | future releases. | | ---------------------------------------------------------------------- | Data descriptors inherited from Bio.Application.AbstractCommandline: | | __dict__ | dictionary for instance variables (if defined) | | __weakref__ | list of weak references to the object (if defined) | | ---------------------------------------------------------------------- | Data and other attributes inherited from Bio.Application.AbstractCommandline: | | parameters = None
For the most basic usage, all you need is to have a FASTA input file, such as opuntia.fasta (available online or in the Doc/examples subdirectory of the Biopython source code). You can then tell MUSCLE to read in this FASTA file, and write the alignment to an output file:
from Bio.Align.Applications import MuscleCommandline
cline = MuscleCommandline(input="data/opuntia.fasta", out="opuntia.txt")
print(cline)
muscle -in data/opuntia.fasta -out opuntia.txt
Note that MUSCLE uses “-in” and “-out” but in Biopython we have to use “input” and “out” as the keyword arguments or property names. This is because “in” is a reserved word in Python.
By default MUSCLE will output the alignment as a FASTA file (using
gapped sequences). The Bio.AlignIO
module should be able to read this
alignment using format=fasta
. You can also ask for ClustalW-like
output:
from Bio.Align.Applications import MuscleCommandline
cline = MuscleCommandline(input="data/opuntia.fasta", out="opuntia.aln", clw=True)
print(cline)
muscle -in data/opuntia.fasta -out opuntia.aln -clw
Or, strict ClustalW output where the original ClustalW header line is used for maximum compatibility:
from Bio.Align.Applications import MuscleCommandline
cline = MuscleCommandline(input="data/opuntia.fasta", out="opuntia.aln", clwstrict=True)
print(cline)
muscle -in data/opuntia.fasta -out opuntia.aln -clwstrict
The Bio.AlignIO
module should be able to read these alignments using
format=clustal
.
MUSCLE can also output in GCG MSF format (using the msf
argument), but
Biopython can’t currently parse that, or using HTML which would give a
human readable web page (not suitable for parsing).
You can also set the other optional parameters, for example the maximum number of iterations. See the built in help for details.
You would then run MUSCLE command line string as described above for
ClustalW, and parse the output using Bio.AlignIO
to get an alignment
object.
Using a MUSCLE command line as in the examples above will write the alignment to a file. This means there will be no important information written to the standard out (stdout) or standard error (stderr) handles. However, by default MUSCLE will write the alignment to standard output (stdout). We can take advantage of this to avoid having a temporary output file! For example:
from Bio.Align.Applications import MuscleCommandline
muscle_cline = MuscleCommandline(input="data/opuntia.fasta")
print(muscle_cline)
muscle -in data/opuntia.fasta
If we run this via the wrapper, we get back the output as a string. In
order to parse this we can use StringIO
to turn it into a handle.
Remember that MUSCLE defaults to using FASTA as the output format:
from Bio.Align.Applications import MuscleCommandline
muscle_cline = MuscleCommandline(input="data/opuntia.fasta")
stdout, stderr = muscle_cline()
from io import StringIO
from Bio import AlignIO
align = AlignIO.read(StringIO(stdout), "fasta")
print(align)
SingleLetterAlphabet() alignment with 7 rows and 906 columns TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273289|gb|AF191663.1|AF191663 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273291|gb|AF191665.1|AF191665 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273290|gb|AF191664.1|AF191664 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273287|gb|AF191661.1|AF191661 TATACATAAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273286|gb|AF191660.1|AF191660 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273285|gb|AF191659.1|AF191659 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273284|gb|AF191658.1|AF191658
The above approach is fairly simple, but if you are dealing with very
large output text the fact that all of stdout and stderr is loaded into
memory as a string can be a potential drawback. Using the subprocess
module we can work directly with handles instead:
import subprocess
import sys
from Bio.Align.Applications import MuscleCommandline
muscle_cline = MuscleCommandline(input="data/opuntia.fasta")
child = subprocess.Popen(str(muscle_cline),
stdout=subprocess.PIPE, stderr=subprocess.PIPE,
shell=(sys.platform != "win32"),
universal_newlines=True)
from Bio import AlignIO
align = AlignIO.read(child.stdout, "fasta")
print(align)
SingleLetterAlphabet() alignment with 7 rows and 906 columns TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273289|gb|AF191663.1|AF191663 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273291|gb|AF191665.1|AF191665 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273290|gb|AF191664.1|AF191664 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273287|gb|AF191661.1|AF191661 TATACATAAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273286|gb|AF191660.1|AF191660 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273285|gb|AF191659.1|AF191659 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273284|gb|AF191658.1|AF191658
We don’t actually need to have our FASTA input sequences prepared in a file, because by default MUSCLE will read in the input sequence from standard input! Note this is a bit more advanced and fiddly, so don’t bother with this technique unless you need to.
First, we’ll need some unaligned sequences in memory as SeqRecord
objects. For this demonstration I’m going to use a filtered version of
the original FASTA file (using a generator expression), taking just six
of the seven sequences:
from Bio import SeqIO
records = (r for r in SeqIO.parse("data/opuntia.fasta", "fasta") if len(r) < 900)
Then we create the MUSCLE command line, leaving the input and output to their defaults (stdin and stdout). I’m also going to ask for strict ClustalW format as for the output.
from Bio.Align.Applications import MuscleCommandline
muscle_cline = MuscleCommandline(clwstrict=True)
print(muscle_cline)
muscle -clwstrict
Now for the fiddly bits using the subprocess
module, stdin and stdout:
import subprocess
import sys
child = subprocess.Popen(str(cline),
stdin=subprocess.PIPE, stdout=subprocess.PIPE,
stderr=subprocess.PIPE, universal_newlines=True,
shell=(sys.platform != "win32"))
That should start MUSCLE, but it will be sitting waiting for its FASTA input sequences, which we must supply via its stdin handle:
SeqIO.write(records, child.stdin, "fasta")
--------------------------------------------------------------------------- BrokenPipeError Traceback (most recent call last) <ipython-input-92-11690e1b720b> in <module>() ----> 1 SeqIO.write(records, child.stdin, "fasta") /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/SeqIO/__init__.py in write(sequences, handle, format) 481 if format in _FormatToWriter: 482 writer_class = _FormatToWriter[format] --> 483 count = writer_class(fp).write_file(sequences) 484 elif format in AlignIO._FormatToWriter: 485 # Try and turn all the records into a single alignment, /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/SeqIO/Interfaces.py in write_file(self, records) 209 """ 210 self.write_header() --> 211 count = self.write_records(records) 212 self.write_footer() 213 return count /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/SeqIO/Interfaces.py in write_records(self, records) 194 count = 0 195 for record in records: --> 196 self.write_record(record) 197 count += 1 198 # Mark as true, even if there where no records /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/SeqIO/FastaIO.py in write_record(self, record) 209 if self.wrap: 210 for i in range(0, len(data), self.wrap): --> 211 self.handle.write(data[i:i + self.wrap] + "\n") 212 else: 213 self.handle.write(data + "\n") BrokenPipeError: [Errno 32] Broken pipe
child.stdin.close()
--------------------------------------------------------------------------- BrokenPipeError Traceback (most recent call last) BrokenPipeError: [Errno 32] Broken pipe During handling of the above exception, another exception occurred: BrokenPipeError Traceback (most recent call last) <ipython-input-93-f5a7c02c7fa3> in <module>() ----> 1 child.stdin.close() BrokenPipeError: [Errno 32] Broken pipe
After writing the six sequences to the handle, MUSCLE will still be waiting to see if that is all the FASTA sequences or not – so we must signal that this is all the input data by closing the handle. At that point MUSCLE should start to run, and we can ask for the output:
from Bio import AlignIO
align = AlignIO.read(child.stdout, "clustal")
print(align)
--------------------------------------------------------------------------- ValueError Traceback (most recent call last) <ipython-input-94-240021347a83> in <module>() 1 from Bio import AlignIO ----> 2 align = AlignIO.read(child.stdout, "clustal") 3 print(align) /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/AlignIO/__init__.py in read(handle, format, seq_count, alphabet) 429 first = None 430 if first is None: --> 431 raise ValueError("No records found in handle") 432 try: 433 second = next(iterator) ValueError: No records found in handle
Wow! There we are with a new alignment of just the six records, without having created a temporary FASTA input file, or a temporary alignment output file. However, a word of caution: Dealing with errors with this style of calling external programs is much more complicated. It also becomes far harder to diagnose problems, because you can’t try running MUSCLE manually outside of Biopython (because you don’t have the input file to supply). There can also be subtle cross platform issues (e.g. Windows versus Linux, Python 2 versus Python 3), and how you run your script can have an impact (e.g. at the command line, from IDLE or an IDE, or as a GUI script). These are all generic Python issues though, and not specific to Biopython.
If you find working directly with subprocess
like this scary, there is
an alternative. If you execute the tool with muscle_cline()
you can
supply any standard input as a big string, muscle_cline(stdin=...)
.
So, provided your data isn’t very big, you can prepare the FASTA input
in memory as a string using StringIO
(see
Section [sec:appendix-handles]):
from Bio import SeqIO
records = (r for r in SeqIO.parse("data/opuntia.fasta", "fasta") if len(r) < 900)
handle = StringIO()
SeqIO.write(records, handle, "fasta")
6
data = handle.getvalue()
You can then run the tool and parse the alignment as follows:
stdout, stderr = muscle_cline(stdin=data)
from Bio import AlignIO
align = AlignIO.read(StringIO(stdout), "clustal")
print(align)
SingleLetterAlphabet() alignment with 6 rows and 900 columns TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273290|gb|AF191664.1|AF19166 TATACATTAAAGGAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273289|gb|AF191663.1|AF19166 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273287|gb|AF191661.1|AF19166 TATACATAAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273286|gb|AF191660.1|AF19166 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273285|gb|AF191659.1|AF19165 TATACATTAAAGAAGGGGGATGCGGATAAATGGAAAGGCGAAAG...AGA gi|6273284|gb|AF191658.1|AF19165
You might find this easier, but it does require more memory (RAM) for the strings used for the input FASTA and output Clustal formatted data.
The EMBOSS suite includes the water
and needle
tools for Smith-Waterman algorithm local alignment, and
Needleman-Wunsch global alignment. The tools share the same style
interface, so switching between the two is trivial – we’ll just use
needle
here.
Suppose you want to do a global pairwise alignment between two sequences, prepared in FASTA format as follows:
>HBA_HUMAN
MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHG
KKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTP
AVHASLDKFLASVSTVLTSKYR
in a file alpha.fasta
, and secondly in a file beta.fasta
:
>HBB_HUMAN
MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPK
VKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFG
KEFTPPVQAAYQKVVAGVANALAHKYH
Let’s start by creating a complete needle
command line object in one
go:
from Bio.Emboss.Applications import NeedleCommandline
needle_cline = NeedleCommandline(asequence="data/alpha.faa", bsequence="data/beta.faa",
gapopen=10, gapextend=0.5, outfile="needle.txt")
print(needle_cline)
needle -outfile=needle.txt -asequence=data/alpha.faa -bsequence=data/beta.faa -gapopen=10 -gapextend=0.5
Why not try running this by hand at the command prompt? You should see
it does a pairwise comparison and records the output in the file
needle.txt
(in the default EMBOSS alignment file format).
Even if you have EMBOSS installed, running this command may not work – you might get a message about “command not found” (especially on Windows). This probably means that the EMBOSS tools are not on your PATH environment variable. You can either update your PATH setting, or simply tell Biopython the full path to the tool, for example:
from Bio.Emboss.Applications import NeedleCommandline
needle_cline = NeedleCommandline(r"C:\EMBOSS\needle.exe",
asequence="data/alpha.faa", bsequence="data/beta.faa",
gapopen=10, gapextend=0.5, outfile="needle.txt")
Remember in Python that for a default string \n
or \t
means a new
line or a tab – which is why we’re put a letter “r” at the start for a
raw string.
At this point it might help to try running the EMBOSS tools yourself by hand at the command line, to familiarise yourself the other options and compare them to the Biopython help text:
from Bio.Emboss.Applications import NeedleCommandline
help(NeedleCommandline)
Help on class NeedleCommandline in module Bio.Emboss.Applications: class NeedleCommandline(_EmbossCommandLine) | Commandline object for the needle program from EMBOSS. | | Method resolution order: | NeedleCommandline | _EmbossCommandLine | _EmbossMinimalCommandLine | Bio.Application.AbstractCommandline | builtins.object | | Methods defined here: | | __init__(self, cmd='needle', **kwargs) | Create a new instance of a command line wrapper object. | | ---------------------------------------------------------------------- | Data descriptors defined here: | | aformat | Display output in a different specified output format | | This controls the addition of the -aformat parameter and its associated value. Set this property to the argument value required. | | asequence | First sequence to align | | This controls the addition of the -asequence parameter and its associated value. Set this property to the argument value required. | | auto | Turn off prompts. | | Automatic mode disables prompting, so we recommend you set | this argument all the time when calling an EMBOSS tool from | Biopython. | | | This property controls the addition of the -auto switch, treat this property as a boolean. | | brief | Display brief identity and similarity | | This property controls the addition of the -brief switch, treat this property as a boolean. | | bsequence | Second sequence to align | | This controls the addition of the -bsequence parameter and its associated value. Set this property to the argument value required. | | datafile | Matrix file | | This controls the addition of the -datafile parameter and its associated value. Set this property to the argument value required. | | debug | Write debug output to program.dbg. | | This property controls the addition of the -debug switch, treat this property as a boolean. | | die | Report dying program messages. | | This property controls the addition of the -die switch, treat this property as a boolean. | | endextend | The score added to the end gap penality for each base or residue in the end gap. | | This controls the addition of the -endextend parameter and its associated value. Set this property to the argument value required. | | endopen | The score taken away when an end gap is created. | | This controls the addition of the -endopen parameter and its associated value. Set this property to the argument value required. | | endweight | Apply And gap penalties | | This controls the addition of the -endweight parameter and its associated value. Set this property to the argument value required. | | error | Report errors. | | This property controls the addition of the -error switch, treat this property as a boolean. | | filter | Read standard input, write standard output. | | This property controls the addition of the -filter switch, treat this property as a boolean. | | gapextend | Gap extension penalty | | This controls the addition of the -gapextend parameter and its associated value. Set this property to the argument value required. | | gapopen | Gap open penalty | | This controls the addition of the -gapopen parameter and its associated value. Set this property to the argument value required. | | help | Report command line options. | | More information on associated and general qualifiers can | be found with -help -verbose | | | This property controls the addition of the -help switch, treat this property as a boolean. | | nobrief | Display extended identity and similarity | | This property controls the addition of the -nobrief switch, treat this property as a boolean. | | options | Prompt for standard and additional values. | | If you are calling an EMBOSS tool from within Biopython, | we DO NOT recommend using this option. | | | This property controls the addition of the -options switch, treat this property as a boolean. | | outfile | Output filename | | This controls the addition of the -outfile parameter and its associated value. Set this property to the argument value required. | | similarity | Display percent identity and similarity | | This controls the addition of the -similarity parameter and its associated value. Set this property to the argument value required. | | snucleotide | Sequences are nucleotide (boolean) | | This controls the addition of the -snucleotide parameter and its associated value. Set this property to the argument value required. | | sprotein | Sequences are protein (boolean) | | This controls the addition of the -sprotein parameter and its associated value. Set this property to the argument value required. | | stdout | Write standard output. | | This property controls the addition of the -stdout switch, treat this property as a boolean. | | verbose | Report some/full command line options | | This property controls the addition of the -verbose switch, treat this property as a boolean. | | warning | Report warnings. | | This property controls the addition of the -warning switch, treat this property as a boolean. | | ---------------------------------------------------------------------- | Methods inherited from Bio.Application.AbstractCommandline: | | __call__(self, stdin=None, stdout=True, stderr=True, cwd=None, env=None) | Executes the command, waits for it to finish, and returns output. | | Runs the command line tool and waits for it to finish. If it returns | a non-zero error level, an exception is raised. Otherwise two strings | are returned containing stdout and stderr. | | The optional stdin argument should be a string of data which will be | passed to the tool as standard input. | | The optional stdout and stderr argument may be filenames (string), | but otherwise are treated as a booleans, and control if the output | should be captured as strings (True, default), or ignored by sending | it to /dev/null to avoid wasting memory (False). If sent to a file | or ignored, then empty string(s) are returned. | | The optional cwd argument is a string giving the working directory | to run the command from. See Python's subprocess module documentation | for more details. | | The optional env argument is a dictionary setting the environment | variables to be used in the new process. By default the current | process' environment variables are used. See Python's subprocess | module documentation for more details. | | Default example usage:: | | from Bio.Emboss.Applications import WaterCommandline | water_cmd = WaterCommandline(gapopen=10, gapextend=0.5, | stdout=True, auto=True, | asequence="a.fasta", bsequence="b.fasta") | print("About to run: %s" % water_cmd) | std_output, err_output = water_cmd() | | This functionality is similar to subprocess.check_output() added in | Python 2.7. In general if you require more control over running the | command, use subprocess directly. | | As of Biopython 1.56, when the program called returns a non-zero error | level, a custom ApplicationError exception is raised. This includes | any stdout and stderr strings captured as attributes of the exception | object, since they may be useful for diagnosing what went wrong. | | __repr__(self) | Return a representation of the command line object for debugging. | | e.g. | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5) | >>> cline.asequence = "asis:ACCCGGGCGCGGT" | >>> cline.bsequence = "asis:ACCCGAGCGCGGT" | >>> cline.outfile = "temp_water.txt" | >>> print(cline) | water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5 | >>> cline | WaterCommandline(cmd='water', outfile='temp_water.txt', asequence='asis:ACCCGGGCGCGGT', bsequence='asis:ACCCGAGCGCGGT', gapopen=10, gapextend=0.5) | | __setattr__(self, name, value) | Set attribute name to value (PRIVATE). | | This code implements a workaround for a user interface issue. | Without this __setattr__ attribute-based assignment of parameters | will silently accept invalid parameters, leading to known instances | of the user assuming that parameters for the application are set, | when they are not. | | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5, stdout=True) | >>> cline.asequence = "a.fasta" | >>> cline.bsequence = "b.fasta" | >>> cline.csequence = "c.fasta" | Traceback (most recent call last): | ... | ValueError: Option name csequence was not found. | >>> print(cline) | water -stdout -asequence=a.fasta -bsequence=b.fasta -gapopen=10 -gapextend=0.5 | | This workaround uses a whitelist of object attributes, and sets the | object attribute list as normal, for these. Other attributes are | assumed to be parameters, and passed to the self.set_parameter method | for validation and assignment. | | __str__(self) | Make the commandline string with the currently set options. | | e.g. | >>> from Bio.Emboss.Applications import WaterCommandline | >>> cline = WaterCommandline(gapopen=10, gapextend=0.5) | >>> cline.asequence = "asis:ACCCGGGCGCGGT" | >>> cline.bsequence = "asis:ACCCGAGCGCGGT" | >>> cline.outfile = "temp_water.txt" | >>> print(cline) | water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5 | >>> str(cline) | 'water -outfile=temp_water.txt -asequence=asis:ACCCGGGCGCGGT -bsequence=asis:ACCCGAGCGCGGT -gapopen=10 -gapextend=0.5' | | set_parameter(self, name, value=None) | Set a commandline option for a program (OBSOLETE). | | Every parameter is available via a property and as a named | keyword when creating the instance. Using either of these is | preferred to this legacy set_parameter method which is now | OBSOLETE, and likely to be DEPRECATED and later REMOVED in | future releases. | | ---------------------------------------------------------------------- | Data descriptors inherited from Bio.Application.AbstractCommandline: | | __dict__ | dictionary for instance variables (if defined) | | __weakref__ | list of weak references to the object (if defined) | | ---------------------------------------------------------------------- | Data and other attributes inherited from Bio.Application.AbstractCommandline: | | parameters = None
Note that you can also specify (or change or look at) the settings like this:
from Bio.Emboss.Applications import NeedleCommandline
needle_cline = NeedleCommandline()
needle_cline.asequence="data/alpha.faa"
needle_cline.bsequence="data/beta.faa"
needle_cline.gapopen=10
needle_cline.gapextend=0.5
needle_cline.outfile="needle.txt"
print(needle_cline)
needle -outfile=needle.txt -asequence=data/alpha.faa -bsequence=data/beta.faa -gapopen=10 -gapextend=0.5
print(needle_cline.outfile)
needle.txt
Next we want to use Python to run this command for us. As explained
above, for full control, we recommend you use the built in Python
subprocess
module, but for simple usage the wrapper object usually
suffices:
stdout, stderr = needle_cline()
print(stdout + stderr)
--------------------------------------------------------------------------- ApplicationError Traceback (most recent call last) <ipython-input-103-c1c0502efb05> in <module>() ----> 1 stdout, stderr = needle_cline() 2 print(stdout + stderr) /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/Application/__init__.py in __call__(self, stdin, stdout, stderr, cwd, env) 515 if return_code: 516 raise ApplicationError(return_code, str(self), --> 517 stdout_str, stderr_str) 518 return stdout_str, stderr_str 519 ApplicationError: Non-zero return code 127 from 'needle -outfile=needle.txt -asequence=data/alpha.faa -bsequence=data/beta.faa -gapopen=10 -gapextend=0.5', message '/bin/sh: 1: needle: not found'
Next we can load the output file with Bio.AlignIO
as discussed earlier
in this chapter, as the emboss
format:
from Bio import AlignIO
align = AlignIO.read("needle.txt", "emboss")
print(align)
--------------------------------------------------------------------------- FileNotFoundError Traceback (most recent call last) <ipython-input-104-cb0b4e27470d> in <module>() 1 from Bio import AlignIO ----> 2 align = AlignIO.read("needle.txt", "emboss") 3 print(align) /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/AlignIO/__init__.py in read(handle, format, seq_count, alphabet) 425 iterator = parse(handle, format, seq_count, alphabet) 426 try: --> 427 first = next(iterator) 428 except StopIteration: 429 first = None /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/AlignIO/__init__.py in parse(handle, format, seq_count, alphabet) 348 raise TypeError("Need integer for seq_count (sequences per alignment)") 349 --> 350 with as_handle(handle, 'rU') as fp: 351 # Map the file format to a sequence iterator: 352 if format in _FormatToIterator: /home/tiago_antao/miniconda/lib/python3.5/contextlib.py in __enter__(self) 57 def __enter__(self): 58 try: ---> 59 return next(self.gen) 60 except StopIteration: 61 raise RuntimeError("generator didn't yield") from None /home/tiago_antao/miniconda/lib/python3.5/site-packages/Bio/File.py in as_handle(handleish, mode, **kwargs) 88 yield fp 89 else: ---> 90 with open(handleish, mode, **kwargs) as fp: 91 yield fp 92 else: FileNotFoundError: [Errno 2] No such file or directory: 'needle.txt'