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test_workflow.py
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test_workflow.py
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# -----------------------------------------------------------------------------
# Copyright (c) 2015, The Deblur Development Team.
#
# Distributed under the terms of the BSD 3-clause License.
#
# The full license is in the file LICENSE, distributed with this software.
# -----------------------------------------------------------------------------
from unittest import TestCase, main
from shutil import rmtree
from tempfile import mkdtemp
from os import listdir, remove
from types import GeneratorType
from os.path import join, isfile, abspath, dirname, splitext
from skbio import DNA
from biom import load_table
import logging
from skbio import Sequence
from deblur.workflow import (dereplicate_seqs,
remove_chimeras_denovo_from_seqs,
remove_artifacts_seqs,
create_otu_table,
get_files_for_table,
trim_seqs,
multiple_sequence_alignment,
launch_workflow,
split_sequence_file_on_sample_ids_to_files,
build_index_sortmerna,
start_log, sequence_generator,
sample_id_from_read_id,
remove_artifacts_from_biom_table,
_get_fastq_variant,
filter_minreads_samples_from_table,
fasta_from_biom)
from deblur.deblurring import get_default_error_profile
class workflowTests(TestCase):
""" Test deblur pipeline and individual methods functionality """
def setUp(self):
""" Create working directory and two FASTA input
files corresponding to two samples (s1 and s2).
Each input file contains 120 sequences, of which
100 are 16S amplicons (ART simulator), 10 are
chimeric sequences (Grinder) and 10 are PhiX
artifacts (ART). The 100 amplicon sequences
intend to evenly represent a community of 10
species.
"""
# test output can be written to this directory
self.working_dir = mkdtemp()
# the data directory for the workflow test files
self.test_data_dir = join(dirname(abspath(__file__)), 'data')
self.seqs_fq_fp = join(self.test_data_dir, 'seqs.fq')
self.seqs_fq_bad_fp = join(self.test_data_dir, 'seqs_bad.fq')
self.seqs_fq_illumina13_fp = join(self.test_data_dir,
'seqs_illumina1.3.fq')
self.seqs_s1_fp = join(self.test_data_dir, 'seqs_s1.fasta')
self.seqs_s2_fp = join(self.test_data_dir, 'seqs_s2.fasta')
self.seqs_s3_fp = join(self.test_data_dir, 'seqs_s3.fasta')
self.orig_s1_fp = join(self.test_data_dir, 'simset.s1.fasta')
self.orig_s2_fp = join(self.test_data_dir, 'simset.s2.fasta')
self.orig_s3_fp = join(self.test_data_dir, 'simset.s3.fasta')
self.no_trim_res = join(self.test_data_dir, 'no_trim_results.fasta')
self.files_to_remove = []
logfilename = join(self.working_dir, "log.txt")
start_log(level=logging.DEBUG, filename=logfilename)
def tearDown(self):
for f in self.files_to_remove:
remove(f)
rmtree(self.working_dir)
def test_get_fastq_variant(self):
exp = 'illumina1.8'
obs = _get_fastq_variant(self.seqs_fq_fp)
self.assertEqual(obs, exp)
exp = 'illumina1.3'
obs = _get_fastq_variant(self.seqs_fq_illumina13_fp)
self.assertEqual(obs, exp)
with self.assertRaises(ValueError):
_get_fastq_variant(self.seqs_fq_bad_fp)
def test_sequence_generator_fasta(self):
exp_len = 135
first_id = "s1_1001203-10"
first_few_nucs = "TACGTAGGTGGCAAGCGTTA"
obs = list(sequence_generator(self.seqs_s1_fp))
self.assertEqual(len(obs), exp_len)
self.assertEqual(obs[0][0], first_id)
self.assertTrue(obs[0][1].startswith(first_few_nucs))
def test_sequence_generator_fastq(self):
exp_len = 2
first_id = "foo"
first_few_nucs = "GCgc"
obs = list(sequence_generator(self.seqs_fq_fp))
self.assertEqual(len(obs), exp_len)
self.assertEqual(obs[0][0], first_id)
self.assertTrue(obs[0][1].startswith(first_few_nucs))
def test_sequence_generator_invalid_format(self):
allres = []
input_fp = join(self.test_data_dir, 'readme.txt')
msg = "input file %s does not appear to be FASTA or FASTQ" % input_fp
with self.assertWarns(UserWarning) as W:
for res in sequence_generator(input_fp):
allres.append(res)
self.assertEqual(len(allres), 0)
self.assertEqual(str(W.warning), msg)
def test_trim_seqs_notrim(self):
seqs = [("seq1", "tagggcaagactccatggtatga"),
("seq2", "cggaggcgagatgcgtggta"),
("seq3", "tactagcaagattcctggtaaagga"),
("seq4", "aggatgcgagatgcgtg"),
("seq5", "gagtgcgagatgcgtggtgagg"),
("seq6", "ggatgcgagatgcgtggtgatt"),
("seq7", "agggcgagattcctagtgga--")]
obs = trim_seqs(seqs, -1, 0)
self.assertEqual(list(obs), seqs)
def test_trim_seqs_notrim_outofbounds(self):
seqs = [("seq1", "tagggcaagactccatggtatga"),
("seq2", "cggaggcgagatgcgtggta"),
("seq3", "tactagcaagattcctggtaaagga"),
("seq4", "aggatgcgagatgcgtg"),
("seq5", "gagtgcgagatgcgtggtgagg"),
("seq6", "ggatgcgagatgcgtggtgatt"),
("seq7", "agggcgagattcctagtgga--")]
with self.assertRaises(ValueError):
list(trim_seqs(seqs, -2, 0))
def test_trim_seqs(self):
seqs = [("seq1", "tagggcaagactccatggtatga"),
("seq2", "cggaggcgagatgcgtggta"),
("seq3", "tactagcaagattcctggtaaagga"),
("seq4", "aggatgcgagatgcgtg"),
("seq5", "gagtgcgagatgcgtggtgagg"),
("seq6", "ggatgcgagatgcgtggtgatt"),
("seq7", "agggcgagattcctagtgga--")]
obs = trim_seqs(seqs, 20, 0)
self.assertTrue(isinstance(obs, GeneratorType))
exp = [("seq1", "tagggcaagactccatggta"),
("seq2", "cggaggcgagatgcgtggta"),
("seq3", "tactagcaagattcctggta"),
("seq5", "gagtgcgagatgcgtggtga"),
("seq6", "ggatgcgagatgcgtggtga"),
("seq7", "agggcgagattcctagtgga")]
self.assertEqual(list(obs), exp)
def test_trim_seqs_left(self):
seqs = [("seq1", "tagggcaagactccatggtatga"),
("seq2", "cggaggcgagatgcgtggta"),
("seq3", "tactagcaagattcctggtaaagga"),
("seq4", "aggatgcgagatgcgtg"),
("seq5", "gagtgcgagatgcgtggtgagg"),
("seq6", "ggatgcgagatgcgtggtgatt"),
("seq7", "agggcgagattcctagtgga--")]
obs = trim_seqs(seqs, 20, 5)
self.assertTrue(isinstance(obs, GeneratorType))
exp = [("seq1", "caagactccatggta"),
("seq2", "gcgagatgcgtggta"),
("seq3", "gcaagattcctggta"),
("seq5", "cgagatgcgtggtga"),
("seq6", "cgagatgcgtggtga"),
("seq7", "gagattcctagtgga")]
self.assertEqual(list(obs), exp)
def test_dereplicate_seqs_remove_singletons(self):
""" Test dereplicate_seqs() method functionality with
removing singletons
"""
seqs = [("seq1", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq2", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq3", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq4", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCT"),
("seq5", "TACCAGCCCCTTAAGTGGTAGGGACGATTATTTGGCCTAAAGCGTCCG"),
("seq6", "CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT"),
("seq7", "CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT")]
seqs_fp = join(self.working_dir, "seqs.fasta")
with open(seqs_fp, 'w') as seqs_f:
for seq in seqs:
seqs_f.write(">%s\n%s\n" % seq)
output_fp = join(self.working_dir, "seqs_derep.fasta")
dereplicate_seqs(seqs_fp=seqs_fp,
output_fp=output_fp)
self.assertTrue(isfile(output_fp))
exp = [("seq1;size=3",
"TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq6;size=2",
"CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT")]
act = [item for item in sequence_generator(output_fp)]
self.assertEqual(act, exp)
def test_dereplicate_seqs(self):
""" Test dereplicate_seqs() method functionality,
keep singletons
"""
seqs = [("seq1", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq2", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq3", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq4", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCT"),
("seq5", "TACCAGCCCCTTAAGTGGTAGGGACGATTATTTGGCCTAAAGCGTCCG"),
("seq6", "CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT"),
("seq7", "CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT")]
seqs_fp = join(self.working_dir, "seqs.fasta")
with open(seqs_fp, 'w') as seqs_f:
for seq in seqs:
seqs_f.write(">%s\n%s\n" % seq)
output_fp = join(self.working_dir, "seqs_derep.fasta")
dereplicate_seqs(seqs_fp=seqs_fp,
output_fp=output_fp,
min_size=1)
self.assertTrue(isfile(output_fp))
exp = [("seq1;size=3",
"TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("seq6;size=2",
"CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT"),
("seq4;size=1",
"TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCT"),
("seq5;size=1",
"TACCAGCCCCTTAAGTGGTAGGGACGATTATTTGGCCTAAAGCGTCCG")]
act = [item for item in sequence_generator(output_fp)]
self.assertEqual(act, exp)
def test_filter_minreads_samples_from_table(self):
""" Test filter_minreads_samples_from_table() function
for removal of samples with small number of reads
using the s4 dataset biom table
"""
input_biom_file = join(self.test_data_dir, 'final.s4.biom')
table = load_table(input_biom_file)
# test basic filtering with 0 reads does not remove ok sample
new_table = filter_minreads_samples_from_table(table)
self.assertEqual(new_table.shape[1], 1)
# test basic filtering with enough reads removes the sample
# and also inplace=False works
new_table = filter_minreads_samples_from_table(table,
minreads=182,
inplace=False)
self.assertEqual(new_table.shape[1], 0)
self.assertEqual(table.shape[1], 1)
# test basic filtering with enough reads removes the sample
# and also inplace=True works
filter_minreads_samples_from_table(table,
minreads=200,
inplace=True)
self.assertEqual(table.shape[1], 0)
def test_remove_artifacts_from_biom_table(self):
""" Test remove_artifacts_from_biom_table() function for
removing non 16s sequences from a biom table and matching
fasta file. This test uses a pre-calculated biom table and
fasta file and tests the output only 16s and only artifacts
tables
s4 dataset is similar to s2 but with two added non-16s
sequences (which are not phix/adapter)
"""
# create the positive reference databases
pos_ref_fp = join(self.test_data_dir, '70_otus.fasta')
pos_ref_db_fp = build_index_sortmerna(
ref_fp=(pos_ref_fp,),
working_dir=self.working_dir)
# remove the artifacts from the s4 biom table
input_biom_file = join(self.test_data_dir, 'final.s4.biom')
input_fasta_file = join(self.test_data_dir, 'final.s4.seqs.fa')
tmp_files = remove_artifacts_from_biom_table(input_biom_file,
input_fasta_file,
[pos_ref_fp],
self.working_dir,
pos_ref_db_fp)
origfilename = join(self.test_data_dir, 'simset.s2.fasta')
trim_length = 150
orig_seqs = [item[1] for item in sequence_generator(origfilename)]
orig_seqs = [item[:trim_length].upper() for item in orig_seqs]
no_artifacts_table_name = join(self.working_dir, 'reference-hit.biom')
no_artifacts_table = load_table(no_artifacts_table_name)
obs_seqs = no_artifacts_table.ids(axis='observation')
self.assertEqual(set(obs_seqs), set(orig_seqs))
# test the fasta file
no_artifacts_fasta_name = join(
self.working_dir, 'reference-hit.seqs.fa')
fasta_seqs = [item[1]
for item in sequence_generator(no_artifacts_fasta_name)]
self.assertEqual(set(fasta_seqs), set(orig_seqs))
# test the non-hit output biom
artifacts_table_name = join(self.working_dir, 'reference-non-hit.biom')
artifacts_table = load_table(artifacts_table_name)
obs_seqs = artifacts_table.ids(axis='observation')
artifact_seqs = [
'aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa'
'aaaaaaaaaaaaaaaaaaaatttttttttttttttttttttttttttttttttttttttttttt'
'tttttttttttttttttttttt',
'AACAATGGGGGCAAGCGTTAATCATAATGGCTTAAAGAATTCGTAGAATtatatatattatata'
'tatatTAGAGTTAATAAATATTAATTAAAGAATTATAACAATGGGGGCAAGCGTTAATCATAAT'
'GGCTTAAAGAATTCGTAGAATT']
artifact_seqs = [item[:trim_length].upper() for item in artifact_seqs]
obs_seqs = [item[:trim_length].upper() for item in obs_seqs]
self.assertEqual(set(obs_seqs), set(artifact_seqs))
# test the fasta file
artifacts_fasta_name = join(
self.working_dir, 'reference-non-hit.seqs.fa')
fasta_seqs = [item[1].upper()
for item in sequence_generator(artifacts_fasta_name)]
self.assertEqual(set(fasta_seqs), set(artifact_seqs))
self.assertEqual(len(obs_seqs), 2)
self.assertEqual(len(tmp_files), 2)
def test_remove_artifacts_seqs(self):
""" Test remove_artifacts_seqs() function for removing
sequences not matching to a reference database
using SortMeRNA. This test forces a new index
construction for the reference sequences.
"""
seqs = [("seq1", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCC"),
("seq2", "CCTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("seq3", "TCGCTATTATTGAGCCTAAAACGTCCGTAGTCGGCTTTGTAAATCCC"),
("seq4", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCC"),
("seq5", "CTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAATAGGGTC"),
("seq6", "TTGAGCCTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAAT"),
("phix1", "TCTAAAGGTAAAAAACGTTCTGGCGCTCGCCCTGGTCGTCCGCAGCC"),
("phix2", "CTGGCGCTCGCCCTGGTCGTCCGCAGCCGTTGCGAGGTACTAAAGGC"),
("phix3", "GCGCATAAATTTGAGCAGATTTGTCGTCACAGGTTGCGCCGCCAAAA")]
exp_seqs = ["seq1", "seq2", "seq3", "seq4", "seq5", "seq6"]
seqs_fp = join(self.working_dir, "seqs.fasta")
with open(seqs_fp, 'w') as seqs_f:
for seq in seqs:
seqs_f.write(">%s\n%s\n" % seq)
ref = [("ref1", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTA"
"GTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref2", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref3", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref4", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref5", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATAGGGT"),
("ref6", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT")]
ref_fp = join(self.working_dir, "ref2.fasta")
with open(ref_fp, 'w') as ref_f:
for seq in ref:
ref_f.write(">%s\n%s\n" % seq)
self.files_to_remove.append(ref_fp)
ref_db_fp = build_index_sortmerna(
ref_fp=(ref_fp,),
working_dir=self.working_dir)
output_fp, num_seqs_left, tmp_files = remove_artifacts_seqs(
seqs_fp=seqs_fp, ref_fp=(ref_fp,), working_dir=self.working_dir,
ref_db_fp=ref_db_fp, negate=False, threads=1)
obs_seqs = []
for label, seq in sequence_generator(output_fp):
obs_seqs.append(label)
self.assertEqual(obs_seqs, exp_seqs)
# validate it creates one tmp file
self.assertEqual(len(tmp_files), 1)
def test_remove_artifacts_seqs_index_prebuilt(self):
""" Test remove_artifacts_seqs() function for removing
sequences not matching to a reference database
using SortMeRNA. This test passes a built index.
"""
seqs = [("seq1", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCC"),
("seq2", "CCTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("seq3", "TCGCTATTATTGAGCCTAAAACGTCCGTAGTCGGCTTTGTAAATCCC"),
("seq4", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCC"),
("seq5", "CTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAATAGGGTC"),
("seq6", "TTGAGCCTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAAT"),
("phix1", "TCTAAAGGTAAAAAACGTTCTGGCGCTCGCCCTGGTCGTCCGCAGCC"),
("phix2", "CTGGCGCTCGCCCTGGTCGTCCGCAGCCGTTGCGAGGTACTAAAGGC"),
("phix3", "GCGCATAAATTTGAGCAGATTTGTCGTCACAGGTTGCGCCGCCAAAA")]
exp_seqs = ["seq1", "seq2", "seq3", "seq4", "seq5", "seq6"]
seqs_fp = join(self.working_dir, "seqs.fasta")
with open(seqs_fp, 'w') as seqs_f:
for seq in seqs:
seqs_f.write(">%s\n%s\n" % seq)
ref = [("ref1", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTA"
"GTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref2", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref3", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref4", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref5", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATAGGGT"),
("ref6", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT")]
ref_fp = join(self.working_dir, "ref3.fasta")
with open(ref_fp, 'w') as ref_f:
for seq in ref:
ref_f.write(">%s\n%s\n" % seq)
self.files_to_remove.append(ref_fp)
# build index
sortmerna_db = build_index_sortmerna([ref_fp], self.working_dir)
output_fp = join(self.working_dir, "seqs_filtered.fasta")
output_fp, num_seqs_left, _ = remove_artifacts_seqs(
seqs_fp=seqs_fp, ref_fp=(ref_fp,), working_dir=self.working_dir,
ref_db_fp=sortmerna_db, negate=False, threads=1)
obs_seqs = []
for label, seq in sequence_generator(output_fp):
obs_seqs.append(label)
self.assertEqual(obs_seqs, exp_seqs)
def test_remove_artifacts_seqs_negate(self):
""" Test remove_artifacts_seqs() function for removing
sequences matching to a reference database
using SortMeRNA.
"""
seqs = [("seq1", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCC"),
("seq2", "CCTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("seq3", "TCGCTATTATTGAGCCTAAAACGTCCGTAGTCGGCTTTGTAAATCCC"),
("seq4", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCC"),
("seq5", "CTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAATAGGGTC"),
("seq6", "TTGAGCCTAAAACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAAT"),
("phix1", "TCTAAAGGTAAAAAACGTTCTGGCGCTCGCCCTGGTCGTCCGCAGCC"),
("phix2", "CTGGCGCTCGCCCTGGTCGTCCGCAGCCGTTGCGAGGTACTAAAGGC"),
("phix3", "GCGCATAAATTTGAGCAGATTTGTCGTCACAGGTTGCGCCGCCAAAA")]
# seq5 is 80% similar, so should be kept for 0.95 default similarity
# to artifacts
exp_seqs = ["seq5", "phix1", "phix2", "phix3"]
seqs_fp = join(self.working_dir, "seqs.fasta")
with open(seqs_fp, 'w') as seqs_f:
for seq in seqs:
seqs_f.write(">%s\n%s\n" % seq)
ref = [("ref1", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTA"
"GTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref2", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref3", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref4", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref5", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATAGGGT"),
("ref6", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT")]
ref_fp = join(self.working_dir, "ref4.fasta")
with open(ref_fp, 'w') as ref_f:
for seq in ref:
ref_f.write(">%s\n%s\n" % seq)
self.files_to_remove.append(ref_fp)
ref_db_fp = build_index_sortmerna([ref_fp], self.working_dir)
output_fp = join(self.working_dir, "seqs_filtered.fasta")
output_fp, num_seqs_left, _ = remove_artifacts_seqs(
seqs_fp=seqs_fp, ref_fp=(ref_fp,), working_dir=self.working_dir,
ref_db_fp=ref_db_fp, negate=True, threads=1)
obs_seqs = []
for label, seq in sequence_generator(output_fp):
obs_seqs.append(label)
self.assertEqual(obs_seqs, exp_seqs)
def test_remove_chimeras_denovo_from_seqs(self):
""" Test remove_chimeras_denovo_from_seqs() method functionality.
Remove chimeric sequences from a FASTA file using the UCHIME
algorithm, implemented in VSEARCH.
"""
seqs = [("s1_104;size=2;", "GTGCCAGCCGCCGCGGTAATACCCGCAGCTCAAGTGGTG"
"GTCGCTATTATTGAGCCTAAAACGTCCGTAGTCGGCTTT"
"GTAAATCCCTGGGTAAATCGGGAAGCTTAACTTTCCGAC"
"TTCCGAGGAGACTGTCAAACTTGGGACCGGGAG"),
("s1_106;size=2;", "GTGTCAGCCGCCGCGGTAATACCAGCTCTCCGAGTGGTG"
"TGGATGTTTATTGGGCCTAAAGCGTCCGTAGCCGGCTGC"
"GCAAGTCTGTCGGGAAATCCGCACGCCTAACGTGCGGGC"
"GTCCGGCGGAAACTGCGTGGCTTGGGACCGGAA"),
("s1_1;size=9;", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAA"
"ACGTCCGTAGTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"
"CGCTTAACGATCCGATTCTGGGGAGACTGCAAAGCTTGGGA"
"CCGGGCGAGGTTAGAGGTACTCTCGGG"),
("s1_20;size=9;", "TACCTGCAGCCCAAGTGGTGGTCGATTTTATTGAGTCTAA"
"AACGTTCGTAGCCGGTTTGATAAATCCTTGGGTAAATCGG"
"GAAGCTTAACTTTCCGATTCCGAGGAGACTGTCAAACTTG"
"GGACCGGGAGAGGCTAGAGGTACTTCTGGG"),
("s1_40;size=8;", "TACCAGCTCTCCGAGTGGTGTGGATGTTTATTGGGCCTAA"
"AGCATCCGTAGCTGGCTAGGTTAGTCCCCTGTTAAATCCA"
"CCGAATTAATCGTTGGATGCGGGGGATACTGCTTGGCTAG"
"GGGACGAGAGAGGCAGACGGTATTTCCGGG"),
("s1_60;size=8;", "TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAA"
"AGCGTCCGTAGCCGGCTGCGCAAGTCTGTCGGGAAATCCG"
"CACGCCTAACGTGCGGGTCCGGCGGAAACTGCGTGGCTTG"
"GGACCGGAAGACTCGAGGGGTACGTCAGGG")]
seqs_non_chimera = ["s1_1;size=9;", "s1_20;size=9;",
"s1_40;size=8;", "s1_60;size=8;"]
seqs_fp = join(self.working_dir, "seqs.fasta")
with open(seqs_fp, 'w') as seqs_f:
for seq in seqs:
seqs_f.write(">%s\n%s\n" % seq)
output_fp = remove_chimeras_denovo_from_seqs(
seqs_fp=seqs_fp,
working_dir=self.working_dir)
seqs_obs = []
for label, seq in sequence_generator(output_fp):
label = label.split()[0]
seqs_obs.append(label)
self.assertEqual(seqs_non_chimera, seqs_obs)
def test_multiple_sequence_alignment(self):
"""Test multiple sequence alignment.
"""
seqs = [DNA('caccggcggcccggtggtggccattattattgggtctaaag',
metadata={'id': 'seq_1'}, lowercase=True),
DNA('caccggcggcccgagtggtggccattattattgggtcaagg',
metadata={'id': 'seq_2'}, lowercase=True),
DNA('caccggcggcccgagtgatggccattattattgggtctaaag',
metadata={'id': 'seq_3'}, lowercase=True),
DNA('aaccggcggcccaagtggtggccattattattgggtctaaag',
metadata={'id': 'seq_4'}, lowercase=True),
DNA('caccgggcccgagtggtggccattattattgggtctaaag',
metadata={'id': 'seq_5'}, lowercase=True)]
seqs_fp = join(self.working_dir, "seqs.fna")
with open(seqs_fp, 'w') as o:
for seq in seqs:
seq.write(o, format='fasta')
alignment_file = multiple_sequence_alignment(seqs_fp)
aligned_seqs = [DNA(item[1], metadata={'id': item[0]}, lowercase=True)
for item in sequence_generator(alignment_file)]
align_exp = [
DNA('caccggcggcccg-gtggtggccattattattgggtctaaag',
lowercase=True, metadata={'id': 'seq_1'}),
DNA('caccggcggcccgagtggtggccattattattgggtcaagg-',
lowercase=True, metadata={'id': 'seq_2'}),
DNA('caccggcggcccgagtgatggccattattattgggtctaaag',
lowercase=True, metadata={'id': 'seq_3'}),
DNA('aaccggcggcccaagtggtggccattattattgggtctaaag',
lowercase=True, metadata={'id': 'seq_4'}),
DNA('caccg--ggcccgagtggtggccattattattgggtctaaag',
lowercase=True, metadata={'id': 'seq_5'})]
self.assertEqual(aligned_seqs, align_exp)
def test_build_index_sortmerna(self):
"""Test functionality of build_index_sortmerna()
"""
ref1 = [("ref1", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTA"
"GTCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref2", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref3", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref4", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT"),
("ref5", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATAGGGT"),
("ref6", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT")]
ref2 = [("ref1", "GTCGTAGCTAGCTGCCCACGATCGTAGCTAGCTAGCTACGTAGCTCATCAC"
"TCGCCGACCCACGTCCCACTGATGCTGTGGG"),
("ref2", "GCGGCGCCCAAAAATGTCGTGTAAAATTTTCTCGTACCCACTTGCTACCCA"
"TGGCCGCCATGCTGCTAACGCAATATATATA"),
("ref3", "TGTGAAAGCGCGCGAGAGAGTCGTATATATGGGCGCGGCGCGATGCTGCCC"
"GTCGATGCTGATCCCCCACGTACGTAGCCCC"),
("ref4", "GTGTGCTCGCGTAGCTAGCTTATATATCGGCGCGTAGTGCTAGCCCCAAAA"
"GTGTCCCCCCCCTCCTTTTTTATATATGCAA"),
("ref5", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATAGGGT"),
("ref6", "TACCCGCAGCTCAAGTGGTGGTCGCTATTATTGAGCCTAAAACGTCCGTAG"
"TCGGCTTTGTAAATCCCTGGGTAAATCGGGT")]
ref1_fp = join(self.working_dir, "ref1.fasta")
with open(ref1_fp, 'w') as ref_f:
for seq in ref1:
ref_f.write(">%s\n%s\n" % seq)
ref2_fp = join(self.working_dir, "ref2.fasta")
with open(ref2_fp, 'w') as ref_f:
for seq in ref2:
ref_f.write(">%s\n%s\n" % seq)
ref_fps = tuple([ref1_fp, ref2_fp])
ref_db_fp = build_index_sortmerna(
ref_fp=ref_fps,
working_dir=self.working_dir)
self.assertEqual(len(ref_fps), len(ref_db_fp))
def test_build_index_sortmerna_fail(self):
"""Test functionality of build_index_sortmerna()
"""
with self.assertRaises(RuntimeError):
build_index_sortmerna(
ref_fp='foo',
working_dir=self.working_dir)
def run_workflow_try(self, simfilename, origfilename,
ref_fp, ref_db_fp, threads=1,
trim_length=100):
"""Test launching the complete workflow using simulated sequences
and compare to original ground truth.
Parameters
----------
simfilename : str
name of the simulated reads fasta file
origfilename : str
name of the fasta file with the ground truth sequences
ref_fp : list of str
list of the reference database files
def_db_fp : list of str
list of the indexed database files or None to create them
threads : int
number of threads to use (default=1)
trim_length : int
length of sequences to trim to (default=100)
"""
seqs_fp = simfilename
output_fp = self.working_dir
mean_error = 0.005
error_dist = get_default_error_profile()
indel_prob = 0.01
indel_max = 3
min_size = 2
left_trim_length = 0
nochimera = launch_workflow(seqs_fp=seqs_fp, working_dir=output_fp,
mean_error=mean_error,
error_dist=error_dist,
indel_prob=indel_prob,
indel_max=indel_max,
trim_length=trim_length,
left_trim_length=left_trim_length,
min_size=min_size,
ref_fp=(ref_fp,),
ref_db_fp=ref_db_fp,
threads_per_sample=threads)
# get the trimmed ground truth sequences
orig_seqs = [item[1] for item in sequence_generator(origfilename)]
orig_seqs = [item[:trim_length].upper() for item in orig_seqs]
output_filename = 'all.biom'
output_table_fp = join(output_fp, output_filename)
create_otu_table(output_table_fp, [(nochimera, seqs_fp)])
table_obs = load_table(output_table_fp)
outseqs = table_obs.ids(axis='observation')
outseqs = list(outseqs)
outseqs.sort()
orig_seqs.sort()
# test we see all ground truth sequences and no other
self.assertEqual(outseqs, orig_seqs)
def test_get_files_for_table(self):
filelist = get_files_for_table(self.test_data_dir)
file1 = join(self.test_data_dir,
'testmerge.fasta.trim.derep.no_artifacts'
'.msa.deblur.no_chimeras')
file2 = join(self.test_data_dir,
'testmerge2.fasta.trim.derep.no_artifacts'
'.msa.deblur.no_chimeras')
file3 = join(self.test_data_dir,
'testmerge.fastq.trim.derep.no_artifacts'
'.msa.deblur.no_chimeras')
self.assertEqual(len(filelist), 3)
fnames, sample_names = zip(*filelist)
self.assertIn(file1, fnames)
self.assertIn(file2, fnames)
self.assertIn(file3, fnames)
self.assertIn('testmerge', sample_names)
def test_create_otu_table(self):
# merge the fasta files
m1 = join(self.test_data_dir,
'testmerge.fasta.trim.derep.no_artifacts'
'.msa.deblur.no_chimeras')
m2 = join(self.test_data_dir,
'testmerge2.fasta.trim.derep.no_artifacts'
'.msa.deblur.no_chimeras')
outfile = join(self.working_dir, 'testmerge.biom')
fasta_outfile = join(self.working_dir, 'testmerge.seq.fa')
create_otu_table(outfile, [(m1, 'testmerge'), (m2, 'testmerge2')],
outputfasta_fp=fasta_outfile)
# test the result
table = load_table(outfile)
tableids = table.ids(axis='observation')
# test a sequence present in both
self.assertEqual(table.get_value_by_ids(
'TACGAGGGGGGCGAGCGTTGTTCGGAATTATTGGGCGTAAAAGGTGCGTAGGCGGTTCG'
'GTAAGTTTCGTGTGAAATCTTCGGGCTCAACTCGAAGCCTGCACGAAATACTGCCGGGC'
'TTGAGTGTGGGAGAGGTGAGTGGAATTTCCGGT', 'testmerge'), 5)
self.assertEqual(table.get_value_by_ids(
'TACGAGGGGGGCGAGCGTTGTTCG'
'GAATTATTGGGCGTAAAAGGTGCGTAGGCGGTTCGGTAAGTTTCGTGTGAAATCTTCGGG'
'CTCAACTCGAAGCCTGCACGAAATACTGCCGGGCTTGAGTGTGGGAGAGGTGAGTGGAAT'
'TTCCGGT', 'testmerge2'), 8)
# and an otu present only in one
self.assertEqual(table.get_value_by_ids(
'TACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGAGCGTAGGCGGTTTCTT'
'AAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGA'
'GTGCAGAAGAGGAGAGTGGAATTCCATGT', 'testmerge'), 7)
self.assertEqual(table.get_value_by_ids(
'TACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGAGCGTAGGCGGTTTCTTA'
'AGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGT'
'GCAGAAGAGGAGAGTGGAATTCCATGT', 'testmerge2'), 0)
# test the output fasta file
allseqs = []
for label, seq in sequence_generator(fasta_outfile):
self.assertTrue(seq in tableids)
allseqs.append(seq)
self.assertEqual(len(allseqs), len(tableids))
# test minimal read filtering ( minreads>0 )
minreads = 7
outfile2 = join(self.working_dir, 'testmerge2.biom')
create_otu_table(outfile2, [(m1, 'testmerge'), (m2, 'testmerge2')],
minreads=minreads)
table2 = load_table(outfile2)
table2ids = table2.ids(axis='observation')
tablesum = table.sum(axis='observation')
for idx, cid in enumerate(table.ids(axis='observation')):
if tablesum[idx] >= minreads:
self.assertIn(cid, table2ids)
else:
self.assertNotIn(cid, table2ids)
self.assertEqual(table2.get_value_by_ids(
'TACGAGGGGGGCGAGCGTTGTTCG'
'GAATTATTGGGCGTAAAAGGTGCGTAGGCGGTTCGGTAAGTTTCGTGTGAAATCTTCGGG'
'CTCAACTCGAAGCCTGCACGAAATACTGCCGGGCTTGAGTGTGGGAGAGGTGAGTGGAAT'
'TTCCGGT', 'testmerge2'), 8)
# and an otu present only in one
self.assertEqual(table2.get_value_by_ids(
'TACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGAGCGTAGGCGGTTTCTT'
'AAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGA'
'GTGCAGAAGAGGAGAGTGGAATTCCATGT', 'testmerge'), 7)
def test_create_otu_table_same_sampleid(self):
# merge the fasta files
m1 = join(self.test_data_dir,
'testmerge.fasta.trim.derep.no_artifacts'
'.msa.deblur.no_chimeras')
m2 = join(self.test_data_dir,
'testmerge2.fasta.trim.derep.no_artifacts'
'.msa.deblur.no_chimeras')
outfile = join(self.working_dir, 'testmerge.biom')
with self.assertWarns(UserWarning):
create_otu_table(outfile, [(m1, 'testmerge'), (m2, 'testmerge')])
def test_launch_workflow(self):
"""Test launching complete workflow using 3 simulated sequence files.
seqs1 - 100 reads using art, original sequences are >0.5 identical.
seqs2 - 200 reads using grinder, original sequences are >0.9 identical,
0.1 chimeras, 35 phix reads
seqs3 - simple - 15 reads from seqs1 (10 reads for 1001203,
5 reads for 694276) for manual test validation
"""
# index the 70% rep. set database
ref_fp = join(self.test_data_dir, '70_otus.fasta')
ref_db_fp = build_index_sortmerna(
ref_fp=(ref_fp,),
working_dir=self.working_dir)
self.run_workflow_try(self.seqs_s1_fp,
self.orig_s1_fp, ref_fp, ref_db_fp)
self.run_workflow_try(self.seqs_s2_fp,
self.orig_s2_fp, ref_fp, ref_db_fp)
self.run_workflow_try(self.seqs_s3_fp,
self.orig_s3_fp, ref_fp, ref_db_fp)
self.run_workflow_try(self.seqs_s3_fp,
self.orig_s3_fp, ref_fp, ref_db_fp,
threads=3)
def test_launch_workflow_skip_trim(self):
# index the 70% rep. set database
ref_fp = join(self.test_data_dir, '70_otus.fasta')
ref_db_fp = build_index_sortmerna(
ref_fp=(ref_fp,),
working_dir=self.working_dir)
seqs_fp = self.seqs_s1_fp
output_fp = self.working_dir
mean_error = 0.005
error_dist = get_default_error_profile()
indel_prob = 0.01
indel_max = 3
min_size = 2
# trim length longer than sequences
trim_length = -1
left_trim_length = 0
threads = 1
output_fp = launch_workflow(seqs_fp=seqs_fp,
working_dir=output_fp,
mean_error=mean_error,
error_dist=error_dist,
indel_prob=indel_prob,
indel_max=indel_max,
trim_length=trim_length,
left_trim_length=left_trim_length,
min_size=min_size,
ref_fp=(ref_fp,),
ref_db_fp=ref_db_fp,
threads_per_sample=threads)
exp = Sequence.read(self.no_trim_res, format='fasta')
res = Sequence.read(output_fp, format='fasta')
self.assertEqual(exp, res)
def test_launch_workflow_incorrect_trim(self):
"""
test if we get the warning when trim length
is too long
"""
# index the 70% rep. set database
ref_fp = join(self.test_data_dir, '70_otus.fasta')
ref_db_fp = build_index_sortmerna(
ref_fp=(ref_fp,),
working_dir=self.working_dir)
seqs_fp = self.seqs_s1_fp
output_fp = self.working_dir
mean_error = 0.005
error_dist = get_default_error_profile()
indel_prob = 0.01
indel_max = 3
min_size = 2
# trim length longer than sequences
trim_length = 151
left_trim_length = 0
threads = 1
with self.assertWarns(UserWarning):
launch_workflow(seqs_fp=seqs_fp, working_dir=output_fp,
mean_error=mean_error,
error_dist=error_dist,
indel_prob=indel_prob,
indel_max=indel_max,
trim_length=trim_length,
left_trim_length=left_trim_length,
min_size=min_size,
ref_fp=(ref_fp,),
ref_db_fp=ref_db_fp,
threads_per_sample=threads)
def get_seqs_act_split_sequence_on_sample_ids(self, output_dir):
"""Parse output of split_sequence_file_on_sample_ids_to_files()
Parameters
----------
output_dir: string
output directory path storing FASTA files
Returns
-------
seqs_act: dict
dictionary with keys being sample IDs and values list of
sequences belonging to sample ID
"""
seqs_act = {}
for fn in listdir(output_dir):
input_fp = join(output_dir, fn)
sample_file = splitext(fn)[0]
for label, seq in sequence_generator(input_fp):
sample = label.split('_')[0]
self.assertEqual(sample_file, sample)
if sample not in seqs_act:
seqs_act[sample] = [(label, seq)]
else:
seqs_act[sample].append((label, seq))
return seqs_act
def test_sample_id_from_read_id(self):
"""Test the fasta readid to sample id
used in split_sequence_file_on_sample_ids_to_files
"""
self.assertEqual(sample_id_from_read_id("Samp1_0 M04771:27:000000000"
"-ARFWH:1:1101:18081:1897 1:"
"N:0:0 orig_bc=CGTTAAGTCAGC n"
"ew_bc=CGTTAAGTCAGC bc_diffs="
"0"), "Samp1")
self.assertEqual(sample_id_from_read_id("S1_1_0 M04771:27:000000000"
"-ARFWH:1:1101:18081:1897 1:"
"N:0:0 orig_bc=CGTTAAGTCAGC n"
"ew_bc=CGTTAAGTCAGC bc_diffs="
"0"), "S1_1")
def test_split_sequence_file_on_sample_ids_to_files(self):
"""Test functionality of split_sequence_file_on_sample_ids_to_files()
"""
seqs_fasta = {"s1": [
("s1_seq1",
"TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("s1_seq2",
"TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG")],
"s2": [
("s2_seq3",
"TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCG"),
("s2_seq4",
"TACCGGCAGCTCAAGTGATGACCGCTATTATTGGGCCTAAAGCGTCCT")],
"s3": [
("s3_seq5",
"TACCAGCCCCTTAAGTGGTAGGGACGATTATTTGGCCTAAAGCGTCCG"),
("s3_seq6",
"CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT")],
"s4": [
("s4_seq7",
"CTGCAAGGCTAGGGGGCGGGAGAGGCGGGTGGTACTTGAGGGGAGAAT")]}
# Test FASTA file split on sample IDs to multiple FASTA files
seqs_fp = join(self.working_dir, "seqs.fasta")
with open(seqs_fp, 'w') as seqs_f:
for sample in seqs_fasta:
for seq in seqs_fasta[sample]:
seqs_f.write(">%s\n%s\n" % seq)
output_dir = mkdtemp()
split_sequence_file_on_sample_ids_to_files(seqs=seqs_fp,
outdir=output_dir)
seqs_act = self.get_seqs_act_split_sequence_on_sample_ids(
output_dir=output_dir)
self.assertEqual(seqs_fasta, seqs_act)
def test_fasta_from_biom(self):
'''Test the fasta file from biom table function fasta_from_biom()
'''
input_biom_file = join(self.test_data_dir, 'final.s4.biom')
table = load_table(input_biom_file)
output_fasta = join(self.working_dir, 'final.s4.seqs.fa')
fasta_from_biom(table, output_fasta)
self.assertTrue(isfile(output_fasta))
table_seqs = table.ids(axis='observation')
expected = [(seq, seq) for seq in table_seqs]
written_seqs = [item for item in sequence_generator(output_fasta)]
self.assertListEqual(written_seqs, expected)
if __name__ == '__main__':
main()