Table of Contents 0. Purpose 0. Goals 0. Hints 0. Functions that you will need to complete the exercise. 0. Question 1 0. Question 2 0. Question 3 0. Question 4 0. Question 5 0. Question 6 0. Question 7
In this assignment you'll use multiple sequence alignment to reconstruct the phylogeny of a group of organisms based on their 16S rRNA sequences. This assignment builds on ideas from the previous assignment, in that in the last assignment you were identifying good primers to use for amplifying 16S from diverse organisms, and in this assignment we're using those sequences to group organisms by their relatedness. Because of the very large numbers of sequences that are commonly obtained in a modern DNA-sequencing-based experiment, grouping similar sequences and then working with representative sequences for each of those groups is common for computational efficiency. We'll be exploring these ideas in more detail throughout the next segments of the class.
From a bioinformatics standpoint, we usually start working with sequences in fasta format, very similar to the sequences in the cell below. See here for an explanation of the fasta format.
At this point, you should be feeling fairly comfortable interacting with the IPython Notebook. This assignment will give you additional practice while you explore the ideas mentioned above.
Continue to work with IPython Notebooks and interact with python code. Understand what multiple sequence alignment is used for, and the concept of grouping sequences into clusters of OTUs. Consider the possible drawbacks to these methods.
Remember to learn about what a function does, you can run:
help(name_of_function)
Try this with the functions below to see what they do.
%pylab inline
import skbio.io
from skbio import DNA
from iab.algorithms import progressive_msa_and_tree, iterative_msa_and_tree, kmer_distance, guide_tree_from_sequences
The cell below contains the sequences that you will be working with throughout the assignment
from io import StringIO
seqs_16s = """>881726
GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGATTCATCCTTCGGGATGGGTTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGCAAGTCCGGGATAACTAACGGAAACGTTAGCTAATACCGGATACGCGGTTGGATCGCATGATCCGATCGGGAAAGACGGCGCAAGCTGCCACTTGTAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGAGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGCAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTCTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGCTCGGGAGAGTAACTGCTCTCGAGGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCGATTAAGTTTGGTGTTTAAGCCCGGGGCTCAACCCCGGTTCGCACTGAAAACTGATCGACTTGAGTGTAGGAGAGGAAAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGCCTATAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGCATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTCAACACAGTAAGCATGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCCCTGAATCCTCTAGAGATAGAGGCGGCCCTTCGGGGACAGGGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCGTAGTTGCCAGCACTTCGGGTGGGCACTCTAGGATGACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGCTGCGAAGCCGCGAGGTGGAGCCAATCCCAGAAAGCCGGTCTCAGTTCAGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCT
>793074
GAATGAACGCTGGCGGCGTGCTTAATAATGCAAGTCGAGCGCGTAGCAATACGAGCGGCGCACGGGTGCGTAACACGTAGGTCATCTGCCTCTAGGTCGGGGATAACTGCGGGAAACTGCAGCTAATACCCGATGATATCGAGAGATCAAAGCTTCGGTGCCTAGAGAGGAGCCTGCGGCTCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCCACGATGAGTAGCCGGCCTGAGAGGGCGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATATTGGGCAATGGGCGAAAGCCTGACCCAGCAACGCCGCGTGAGTGATGAAGCCTTTCGGGGTGTAAAGCTCTTTTGGCAGGGACGAATCAATGACGGTACCTGCGTAATAAGCCCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGGGGGGGCAAGCGTTATTCGGAATTACTGGGCGTAAAGCGCGCGTAGGCGGCTTCTTAAGTCGGGTGTTTAATGTCGGGGCTCAACTCCGGCGCTGCACTCGATACTGGGAGGCTAGAGTACTCGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTTAGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGAGAGTAACTGACGCTCAGAGCGCGAAAGCCAGGGGATCGAACGGGATTAGATACCCCGGTAGTCCTGGCTGTAAACGATGGGTACTAGATGTCGCCGGTATCAATCCCGGCGGTATCGTCGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGACTTGACATACCTCGGACCGGACCTAGAGATAGGACCTTCTCCCGTAAGGGAGCCGGGGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCCATCCCTAGTTGCCAGCGAGTCATGTCGGGAACTCTAGGGAGACTGCCGTTGATAAAACGAGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGTCCAGGGCTACACACGTGCTACAATGGCCACCACAAAGGGTCGCAATACCGTGAGGTGGAGCTAATCCCAAAAAGGTGGCCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAACGCCGCGGTGAATACAGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGCTGGTTGCGTTAGAAGTCGCCAGGCCAACCGCAAGGGGGCAGGCGCCGAATGCGTGATGAGTGATTGGGGT
>669210
AGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGACTAGCCCCTTCGGGGGCGAAGTTAGTGGCGAACGGGTGAGTAACGCGTAAGTAACCTGCCCCCGGGACTGGGATAACAGCTCGAAAGAGCCGCTAATACCGGATAATTGTTGCAACACTTAGGAGTTGTAACTAAAGAAGGCCTCTGTTTCAAGCTTTCACCTGGGGATGGGCTTGCGTCCCATTAGCTTGTTGGTGAGGTAACGGCTCACCAAGGAAACGATGGGTAGCCGGCCTGAGAGGGTGGTCGGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGAGGAATCTTGCGCAATGGGCTAACGCCTGACGCAGCGACGCCGCGTGGACGATGAAGCTTTTCGGAGTGTAAAGTCCTTTCAGGAGGGAAGAAATGCCGGTAGTGTGAATAACACACCGGTTTGACGGTACCTCAAGAAGAAGCCCCGGCTAACTCCGTGCCAGCAGCCGCGGTAACACGGAGGGGGCAAGCGTTGTTCGGAATCACTGGGCGTAAAGAGCGCGTAGGTGGTTGTGTAAGTCGGATGTGAAATCCCTCGGCTCAACCGAGGAACTGCGTTCGAAACTACATAGCTAGAGGGCAGGAGAGGAGAGCGGAATTCCCAGTGTAGCGGTGAAATGCGCAGATATTGGGAAGAACACCGGTGGCGAAGGCGGCTCTCTGGACTGTTCCTGACACTGAGGCGCGAAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGGCACTAGGTGTGGGGGGTGTCGATCCCCCCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGAAGTACGATCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCGGAATTTGACATGTTTCTGACGGCCTGCAGAAATGCAGGCTTCCCCTCGGGGCAGATACACAGGAGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCCTTAGTTGCCATCGGTTCGGCCGGGAACTCTAAGGGGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCAGTATGGCCTTTATGTTCCGGGCTACACACGTGCTACAATGGCTGGTACAAAGGGTCGCGATGCCGTGAGGTGGAGCCAATCCCAAAAAGCCAGTCTTAGTTCGGATTGGAGTCTGCAACTCGACTCTATGAAGCCGGAATCGCTAGTAATCGTGGATCAGCACGCCATGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTTGGTTGTACCAGAAGTCATTGGGCTAACCCTTTTGGGGGGCAGATGCCGAAGGTATGGTCAGCGATTGGGGTGAAGTCGTAACAAGGTAACC
>583705
ACGGGTGAGTAACGCGTATGCAACCTACCTCGGAAAAGGGGATGACTGGTGGAAACGGGGATTAATGCCCCCTAGGGTTGTTTCTCTGCCTGGGTGAGCCGTTACTATTGGAACCGATTGAGATGGCCATGTTGGTCATTTCCTGGTTGGTGAGGTTACCTCACACCAAGGCGACGATGACTACGGGGTCTAAAAGGATGGTCCCGCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGGGCAACCCTGAACCAGCCATGCCGCGTGAAGGAAGACGGCCCTATGGGTTGTAAACTTCTTTTATATGGGAATAAAGAGAGGTACGTGTACCTCAGTGAATGTACCATATGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCAGGCGTTATCCGGATTTATTAGGTTTAAAGGGTGCGTAGGCGGGATACTAAGTCAGTGGTGAAAGTTTGCGGCTCAACCGTAAAATCGCCATTGATACTGGTATTCTTGAGTATACAGGAAGTAGGCGGAATGTGTAGTGTAGCGGTGAAATGCATAGATATTACACAGAACACCGATTGCGAAGGCAGCTTACTATAGTATAACTGACGCTGATGCACGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGATTACTGGTTGTGCGCGATACACAGTGCGCGACTGAGCGAAAGCATTAAGTAATCCACCTGGGGAGTACGGCGGCAACGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCTGGGCTTAAATGTAGAGTGCATGGAGTGGAAACATTCCTTTCCTTCGGGACTCTTTACAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGGTTAAGTCCCATAACGAGCGCAACCCCTATCATTAGTTGCTAACAGGTCAAGCTGAGGACTCTAGCGAAACTGCCGGTGTAAACCGTGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTATGTCCAGGGCTACACACGTGTTACGATGGCCAGTACAAAGGGTAGCTACCTGGTGACAGGATGCTAATCTCAAAAGCTGGTCTCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTTGGATTCGCTAGTAATCGTATATCAGCCATGATACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAACCATGGAAGCTGGGGGTACCTGAAGTACGTCACCGCAAGGAGCGTCCTAGGGTAAATCTAGTGACTGGGGTTAAGTGGTAACAAGGTAACC
>524860
AGAGTTTGATCCTGGCTCAGAACGAACGTTGGCGGCATGGATGAGGCATGCAAGTCGCGGGAATCCCCAGCAATGGGGGGAACCGGCGTAAGGGGCAGTAAGGCGTAGGTACCTACCCCCAGGTCCGGGATAGCCCGCCGAGAGGCGGGGTAATACCGGATGACCTCGGGAGAGCAAAGCTCCGGCGCCTGAGGCGGGGCCTACGTGATATTACCTAGTTGGCGGGGTAACGGCCCACCAAGGGGGAGATGTCTAGCGGGTGTGAGAGCACGACCCGCGCCACTCGCACTGAGACACTGGCGAGACACCTACGGGTGGCTGCAGTCGAGGATCTTCGGCAATGGGGGCAACCCTGACCGAGCGATGCCGCGTGGGCGACGAAGGCCTTCGGGTTGTAAAGCCCTGTCGAGGGGGAGAAAGCCGCAAGGCGGATCCATCCCTGGAGGAAGCTCGGGCTAAGTTCGTGCCAGCAGCCGCGGTAAGACGAACCGAGCGAACGTTGTTCGGAATCACTGGGCTTAAAGGGCGCGTAGGCGGGCTGCCGCGTCCGGGGTGAAATCCCACGGCTCAACCGTGGAACGGCCCCGGGTACGGGCGGCCTCGAGGGGGATAGGGGCGTGCGGAACTGTGGGTGGAGCGGTGAAATGCGTTGATATCCACAGGAACTCCGGTGGCGAAGGCGGCACGCTGGATCCTCTCTGACGCTGAGGCGCGGAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCCTAAACGATGAGAACTGGGTAGTAGCCCTGGCATGGGGTTACTGCCGCAGCCAAAGTGCTAAGTTCTCCGCCTGGGGAGTATGGTCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCTCACACAAGCGGTGGAGCATGTGGCTTAATTCGAGGCTACGCGAAGAACCTTATCCTGGACTTGACATGTGCGAAAGCGCCAGCAGGTAGGACCCGGAAACGGGAACGAACGGTATCCAACCCGGAAGCTGGTACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTCGGGTTAAGTCCCATAACGAGCGAAACCCTTACCCCTTGTTGCAACCCGAAAGGGGCACTCGAGGGGGACTGCCGGTGTCAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCTTTATGTCCAGGGCTGCACACGTGCTACAATGGCGTGGACAGAGGGACGCGACTGCGCGAGCAGAAGCCGACCCCCGAAAGCACGCCCCAGTTCAGATCGCAGGCTGCAACCCGCCTGCGTGAAGCCGGAATCGCTAGTAATCGCGGGTCAGCAACACCGCGGTGAATGTGTTCCTGAGCCTTGTACACACCGCCCGTCAAGCCACGAAAGGGAGGGACGTCCGAAGTCGCCTCGCGGCGCCGAAGACGGACTTCCTGATTGGGACTAAGTCGTAACAAGGTAACC
>501793
GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGGAGTGGTTGAAGGAGCTTGCTCTTTTGATCGCTTAGTGGCAGACGGGTGAGTAACACGTAGGCAACCTGGCTGTAAGACGGGGATAACTGGCGGAAACGTGAGCTAAAACCGGATGGTCGGCTTGAGGGCATCCTCGAGTCGGGAAAGGACGGAGCAATCTGTCGCTTACAGATGGGCCTGCGGCGCATTAGCTAGTTGGTAGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGAACGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAACGCCAGGGAGAGTAACTGCTCTCTGGGTGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCGTTTAAGTCTCATGTCTAAACCCCGGGGCTCAACCTCGGGGTGCATGGGAAACTGGGCGACTGGAGTGCATGTGAGGAAAGTGGAATTCCACGTGTAGCGGTGGAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGCCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACATTAAGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGGGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGAAACATGCAGAGATGTGTGCCCTCTTCGGAGCATTGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAGGTTAGTTGCCAGCAGGTGAAGCTGGGCACTCTAACATGACTGCCGGTGACAAACCGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCAGTACAACGGGAAGCGAAGTGGCGACACGGAGCCAATCTTAGAAAGCTGGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCT
>296752
AGAGTTTGATCTCTGGCTCAGAACAAACGCTGGCGGTGCGTCTTAAGCATGCAAGTCGAGCGATGGTAGGGGGCTTGCTCCCTATTCATAGCGGCGGACTGGTGAGTAACGCGTAGATGACATACCTTTTGCTGGGGGATAGCTTGTGGAAACACAGGGTAATACCGCATACGATTGAGGCGGTTAGAGCGCTTCAATCAAAGCCTTGTATGGGGCGGCAGTTGAGTGGTCTGCGTACTATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGATGATAGTTATCCGGCCTGAGAGGGTGAACGGACACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGCTAAGAATATTCCGCAATGGGCGAAAGCCTGACGGAGCGACACCGCATGAGTGATGAAGGTCGAAAGATTGTAAAATTCTTTTTGAGAGTGATGAATAAAGTCGAGCAGTAATGCTCGGTGATGACGGTAACTTTTGAATAAGGGGTGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAAGCCCCAAGCGTTGTTCGGAATTATTGGGTGTAAAGGGCATGTAGGTGGTCTTGCAAGCTTGATGTGAAATCTTACAGCTTAACTGTAAAACTGCATTGAGAACTGCATAACTTAAGAATAACTGAGGCGCAACTGGAATTCCAGGTGTAGGGGTGAAATCTGTAGATATCTGGAAGAACACCAATGGCGAAGGCAAGTTGCGAGCAGATTATTGACACTGAGGTGCGAAGGTGCGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCGCACAGACAACGATGTACACTGGGCGTCTGGCTTTATGCTGGGTGCCGTAGTAAACGCGATAAGTGTACCGCCTGGGGAGTATGCTCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGAAATCTTACCTGGGTTTGACATTTAGTGGAATTGTATAGAGATATGCAAGGTACTTGTACCCGCTAAACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTACTGCTAGTTACTAACAGGTTATGCTGAGGACTCTAGCGGAACTGCCGGTGACAAACCGGAGGAAGATGGGGATGACGTCAAGTCATCATGGCCCTTATGTCCAGGGCAACACACGTGCTACAATGGTTGTAACAAAGTGATGCGAAATCGCAAGATGAAGCAAAACGCAGAAATGCAATCGTAGTTCGGATTGGAGTCTGAAACTCGACTCCATGAAGTTGGAATCGCTAGTAATCGCATATCAGCACGATGCGGTGAATACGTTCCCGGGCCTTGCACACACCGCCCGTCA
>293514
AGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGTGCGTCTTAAGCATGCAAGTCGAGCGATGGTAGGGGGCTTGCTCCCTATTCATAGCGGCGGACTGGTGAGTAACGCGTAGATGACATACCTTTTGCTGGGGGATAGCTTGTGGAAACACAGGGTAATACCGCATACGATTGAGGCGGTTAGAGCGCTTCAATCAAAGCCTTGTATGGGGCGGCAGTTGAGTGGTCTGCGTACTATTAGCTTGTTGGTGGGGTAACGGCTCACCAAGGCGATGATAGTTATCCGGCCTGAGAGGGTGAACGGACACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGCTAAGAATATTCCGCAATGGGCGAAAGCCTGACGGAGCGACACCGCGTGAGTGATGAAGGTCGAAAGATTGTAAAACTCTTTTGAGAGTGATGAATAAGTCGAGCAGTAATGCTCGGTGATGACGGTAACTTTTGAATAAGGGGTGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAAGCCCCAAGCGTTGTTCGGAATTATTGGGCGTAAAGGGCATGTAGGTGGTCTTGCAAGCTTGATGTGAAATCTTACAGCTTAACTGTAAAACTGCATTGAGAACTGCATAACTTAAGAATAACTGAGGCGCAACTGGAATTCCAGGTGTAGGGGTGAAATCTGTAGATATCTGGAAGAACACCAATGGCGAAGGCAAGTTGCGAGCAGATTATTGACACTGAGGTGCGAAGGTGCGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCGCACAGTCAACGATGTACACTGGGCGTCTGGCTTTATGCTGGGTGCCGTAGTAAACGCGATAAGTGTACCGCCTGGGGAGTATGCTCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGAATCTTACCTGGGTTTGACATACACATTATCTTTGCAGAGATGTAAAGCGGGGGTAACCCCAATGTGAACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGCCAGTTACTAACAAGTTAAGTTGAGGACTCTGGCGAAACTGCCGGTGACAAATCGGAGGAAGATGGGGATGACGTCAAGTCATCATGGCCCTTATGTCCAGGGCAACACACGTGCTACAATGGTTGAGACAGAGTGATGCTAAGTCGCAAGATGGAGCAAAACGCAGAAATTCAATCGTAGTTCGGATTGGAGTCTGAAACTCGACTCCATGAAGTTGGAATCGCTAGTAATCGCATATCAGCACGATGCGGTGAATACGTTCCCGGGCCTTGCACACACCGCCCGTCA
>292553
AGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGTGCGTCTTAAGCATGCAAGTCGAGCGATGAATGAGGGGCTTGCTCCTTATTCATAGCGGCGGACTGGTGAGTAACGCGTAGATGACATGTCGATGGCAGGGGGATAGCCAGTAGAAATATTGGGTAATACCGCGTATCCTTCTTGTTGTTAGAGGACAAGAAGAAAAGCCTTGTATGGGGCGGCTATTGAGTGGTCTGCGTACTATTAGTTTGTTGGTGGGGTAACGGCCTACCAAGACTATGATAGTTATCCGGCCTGAGAGGGTGAACGGACACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGCTAAGAATATTCCGCAATGGGCGAAAGCCTGACGGAGCGACACCGCGTGAGTGATGAAGGTCGAAAGATTGTAAAACTCTTTTGAATATGATGAATAAGTCAAGCAGTAATGCTTGGCGATGACGGTAGTGTTTGAATAAGGGGTGGCTAATTACGTGCCAGCAGCCGCGGTAACACGTAAGCCCCAAGCGTTGTTCGGAATTATTGGGCGTAAAGGGCATGTAGGTGGTTTTGTAAGCTTGATGTGAAATCTTACAGCTTAACTGTAAAACTGCATTGAGAACTGCAGAACTAGAGTAACTGAGGTGCAACTGGAATTCCAGGTGTAGGGGTGAAATCTGTAGATATCTGGAAGAACACCAATGGCGAAGGCAAGTTGCAAGCAGATTACTGACACTGAGGTGCGAAGGTGCGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCGCACAGTCAACGATGTACACTGGGCGTCTGGCTTTATGCTGGGTGCCGTAGTAAACGCGATAAGTGTACCGCCTGGGGAGTATGCTCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGAAATCTTACCTGGGTTTGACATTTAGTGGAATTGTATAGAGATATGCAAGGTACTTGTACCCGCTAAACAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTACTGCTAGTTACTAACAGGTTATGCTGAGGACTCTAGCGGAACTGCCGGTGACAAACCGGAGGAAGATGGGGATGACGTCAAGTCATCATGGCCCTTATGTCCAGGGCAACACACGTGCTACAATGGTTGTAACAAAGTGATGCGAAATCGCAAGATGAAGCAAAACGCAGAAATGCAATCGTAGTTCGGATTGGAGTCTGAAACTCGACTCCATGAAGTTGGAATCGCTAGTAATCGCATATCAGCACGATGCGGTGAATACGTTCCCGGGCCTTGCACTAACGGCCCGTCA
>266495
AGTTTGATCCTGGCTCAAGATGAACGCTAGCGGCAGGCTTAACACATGCAAGTCAAAGGGCAACGGGGAGAGTGCTTGCACTCTCTGCCGGCGACTGGCGCACGGGTGAGTAACACTTATGCAGACACTGCCTTCCACAGGGCGGACAACCTCTCCCAAAGGGAGGCTAATCCCGCGTATATCCCTTGGGGGCATCCCCGGGGGAGGAAAGGATTACCGGTGTGCAGGATGGGCATGCGGCGCATTACGCAGTAGGCGGGGTAACGGCCCACCTAACCGACCATGCGTATGGGTTCTGAGAGGAAGGCCCCCCACACTGGTACTGAGACACTGACCAGACTCCTACTGGAGGCAGCAGTGAGGAACATTGGTCAATGGGCGGGAGCCTGAACCAGCAAACCCGCGTGAAGGAAGAAGGCGCCGAACGTCGTAAACTTCTTTTGTCCGGGATCAAAGGGCGCCACGTGTGGCGTTGTGAGTGTACCTGTAGAGAAAGCTTCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGTAGGGAGCGAGCGTTGTCCGGATTTATTGGGTGTAAAGGGCGCGTAGGTGGTCGGTTAAGTCAGGTGTGAAAGCTCGGGGCTCAACCCGGAGGATCCGCTGGAACTTTGGTGTCATGAGGCGCAGGAGAAGTAAAGTGGAATTCGTGGTGTAGCGGTGAAATGCATAGATATTGGGCGGAACTCCGGTGGCGAAGGCAGCGTTCTGGCGCGTGCCTGACGCTGAGGCGCGAAAGCGTGGGTATCGAACGGGATTAGATACCCCGGTAGTCCACGCAGTAAACGATGAATACTGGGTGTCGGACCCATAGAACGTTTGGGTGCGCGCAGCGAAAGCGATAAGCATTCCAAGTGGGGAGTACACCGGCAGTGATGAGACTCAAAGGAATCGACGGGGGTTCGCACAAGTGGAGGGATATGTGGTTTAATTAGACGATAAGTGAGGAACGTGACCCGGGTTCAACAGGGAGTCGACAGGGGCAGAGATTCCCTCTTCCACGGACGTCTTCCGAGGTGGGGCATGGTTGTCAGTCAGCTACGTGCCGTGAGGTGTCGGCTTAAGTGCCATAAGGTGTGCAACACGGGCAGACAGTTGCTAACGGGTAGAGCAGTGGAATGTGTAGTGATTGCAGGGGCAAGCCGCGAGGAAGGGGGGGATGATGTCAAATCAGCGCGGCCCTTAGGTCAGGGGTGACACACGTGCTGCAATGGCGGGGACAGAGGGATGTGAAGAGGCGACGTGGAGCGAACCCCAAAAACCCCGCCCCAGTTAGGATTGTAGTATGCAACCCGAATACATGAAGCCGGAATAGGTAGTAATCGCGGATCAGAATGCAGCGGTGAATAAGTTCCCGGCTCTAGCACACACCGCCCGTCA
>229854
GAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCTTAACACATGCAAGTCGAACGGCAGCATGACTTAGCTTGCTAAGTTGATGGCGAGTGGCGAACGGGTGAGTAACGCGTAGGAATATGCCTTAAAGAGGGGGACAACTTGGGGAAACTCAAGCTAATACCGCATAAACTCTTCGGAGAAAAGCTGGGGACTTTCGAGCCTGGCGCTTTAAGATTAGCCTGCGTCCGATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCAGTGGGGAGGAGGGTTTCCCGGTTAAGAGCTAGGGGCATTGGACGTTACCCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCCGCGGTAATACGGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCCGTTAAAANGGTGCCTAAGGTGGTTTGGATNAGTTATGTGTTAAATTCCCTGGCGCCTCCACCCTGGNGCCAGGTCCATANTAAAAACTGTTAAACTCCGAAGTATGGGCACAAGGTAANTTGGAAANTTCCGGTGGTNANCCGNTGAAAATGCGCTTAGAGATNCGGGAAGGGACCACCCCAGTGGGGAAGGCGGCTACCTGGCCTAATAACTGACATTGAGGCACGAAAAGCGTGGGGAGCAACCAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCAACTAGCTGTNGGTTATATGAATATAATTAGTGGCGAAGCTAACGCGATAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATNGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATACAGTAAATCTTTCAGAGATGAGAGAGTGCCTTCGGGAATACTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTATCTCTAGTTGCCAGCGAGTAATGTCGGGAACTCTAAAGAGACTGCCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCCGATACAGAGGGGCGCGAAGGAGCGATCTGGAGCAAATCTTATAAAGTCGGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGTCTAACCGCAAGGGGGACGTTTACCACGGTGTGGTTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCG
>182569
AGAGTTTGATCCTGGCTCAGGATGAACGCTAGCTACAGGCTTAACACATGCAAGTCGAGGGGCAGCATGGTGTATCAATATATCTATGGCGACCAGCGCACCGGTGATGCACACCTCTCCTACCTGCCCCTTACTCCGGGATGATCTTTCTAAAAAAATATTACTACTCCATGGTATTACCGAAAAACGTCTTTTTGTTGTTTAAAAACTTCGATGGTGGAAGGTGATGCTTTCTATTATATACTTGGTGGGGTAACAGCCCACCACCTCAGCGATGAATAGGGGTTCTAATAAGAAGGTCCCCCCCATGGTAACTGGGCCCCGGTCCAAATTCTTCGGGAAGCCACCAGTGAGGATTATTGTTCAATGGCGGAGATTTTGACCCAGCCCAAGTAGCGTGAAGGATGACTGCTCCCATAGGTGGTAAACTTCTTTTATATGGGAATAAAGTGAGTCACGTGTGTCTTTTTGTATGTATCATATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATTCGAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCGGTTTGTTAAGTCAGTGGTGAAAGTTTGGGGCTCAACCGTGAAATTGCATTTGATACTGGCGGTCTTGAGTGCAGTAGAGGTGGGCGGAATTTGTGGTGTAGCGGTGAAATGCTTAGATATCATGCAGAACTCCGATTGCGAAGGCAGCTCACCGGAGTGTATCTGACGTTGAGGCTCGAAAGTGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCACACAGTAAAGAAGGAATATTGTCGTTGTGGGATCTCCATTAAGGGGTCAAGGGAAAGCATTAATTATTCCCCTGGGGGAGTAGTCCGCCAGAGGTGAAATTAAAAGAAATGGAGGGGGGCCGGCCCAAGGGAAGGACCATGTGGTTTAATTGGAGGATAGGGGAGGACCTTTCCCGGGGTTGAAAGTGCAAATGAATTATGGGGAGAGCCATTCCCTTCAAGGCATGAGAGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGGTTAAGTCCCATAACGAGCGCAACCCTTATCTTCAGTTACTATCAGGTCAAGCTGAGCACTCTGGAGAGACTGCCGTTGTAAGATGAGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGGGGTACAGAAGGCAGCTACCCAGCGACAGGATGCCAATCCCAAAAACCTATCTCAGTTCGGATTGAAGTCTGCAACCCGCCTTCGTGAAGTTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGTGAATACGTTCCCGGGCCTTGCACACACCGCCCGTCA
>1719550
TCCTGGCTCAGAACGAACGTTGGCGGCGTGGATTAGGCATGCAAGTCGCGCGAATCCCCGCAAGGGGGGAAGCGGCGTAAGGGGCAGTAAGGCGTGGGTACCTACCCGGGGGTCGGGGATAGCCCGTCGAGAGACGGGGTAATACCCGATGACGTGGAGACACCAAAGGTCCGCCGCCCTCGGCGGGGCCCACGTGATATTAGCTAGTTGGCGGGGTAACGGCCCACCAAGGCGGGGATGTCTAGCGGGTGTGAGAGCACGACCCGCGCCACTGGCACTGAGACACTGGCCAGACACCTACGGGTGGCTGCAGTCGAGGATCTTCGGCAATGGGGGAAACCCTGACCGAGCGACGCCGCGTGGGCGACGAAGGCCTTCGGGTTGTAAAGCCCTGTCGAGGGGGAGAAAGCCTTAACCGGGTGATCTATCCCTGGAGGAAGCACGGGCTAAGTTCGTGCCAGCAGCCGCGGTAAGACGAACCGTGCGAACGTTGTTCGGAATCACTGGGCTTAAAGGGCGCGTAGGCGGGTTGCCGCGTCCGGGGTGAAATCCCACGGCTCAACCGTGGGGCGGCCCCGGGTACGGGCAGCCTCGAGGAGAGTAGGGGCATGCGGAACTCTGGGTGGAGCGGTGAAATGCGTTGATATCCAGAGGAACTCCGGTGGCGAAGGCGGCATGCTGGACCCTTCCTGACGCTGAGGCGCGAAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCCTAAACGATGAGAACTAGGTAGCCGGCCGGACATGGGCTGGCTGCCGGAGCCAAAGTGCTAAGTTCTCCGCCTGGGGAGTATGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCTCACACAAGCGGTGGAGCATGTGGCTTAATTCGAGGCTACGCGAAGAACCTTATCCCGGGCTTGACATGTTCGAAAGAGGCTCGAAGTAGCCCGCGGAAACGTGGGGCCAACGGTATCCAGTCCGGAGCGAGCTACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTCGGGTTAAGTCCCATAACGAGCGAAACCCTTACCCTCAGTTGCTTACTAGGACTCTGGGGGGACTGCCGGTGTCAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCTTTATGCCCGGGGCTGCACACGTGCTACAATGGCGTGGACAAAGAGACGCGAGCCCGCGAGGGGGAGCCAATCTCAGAAAGCACGCCCCAGTTCAGATCGCAGGCTGCAACTCGCCTGCGTGAAGCCGGAATCGCTAGTAATCGCGGGTCAGCAACACCGCGGTGAATGTGTTCCTGAGCCTTGTACACACCGCCCGTCAAGCCACGAAAGAGAGGGACGTCCGGAGTCGCCTTCACCGGTGCCGAAGACGGACTTCTTGATTGGGACTAAGTCGTAACAAGGTAACC
>1794723
TTAGAGTTTGATCCTGGCTCAGAACGAACGTTGGCGGCGTGGATTAGGCATGCAAGTCTCGCGAATCCCCGCAAGGGGGGAAGCGGCGTAAGGGGCAGTAAGGCGTGGGTAACCCACCCCGGGGCCCGGGATAGCCCGTCGAGAGACGGGGTAATACCGGGCGACGCAGCGTGCCGGCATCGGTGTGCTGCCAAAGGTCCGCCGCCCCGGGCGGGGCCCACGTGGTATTAGCTAGTTGGTGGGGTGACGGCCCACCAAGGCGGAGATGCCTAGCGGGTGTGAGAGCACGACCCGCGCCACTGGCACTGAGACACTGGCCAGACACCTACGGGTGGCTGCAGTCGAGGATCTTCGGCAATGGGGGCAACCCTGACCGAGCGACGCCGCGTGGGCGACGAAGGCCTTCGGGTTGTAAAGCCCTGTCGAGGGGGAGAAACGTCCCGCAAGGGGCCTGATCTATCCCTGGAGGAAGCACGAGCTAAGTTCGTGCCAGCAGCCGCGGTAAGACGAACCGTGCGAACGTTGTTCGGAATCACTGGGCTTAAAGGGCGCGTAGGCGGGCTGCCGAGTCCGGGGTGAAATCCTCCCGCTCAACGGGAGAACGGCCCCGGGTACTGGCGGCCTCGAGGCGGGTAGGGGCGTGCGGAACACTGGGTGGAGCGGTGAAATGCGTTGATATCCAGTGGAACTCCGGTGGCGAAGGCGGCACGCTGGACCCGTCTGACGCTGAGGCGCGAAAGCCAGGGGAGCGAACGGGATTAGATACCCCGGTAGTCCTGGCCCTAAACGTTGAGAACTAGGTAGTCGGCCGGACATGGGCTGACTGCCGGAGCGAAAGTGCTAAGTTCTCCGCCTGGGGAGTATGGTCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCTCACACAAGCGGTGGAGCATGTGGCTTAATTCGAGGCAACGCGAAGAACCTTATCCCGGGCTTGACATGTGCGAAAGCGTCTGGGGGTACCCGCCGGAAACGGCCGGGGAAGGTATCCAGTCCTGAACCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTCGGGTTAAGTCCCATAACGAGCGAAACCCTTACCCTCAGTTGCCAGCGGGTCACGCCGGGGACTCTGGGGGGACTGCCGGTGTCAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCTTTATGCCCGGGGCTGCACACGTGCTACAATGGCGTGGACAAAGGGGCGCGAACGCGCGAGCGGGAGCCGACCCCGGAAAGCACGCCCCAGTTCAGATCGCAGGCTGCAACTCGCCTGCGTGAAGTCGGAATCGCTAGTAATCGCGGGTCAGCAACACCGCGGTGAATGTGTTCCTGAGCCTTGTACACACCGCCCGTCAAGCCACGAAAGGGAGGGACGGCCGAAGTCGCGCCCCGCGCGCCGACGCCGGACTTCCCGATTGGGACTAAGTCGTAACAAGGTAACC
>1142181
CACGTGGGTCATTTGCCCCGAAGCCCGGGATAGCCCATGGAAACATGGATTAATACCGGATGTGGTTGGAGTACACAGGTGCTCCGTATTAAACGGTAGGTAGCAATACCTTCCGCTTCGGGATAAGCCCGCGGCCCATTAGCTAGTTGGTGGGGTAAGACCCAACCAAGGAGACAACCGGGAGCCGGACAGAAAGGGTGACGGCCACATTGGGACTGAGAAACGGCCCGATCCTACGGAGGCAGCAGTAAGAATCTTCCGCATGAACGAAGTCCGACCGAGCGACGCGCTGAGTGATGAAGGTGTTATGCATCGTAAAGCTCCTTCGGGGAGGAGAATAAGCATAGTCCAAAAGGCTATGTGATGACGACCCTCCCTAAAGAAGCCCCGGCTAATTACGTGCAGCAGCGCGGCAATACGTAAGGGGTAAGCGTTGTTCGGAATTACTGGGCGTAAAGGGTGTGCAGGCGGGAGAGTAAGTTGGGGGTGAAATCTACGGGCCCAACCCGTAAACTGCCCTCAAAACTGCTTTTCTTGAGTGCAGGAGAGGAGACTGGAATTCCTAGTGTAGGAGTGAAATCTGTAGATATTAGGAAGAACACCGGTGGCGAAGGCGAGTCTCTGGCCTGACACTGACGCTGATACACGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGTGCACTAGATGTTGGGGGTGTCAATCCCCTCAGTGTCGCAGTTAACGCATTAAGTGCACCGCCTGGGGAGTATGCTCGCAAGGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCAGGGCTTGACATACAGGTGCCGGGCTGTGAAAGCAGTCCTCTCTTCCGAGCGCCTGTACAGGTGTTGCACGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGTTAAGTCCCCCAACGAGCGCAACCCCTATTGTCTGTGCCATCATTAAGTTGGCACTCGAACGAAACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATATGGCCCTTAGGCCTGGCTACACGTGCTACAATGGACAGTACAAGAGTCGCAAGACCGAAAGGTGGACCATCCAAAAGCTGTCCTCAGTTCCGATTGAAGTCTGAAACTCGACTCCATGAAGTTGGAATCGCTAGTAATCGCGCATCAGAATGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACCCGAGTTGGAAGTACTTGAAGTCGCTGATCTAACCTTCGGGAGGAAGGCGCCGATTGTACGTCTGATAAGGGGGGTGAAGTCGTAACAAGGTAACC
>2683209
CTGGCGGCGTGGTTTAGGCATGCAAGTCGAACGCGAAAGATTTACTTCGGTAAATTGAGTAGAGTGGCGAACGGGTGAGTAATACGTACGAATCTACCTTAAAGACAGGGATAGTCCCGGGAAACTGGGTTTAATACCTGATGGTATCCGGCTTTGCCGGATTAAAGACGGCCTCTATTTATAAGCTGTTACTTTTAGATGAGCGTGCGCTCCATTAGTTAGTTGGTAAGGTAAGAGCTTACCAAGGCGATGATGGATAGGCGTCCTTAACGGGTGGTCGCCCACACTGGGATTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTCGAGAATCGTCTACAATGAACGCAAGTTTGATAGTGCGACGCCGCGTGAATGAAGAAGCATTTCGGTGTGTAAAATTCTTTTATATAAGAACAGTGCATGTATGGTAAATAATTATACGTGAGAGATAGTACTATATGAATAAGCTCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGAGCAAGCGTTGTCCGGAATTACTAGGTGTAAAGGGTAAGTAGGCGGAAATTTAAGTCTCCGGTTAAATCTTCGGGCTCAACCCGAAATCTGCCTGAGATACTGGATTTCTAGAGTAAAGCAGATGAAGGCGGAATTCCTGGAGTAGCGGTGGAATGCGTAGATATCAGGAAGAACACCCATAGCGAAGGCAGCTTTCAATGCTATTACTGACGCTCAATTACGAAGGTGCGGGTATCGAACAGGATTAGATACCCTGGTAGTCCGCACAGTAAACGATATGTACTTGATATTGGATGTTGAAAATTCAGTGTCGTAGCTAACGCGTTAAGTACATCACCTGGGGACTAACGGCCGCAAGGTTAAAACTCAAAGGAATTGACGGGGGCCCACACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGAACTTGACATGCCGAGAATCCTGTAGAAATATGGGAGTGCCTTTTTTGGAGCTCGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTTAGTTGCTACCATTAAGTTGAGGACTCTAAAGAGACTGCCAGAGTACAAATCTGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGTCCTTATGTTCAGGGCTACACACGTGCTACAATGGTTGGAACAAAAGGCAGCGAAGGGGCGACCCGGAGCTAATCTCCAAACCCAATCTTAGTCCGGATTGCAGTCTGCAACTCGACTGCATGAAGTTGGAATCGCTAGTAATCGTGAGTCAGCATATCACGGTGAACATGTTCCTGGGCCTTGTACACACCGCCCGTCAAGTCAGCCGAATCGAGTGCACCCGAAGAAGGTGAGTTAATTAGACAGCTTTCGAAGGTGTGCTTGTAAGGGGGACTAAGTC
>2784824
AGTGGCGCACGGGTGAGTAACGCGTGGGTAACTTGCCTTTAAGTGAGGGATAACCCACTGAAAGGTGGACTAATACCTCATAAGACCACAGTGCTACGGCAGCGTGGTCAAAGGTGGCTTTATTAAAAGCTGCCGCTTGGAGAGAGACCCGCGTCCCATCAGCTTGTTGGTAAGGTAATGGCTTACCAAGGCCGAGACGGGTAGCTGGTCTGAGAGGATGGCCAGCCACACTGGAACTGAAACACGGTCCAGACTCCTACGGGAGGCAGCAGTGAGGAATCTTGCGCAATGGGGGGAACCCTGACGCAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGGTCGTAAAGCCCTGTCGGGAGGGAAGAAACAGTTATGCATGAATAATGCATAACCTTGACGGTACCTCCNGAGGAAGCACCGGCCAACTCCGTGCCAGCAGCCGCGGTAAAACGGAGGGTGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGAGCGTGTAGGCGGATAGATAAGTCGAGTGTGAAAGCCCTCAGCTTAACTGAGGAAGTGCATTCGAAACTATCTTTCTTGGGTACGGAAGAGGGAAGTGGAATTCCCGGTGTAGGGGTGAAATCCGTAGATATCGGGAGGAATACCAGTGGCGAAGGCGACTTCCTGGACCGTCACTGACGCTGAGACGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGGGCACTAGGTGTATCTCGCTTAGCGGGATGTGCCGTAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGTGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCTGGGTTTGACATGCCGAGAATCTGCCAGAAATGGTGGAGTGCCCCGTTAGGGGAACTCGGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCACCTTTAGTTGCCAGCATTAAGTTGGGCACTCTAAAGGGACTGCCGGTGTTAAACCGGAGGAAGGTGGGGACGACGTCNAGTCCTCATGGCCTTTATACCCAGGGCTACACACGTGCTACAATGGCCAGTACAAAGGGCTGCAATCCCGCGAGGGGGAGCCAACCCCAAAAATCTGGTCTTAGTTCGGATTGGAGTCTGCAACTCGACTCCATAAAGGTGGAATCGCTAGTAATCGTGAATCAGCACGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAAAGTTGGCTGTACCAGAAGTTGCTGAGCTAACTCGCCTCGGCGGGAGGCAGGCACCTAAGGTGTGGTTGATGATTGGGGTGAAGT
>2941516
TTAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGATAGGCCTAACACATGCAAGTCGAGGGGTAACAGGGTAGCAATACCGCTGACGACCGGCAAATGGGTGAGTAACGCGTATGCAACCTACCGATAACAGTTGGATAGCTCCCTGAAAGGGGAATTAAACCGGCATGACACTATGAGATCGCCTGTTTTCATAGTTAAATATTTATAGGTTATTGATGGGCATGCGTGACATTAGCAAGTTGGTGAGGTAACGGCTCACCAATGCTACGATGTCTAGGGGTTCTGAGAGGAAGGTCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGACGGAAGTCTGAACCACCCACTTCGCGTGCAGGATGACTGCCCTATGGGTTGTAAACTGCTTTTATATAAGAGGAACAGTATTTATGTATAGATATTTGCCAGTATTATATGAATAAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGTTATCCGGATTTATTGAGTTTAAAGGGTGAGTACGCGGTAGTATAAGTCAGCGGTGATAACTCGCAGCTCATCTGTAAGCTTGCCGTTGACACTGTATTACTTGACTTAACGTTGAGGTATGCTGAATGGGGGGGGGTTACCCGTTGAAATGCATTAATCAAAACAACAGACCACCCGATTTGCGGACGGCAGCAAAACTACACTGTCCACTGACGCTGATGCACAAAAGGCGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGATTACTGGTTGTTTGTGATACACTGCAAGTGACTGAGCGAAAGCACTAAGTAATCCACTTGGCGAGTACGTCGGCAACGATGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTCTAATTCGAGGCAACGCGAAGAACCTTACCCAGACTTGACATCTAGGAAAGGTCCTTGAAAGAGGATCGTGCCCGCAAGGGAATCCTAAGACAGGTGTTGCATGGCTGTCGTCAGCTCCTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGCTTACAGTTACCATCGGTTCGGCCGGGGACTCTGTAAGGACTGCCGCTGATAAAGCGAAGGAAGGCGGGGACGACGTCAAGCAATCACGGCCCTTACGTCTGGGGCTACACACGTGCTACAATGGCCGGTACAATGAGTCGCAAAACCGCGAGGTCAAGCTAATCTCAAAAAACCGGTCTCAGTTCGGATTGGAGTCTGCAACCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCATGGCGCGGCGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA
>998428
GACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGAGTTGTTCCTTCGGGGACAGCTTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGCAGGACCGGGATAACCCACGGAAACGTGAGCTAATACCGGATAGATGGTTCCCTCGCATGAGGGGATCAGGAAAGACGGGGCAACCTGTCACTTGTAGATGGGCCTGCGGCGCATTAGCTAGTTGGCGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGAACGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAGGAAGGTCTTCGGATCGTAAAGCTCTGTTGCCAAGGAAGAACGCTTGGTGGAGTAACTGCCATCAAGGTGACGGTACTTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATGGGCGTAAGCGCGCGCAGCGGTTCTTTAAGTCTGAGGTTAAATGCAGGGCTCAACCTTGTAACGCCTTGGAAACTGGGGGACTGGAGTGTAGGAGAGGAAAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGCCTATAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTTAACACAGTAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGACCCGCACAGGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGATGAAACGTCTAGAGATAGGCGCCCTCTTCGGAGCATTGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGAATTCAGTTGCCAGCACTTCGGGTGGGCACTCTGAATTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCGTGCCCCTTATGACCTGGGCTACACACGTACTACAATGGTCGGTACAACGGGCAGCGAAGCCGCGAGGCGGAGCCAATCCTAGAAAAGCCGATCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCT
>4343117
AACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGCGTGCGCGGTTCACGAACTTGTACGTGGATGGGCGCACGGCGCAGGGGGGCGTAACACGTGGGCACTCTGCCCTCCGATGGGGAATACTCCCGCGAACCGGGGGCTAATACCGCATAACATTCCGAGGACTTGGGTTCTTGGATTCAAAGCAGTGATGCCTGTGAGGAGGAGCCCGCGCCCGATTAGCTAGTTGGTAGGGTAACGGCCTACCTCGGCAATGATCGGTAGCTGGTCTGAGAGGATAATCAGACACACTGCAACTGAAACGAGGCCCAGACTCCTACCGTAGGGAACGCTGGGGAATCTTGCCTTCTGGGCGAAAGCATGACCCAACGACGCCGCGTGGGGGATGAAGCTTTTGCTAGTGTAAACCCCTTTTCACTGGTAAGAATGCACGCAAGGGAGCGACAGTACCCTGGCAAGAAGCCCCGGCTAACTACGTGCCACCCGCCTCGGTAAGACCTAGGGGGCCAGCGTTGTTCGGAATTACTGGGTGTATAGGGTACTTATGCGGTGCGACAAGTTGGGAGTGAAATCTCTGGGCTTAACCCAGAGGCTGCTTCTCAAACTGCTATGCTTGATTGTGACAGAGGCTCTTGAAATTGCAGGAGTAGCGTTGAAATGCATGTATATCTGCAAGATCACCCGAGATATGGACGAACAGCTGGATCACAAGTGACGCTGAGGAACGAAAGCTACGCTGAGCGAACAGGATTATATACACTGGTAGTCCTAGCACTAAACGATCATGACTTGCGGTGACGACCGTTCGGACGTCTCCCGGAGCTAACGCGTTAAGTCCTGCACCTGGGGAGTACGGTCGCAGACTGGAAGTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAACATGTGGTTCAATTCGACGCTACGCGAGGAACCTTACCTGGTTCGAAATTCTTATGACCAGCTGTAGAATTACGGCTTTCCTTCAAGAGACATGAGTCTAGGCGCTCCATGGCTGTCGTCAGTTCGTTCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGCACGTAGTTACTACTCGCAAGAGAGGACTCTACGTGGACTGCTCCGGATAACGGAGAGGAAGGTGGGAATGACGTCAAGTCCGCATGGCCTTTATGTCCAGGGCTACACACGTGTTACAATGCAGGGTACAAACCGTTGCCAACCCGCGAGGGGGAGCTAATCGGATAAAACTGTGCTCAGTTCGGATTGCAGTCTGCAACTCGACTGCATGAAGCTGGAATCGCTAGTAATGGGGATCAGCTTGACGCCGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACATCACGAAAGTGAGCTCACCTAGAAGTCGCCACGCTAACCGCAAGGGGGCAGGCGCCCAAGGTATGACTCATGATTGGGGTG
>4353661
GGATGAACGCTAGCGGGAGGCTTAATACATGCAAGTCGAGGGTGAAGCTTTCTTCGGAAAGTGGAAACCGGCGAACGGGTGCGTAACGCGTACGCAACTTACCCCTTGCTGGAGAATAGCCCCGGGAAACTGGGATTAATGCTCCATGGTATGGTGAAATCGCATGATTTTATCATTAAAGGTTACGGCAAGGGATAGGCGTGCGTCCCATTAGCTTGTTGGTGAGGTAACGGCTCACCAATGCAAACGATGGGTAGCTGGTCTGAGAGGATGATCAGCCACACGGGCACTGAGACACGGGCCCGACTCCTACGGGAGGCAGCAGTAGGGAATATTGGACAATGGACGAAAGTCTGATCCAGCCATCCCGCGTGCAGGACGAATGCCCTATGGGTTGTAAACTGCTTTTCTAAGGAAAGAAATATCTCATTCATGAGGTGCTGACGGTACCTTAGGAATAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTATCCGGATTCACTGGGTTTAAAGGGTGCGTAGGCGGTATGATAAGTCAGTGGTGAAAGCCCGGGGCTCAACTCCGGAACTGCCGTTGATACTGTCATACTTGAGTCCAGTTGAGGTGGGCGGAATGATACATGTAGCGGTGAAATGCTTAGATATGTATCAGAACACCGACTGCGAAGGCAGCTCACTAAACTGGTACTGACGCTGAGGCACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCTAACTCGGTATGTGCGATATACTGTACGTGCCTGAGGGAAACCGTTAAGTTAGCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCTGGGCTCTAATGTACCACGCCCGACCCTGAAAGGGGTCTTCTTCTTCGGAAGCGGGGTACAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCCTTAGTTGCCAGCGGTCCGGCCGGGGACTCTAAGGAGACTGCCTTCGCAAGGAGTGAGGAAGGAGGGGACGACGTCAAATCATCATGGCCTTTATGCCCAGGGCTACACACGTGCTACAATGGTGAGGACAAAGGGCAGCCACTTAGCGATAAGGAGCAAATCCCAAAAACCTCACCTCAGTTCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAACCGCTAGTAATCGCAGATCAGACATGCTGCGGTGAATACGTTCCCGGACCTTGTACACACCGCCCGTCAAGCCATGGAGCCGGGTGTACCTTAAGGCGATAACCGAAAGGAGTTGCCCAAGGTA"""
seqs_16s = StringIO(seqs_16s)
seqs_16s = [s[:50] for s in skbio.io.read(seqs_16s, constructor=DNA, format='fasta')]
seq_lookup = {seq.metadata['id'] : seq for seq in seqs_16s}
tax = """669210 k__Bacteria; p__; c__; o__; f__; g__; s__
881726 k__Bacteria; p__Firmicutes; c__Bacilli; o__Bacillales; f__Paenibacillaceae; g__Paenibacillus; s__
296752 k__Bacteria; p__Spirochaetes; c__Spirochaetes; o__Spirochaetales; f__Spirochaetaceae; g__Treponema; s__
1794723 k__Bacteria; p__Planctomycetes; c__Planctomycetia; o__Gemmatales; f__Gemmataceae; g__Gemmata; s__
2941516 k__Bacteria; p__Bacteroidetes; c__Bacteroidia; o__Bacteroidales; f__Marinilabiaceae; g__; s__
793074 k__Bacteria; p__; c__; o__; f__; g__; s__
4353661 k__Bacteria; p__Bacteroidetes; c__[Saprospirae]; o__[Saprospirales]; f__; g__; s__
292553 k__Bacteria; p__Spirochaetes; c__Spirochaetes; o__Spirochaetales; f__Spirochaetaceae; g__Treponema; s__
2784824 k__Bacteria; p__Proteobacteria; c__Deltaproteobacteria; o__Syntrophobacterales; f__Syntrophaceae; g__; s__
1719550 k__Bacteria; p__Planctomycetes; c__Planctomycetia; o__Gemmatales; f__Gemmataceae; g__Gemmata; s__
182569 k__Bacteria; p__Bacteroidetes; c__Bacteroidia; o__Bacteroidales; f__Bacteroidaceae; g__Bacteroides; s__
266495 k__Bacteria; p__Bacteroidetes; c__Bacteroidia; o__Bacteroidales; f__S24-7; g__; s__
524860 k__Bacteria; p__Planctomycetes; c__Planctomycetia; o__Gemmatales; f__Gemmataceae; g__Gemmata; s__
293514 k__Bacteria; p__Spirochaetes; c__Spirochaetes; o__Spirochaetales; f__Spirochaetaceae; g__Treponema; s__
2683209 k__Bacteria; p__WWE1; c__[Cloacamonae]; o__[Cloacamonales]; f__; g__; s__
501793 k__Bacteria; p__Firmicutes; c__Bacilli; o__Bacillales; f__Paenibacillaceae; g__Paenibacillus; s__
229854 k__Bacteria; p__Proteobacteria; c__Gammaproteobacteria; o__Legionellales; f__Legionellaceae; g__Legionella; s__
583705 k__Bacteria; p__Bacteroidetes; c__Bacteroidia; o__Bacteroidales; f__; g__; s__
1142181 k__Bacteria; p__Spirochaetes; c__GN05; o__SBYZ_6080; f__; g__; s__
998428 k__Bacteria; p__Firmicutes; c__Bacilli; o__Bacillales; f__Paenibacillaceae; g__Paenibacillus; s__
4343117 k__Bacteria; p__Acidobacteria; c__DA052; o__Ellin6513; f__; g__; s__"""
tax_lookup = dict([e.strip().split('\t') for e in tax.split('\n')])
What are the fraction of 3 base k-words unique to the following two sequences?
seq1 = DNA('AGCTAGCATCGATCGATCGATGCATGCAT')
seq2 = DNA('AGCTCGGCATCGAGGGCAGTCAATCGATCT')
help(kmer_distance)
# Compute the kmer distance in this cell
Display the guide tree for the sequences in the cell below.
query_seqs = [DNA("ACGATGACCAGTGCTACCAGT", metadata={'id': 's1'}),
DNA("AACGATCGATCGATCGTGCTA", metadata={'id': 's2'}),
DNA("AACGATCTGCTA", metadata={'id': 's3'}),
DNA("CGATCGATGACATGCATG", metadata={'id': 's4'}),
DNA("CGATCTGCAT", metadata={'id': 's5'})]
help(guide_tree_from_sequences)
# Display the guide tree in this cell.
# hint: Pass metric=kmer_distance
What are the differences in the guide tree from Question 2, the tree that is generated after 1 iteration of iterative multiple sequence alignment, and the tree that is generated after 5 iterations of iterative multiple sequence alignment? Display the trees for both 1 and 5 iterations of iterative multiple sequence alignment.
help(iterative_msa_and_tree)
from skbio.alignment import global_pairwise_align_nucleotide
# add your command for 1 iterations of iterative multiple sequence alignment here
# hint: pass pairwise_aligner=global_pairwise_align_nucleotide
# hint: Pass metric=kmer_distance
# add your command for 5 iterations of iterative multiple sequence alignment here
# hint: pass pairwise_aligner=global_pairwise_align_nucleotide
# hint: Pass metric=kmer_distance
Generate and display a tree based on progressive alignment of the sequences from the second cell (the ones in the seqs_16s
variable). This step can take about 10 minutes to complete.
help(progressive_msa_and_tree)
# Add your command for progressive alignment and tree building here
# hint: pass pairwise_aligner=global_pairwise_align_nucleotide
# hint: Pass metric=kmer_distance
Using the tree representing the sequences from question four as a guide, define clusters (i.e., groups) of sequences at 90% and 70% identity. There is not a single right answer for this, or a single method for grouping sequences. Go about this systematically, and describe the process that you're going through in a couple of paragraphs. These groups are usually referred to as operational taxonomic units, or OTUs, because they represent a hypothesis about the taxonomic relatedness of a group of sequences (which is a proxy for a hypothesis about the relatedness of the group of organisms containing those sequences in their genomes).
If you want to obtain a given sequence, you can now do so by looking up its identifier with seqs_16s.get_seq
:
seq_lookup['4343117']
seq_lookup['4353661']
To compute the pairwise identity for two sequences, use pairwise_percent_id
as follows. IMPORTANT: While this calculation is fast for a pair of sequences (i.e., a pairwise calculation), running it for all pairs of sequences (e.g., in a nested for-loop) could take a couple of hours, depending on the hardware where it's being run. You only need to do pairwise comparisons to answer this question.
from skbio.alignment import global_pairwise_align_nucleotide
def pairwise_percent_id(seq1_id, seq2_id, seq_lookup):
seq1 = seq_lookup[seq1_id]
seq2 = seq_lookup[seq2_id]
aln, _, _ = global_pairwise_align_nucleotide(seq1, seq2)
return 1. - aln[0].distance(aln[1])
print(pairwise_percent_id('793074', '4353661', seq_lookup))
# Compute additional pairwise identities, as necessary, to answer this question here. Show all of your commands!
Discuss your results here.
Using tax_lookup
by their ids, get the taxonomy of each sequence, and include those in the results below. When you see a key that ends with __
, that means that there is no known taxonomic assignment for that sequence at that level. Examine the taxonomy for each of your OTUs at 70% and 90%. Does the taxanomic assignment for these OTUs make sense? Additionally, choose one representative sequence from each of the clusters you defined in question 5. Is the taxonomy of the representative sequences consistent with phylogenetic tree you generated in question 4?
>>> print(tax_lookup['4343117'])
print(tax_lookup['4353661'])
# Perform addition taxonomy look-ups here
Discuss your results here.
For your 90% and 70% OTUs, list three taxa (e.g., at the phylum, class, or species level) that are monophyletic, if any, and three taxa that are not monophyletic, if any. Discuss two specific reasons why some taxa might appear to not be monophyletic based on your tree.
Discuss your results here.