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single_quant_toolbox.py
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# coding=utf-8
# -*- coding: utf-8 -*-
import os
import subprocess
import shlex
import pandas as pd
import argparse
from multiprocessing import Pool
__author__ = 'gdq'
def run_cmd(cmd):
# subprocess.check_call should not be used
# for cannot terminate it when error happens during multiprocessing.
subprocess.call(cmd)
class QuantExpToolbox(object):
"""
A toolbox contains several expression quantification tools for RNAseq data.
salmon: only quasi-mapping-based mode of Salmon used here.
http://salmon.readthedocs.io/en/latest/salmon.html
fastq：a ";" separated string with 3 columns, but 3th column is optional.
------------------------------------
sample1;lfq,lfq2,lfq3;rfq,rfq2,rfq3
------------------------------------
"""
def __init__(self, transcriptome, fastq, method='salmon', libtype=None,
transcript2gene=None, pool=6, threads=10, output=None,
salmon=None, rsem=None, kallisto=None,
read_len=149, read_len_sd=30,
map_tool='bowtie2', map_tool_path=None, samtools=None,
):
self.method = method
self.transcriptome = transcriptome
self.output = os.getcwd() if output is None else output
self.t2g = transcript2gene
self.salmon = salmon
self.rsem = rsem
self.kallisto = kallisto
self.pool = pool
self.threads = threads
self.fastq = self.parse_fastq(fastq)
self.libtype = libtype
self.read_len = read_len
self.read_len_sd = read_len_sd
self.map_tool = map_tool
self.map_tool_path = map_tool_path
if samtools is None:
self.samtools = "samtools"
else:
self.samtools = samtools
@staticmethod
def parse_fastq(fastq):
fastq_info = dict()
sample_fastqs_list = fastq.strip().split()
for each_sample in sample_fastqs_list:
fastq_detail = each_sample.split(';')
sample = fastq_detail[0]
fastq_info[sample] = list()
for each in fastq_detail[1:]:
fastqs = each.split(',')
fastq_info[sample].append(fastqs)
return fastq_info
def build_index(self, kmer=31):
# if os.path.exists('{}/transcripts_index '.format(self.output)):
# if not len(os.listdir('{}/transcripts_index '.format(self.output))) >= 2:
# pass
# else:
# print('Index files seems to be existed in transcripts_index, we will not build index again.')
# return
if self.method.lower() == 'salmon':
cmd = '{}/salmon index '.format(self.salmon)
cmd += '-t {} '.format(self.transcriptome)
cmd += '-i {}/transcripts_index '.format(self.output)
cmd += '-k {}'.format(kmer)
print(cmd)
subprocess.check_call(shlex.split(cmd))
elif self.method.lower() == 'kallisto':
cmd = '{}/kallisto index '.format(self.kallisto)
cmd += '-i {}/transcripts_index '.format(self.output)
cmd += '-k {} '.format(kmer)
cmd += self.transcriptome
print(cmd)
subprocess.check_call(shlex.split(cmd))
elif self.method.lower() == 'rsem':
if self.map_tool not in ['bowtie', 'bowtie2', 'star']:
raise Exception('{} is not supported for rsem'.format(self.map_tool))
cmd = '{}/rsem-prepare-reference '.format(self.rsem)
if self.t2g is not None:
t2g = pd.read_table(self.t2g, header=None, index_col=None)
g2t = t2g.iloc[:, [1, 0]]
g2t_path = os.path.join(self.output, 'g2t.pair')
g2t.to_csv(g2t_path, index=False, sep='\t', header=False)
cmd += "--transcript-to-gene-map {} ".format(g2t_path)
cmd += "--{} ".format(self.map_tool)
if self.map_tool_path is not None:
cmd += "--{}-path {} ".format(self.map_tool, self.map_tool_path)
cmd += '-p {} '.format(self.threads)
cmd += '{} '.format(self.transcriptome)
index_path = '{}/transcripts_index'.format(self.output)
if not os.path.exists(index_path):
os.mkdir(index_path)
cmd += '{}/ref_index'.format(index_path)
print(cmd)
subprocess.check_call(shlex.split(cmd))
else:
raise Exception('unexpected method: ' + self.method)
def get_salmon_cmd(self):
"""
fastq: {sample name: [[fq1,fq2],[fq1,fq2]], }
:return: cmd list
"""
cmd_list = list()
for sample in self.fastq:
fq_list = self.fastq[sample]
cmd = '{}/salmon quant '.format(self.salmon)
cmd += '-i {}/transcripts_index '.format(self.output)
cmd += '-l A '
if len(fq_list) == 2:
cmd += '-1 {} -2 {} '.format(' '.join(fq_list[0]), ' '.join(fq_list[1]))
else:
cmd += '-r {} '.format(' '.join(fq_list[0]))
cmd += '-o {}/{}_quant '.format(self.output, sample)
cmd += '--gcBias '
cmd += '-p {} '.format(self.threads)
if self.t2g is not None:
cmd += ' -g ' + self.t2g
cmd_list.append(shlex.split(cmd))
else:
return cmd_list
def get_kallisto_cmd(self):
cmd_list = list()
for sample in self.fastq:
fq_list = self.fastq[sample]
cmd = '{}/kallisto quant '.format(self.kallisto)
cmd += '-i {}/transcripts_index '.format(self.output)
cmd += '-o {}/{}_quant '.format(self.output, sample)
# cmd += '--plaintext '
if len(fq_list) == 1:
cmd += '--single '
cmd += '-l {} '.format(self.read_len)
cmd += '-s {} '.format(self.read_len_sd)
if self.libtype is not None:
if self.libtype == 'fr':
cmd += '--fr-stranded '
elif self.libtype == 'rf':
cmd += '--rf-stranded '
else:
print('the library type {} is invalid and will be ignored'.format(self.libtype))
# raise Exception('library type can only be either "fr" or "fr"')
pass
cmd += '-t {} '.format(self.threads)
if len(fq_list) == 1:
cmd += ' '.join(fq_list[0])
else:
fq_list = list(zip(fq_list[0], fq_list[1]))
fq_list = [x for y in fq_list for x in y]
cmd += ' '.join(fq_list)
cmd_list.append(shlex.split(cmd))
else:
return cmd_list
def get_rsem_cmd(self):
cmd_list = list()
for sample in self.fastq:
fq_list = self.fastq[sample]
cmd = '{}/rsem-calculate-expression '.format(self.rsem)
cmd += '-p {} '.format(self.threads)
if self.libtype is not None:
if self.libtype == 'fr':
cmd += '--forward-prob 1 '
elif self.libtype == 'rf':
cmd += '--forward-prob 0 '
cmd += '--sort-bam-memory-per-thread 2G '
cmd += '--estimate-rspd '
cmd += '--ci-memory 2048 '
cmd += "--{} ".format(self.map_tool)
if self.map_tool_path is not None:
cmd += "--{}-path {} ".format(self.map_tool, self.map_tool_path)
if len(fq_list) == 1:
cmd += "--fragment-length-mean {} ".format(self.read_len)
cmd += "--fragment-length-sd {} ".format(self.read_len_sd)
cmd += "{} ".format(','.join(fq_list[0]))
else:
cmd += '--paired-end '
cmd += '{} {} '.format(','.join(fq_list[0]), ','.join(fq_list[1]))
cmd += "{} ".format('{}/transcripts_index/ref_index'.format(self.output))
out_path = '{}/{}_quant'.format(self.output, sample)
if not os.path.exists(out_path):
os.mkdir(out_path)
cmd += "{}/{}".format(out_path, sample)
cmd_list.append(shlex.split(cmd))
else:
return cmd_list
def run_quant(self):
if self.method.lower() == 'salmon':
cmd_list = self.get_salmon_cmd()
elif self.method.lower() == 'kallisto':
cmd_list = self.get_kallisto_cmd()
elif self.method.lower() == 'rsem':
cmd_list = self.get_rsem_cmd()
else:
raise Exception(self.method + ' is not supported')
if len(self.fastq) <= self.pool:
pool = Pool(len(self.fastq))
else:
pool = Pool(self.pool)
pool.map(run_cmd, cmd_list)
pool.close()
pool.join()
if len(cmd_list) >= 2:
self.generate_exp_table()
def run_sort_bam(self):
if self.method.lower() == 'rsem':
samples = sorted(self.fastq.keys())
bam_list = [os.path.join(self.output, x + '_quant', x + '.transcript.bam') for x in samples]
cmd_list = list()
sorted_bam_list = list()
for each in bam_list:
out_file = each[:-4] + '.sorted.bam'
sorted_bam_list.append(out_file)
cmd = '{} sort --threads {} -o {} {}'.format(self.samtools, self.threads, out_file, each)
cmd_list.append(shlex.split(cmd))
bam_list_file = os.path.join(self.output, 'bam.list')
print(cmd_list)
with open(bam_list_file, 'w') as f:
for each in sorted_bam_list:
f.write(each + '\n')
if len(self.fastq) <= self.pool:
pool = Pool(len(self.fastq))
else:
pool = Pool(self.pool)
pool.map(run_cmd, cmd_list)
pool.close()
pool.join()
def generate_exp_table(self):
def merge_file(results, target_cols, new_col_names, out):
# target_cols = ['TPM', 'count']
# new_col_names = ['tpm', 'count']
for each_col, new_name in zip(target_cols, new_col_names):
column_list = list()
for sample, quant in results:
tmp_col = pd.read_table(quant, index_col=0, header=0)[each_col]
# tmp_col.name = sample + '_' + new_name
tmp_col.name = sample
tmp_col.index.name = 'seq_id'
column_list.append(tmp_col)
result_table = pd.concat(column_list, axis=1)
result_table.to_csv(out+'.'+new_name+'.matrix', sep='\t')
samples = sorted(self.fastq.keys())
join = os.path.join
if self.method.lower() == 'salmon':
isoform_results = [join(self.output, x+'_quant', 'quant.sf') for x in samples]
gene_results = [join(self.output, x+'_quant', 'quant.genes.sf') for x in samples]
merge_file(zip(samples, isoform_results), ['TPM', 'NumReads'], ['tpm', 'count'],
join(self.output, 'transcript'))
merge_file(zip(samples, gene_results), ['TPM', 'NumReads'], ['tpm', 'count'],
join(self.output, 'gene'))
elif self.method.lower() == 'rsem':
iso_results = [join(self.output, x+'_quant', x+'.isoforms.results') for x in samples]
gene_results = [join(self.output, x+'_quant', x+'.genes.results') for x in samples]
merge_file(zip(samples, iso_results), ['TPM', 'expected_count'], ['tpm', 'count'],
join(self.output, 'transcript'))
merge_file(zip(samples, gene_results), ['TPM', 'expected_count'], ['tpm', 'count'],
join(self.output, 'gene'))
# get mapping rate
cnt_files = [join(self.output, x+'_quant', x+'.stat', x+'.cnt') for x in samples]
with open(join(self.output, 'alignment_rate.txt'), 'w') as f:
f.write('sample\ttotal_reads\taligned_reads\taligned_rate\n')
for s, each in zip(samples, cnt_files):
with open(each) as f2:
# ['un-alignable', 'alignable', 'too_many_align', 'total']
tmp_list = f2.readline().strip('\n').split()
map_num = int(tmp_list[1]) + int(tmp_list[2])
map_rate = float(map_num)/int(tmp_list[3])
f.write('{}\t{}\t{}\t{}\n'.format(s, tmp_list[3], map_num, map_rate))
elif self.method.lower() == 'kallisto':
isoform_results = [join(self.output, x+'_quant', 'abundance.tsv') for x in samples]
merge_file(zip(samples, isoform_results), ['tpm', 'est_counts'], ['tpm', 'count'],
join(self.output, 'transcript'))
# kallisto do not generate gene expression table, thus t2g file will be needed.
t2g_pd = pd.read_table(self.t2g, index_col=0, header=None, usecols=[0, 1], names=['seq_id', 'gene'])
gene_exp_list = list()
gene_count_list = list()
for sample, result in zip(samples, isoform_results):
iso_table = pd.read_table(result, index_col=0, header=0)
iso_table = pd.concat([iso_table, t2g_pd], axis=1)
tmp_table = iso_table['est_counts'].groupby(iso_table['gene']).sum()
tmp_table.name = sample
tmp_table.index.name = 'seq_id'
gene_count_list.append(tmp_table)
# exp
tmp_table = iso_table['tpm'].groupby(iso_table['gene']).sum()
tmp_table.name = sample
tmp_table.index.name = 'seq_id'
gene_exp_list.append(tmp_table)
gene_counts = pd.concat(gene_count_list, axis=1)
gene_counts.to_csv(os.path.join(self.output, 'gene.count.matrix'), sep='\t')
gene_exp = pd.concat(gene_exp_list, axis=1)
gene_exp.to_csv(os.path.join(self.output, 'gene.tpm.matrix'), sep='\t')
else:
pass
if __name__ == "__main__":
parser = argparse.ArgumentParser()
parser.add_argument('-t', type=str, metavar="transcript", required=True,
help="transcripts fasta file")
parser.add_argument('-fq', type=str, metavar="fastq", required=True,
help="str contains the fastq path info, such as: "
"'sample1;lfq,lfq2,lfq3;rfq,rfq2,rfq3 sample2;...'")
parser.add_argument('-m', type=str, metavar="method", default='salmon',
help="salmon[default], kallisto and rsem are supported.")
parser.add_argument('-o', type=str, metavar="out_dir", default=None,
help="Output directory. Default: current working directory.")
parser.add_argument('-t2g', type=str, metavar="transcript2gene",
help="transcript-->gene, two column: Transcript\tGene. "
"But, Salmon also supports gtf or gff input", )
parser.add_argument('-strand', type=str, metavar="library_type", default=None,
help="'fr' or 'rf'. Default: None. Strand-specific protocol type.")
parser.add_argument('-pool', type=int, metavar="pool_size", default=6,
help="Process number for batch analysis of many samples. Default: 6")
parser.add_argument('-thread', type=str, metavar="thread_number", default=10,
help="Threads number for analysis of every single sample.")
parser.add_argument('-salmon', type=str,
default="/mnt/ilustre/users/sanger-dev/app/bioinfo/rna/Salmon-0.8"
".2_linux_x86_64/bin/",
help="where is salmon installed")
parser.add_argument('-rsem', type=str,
default="/mnt/ilustre/users/sanger-dev/app/bioinfo/rna/RSEM-1.2.31/bin",
help="where is rsem installed")
parser.add_argument('-kallisto', type=str,
default="/mnt/ilustre/users/sanger-dev/app/bioinfo/rna/kallisto_linux-v0"
".43.1/",
help="where is kallisto installed")
parser.add_argument('-rl', type=int, metavar="read_length", default=149,
help="Only used for single-end mode of kallisto/rsem. "
"Estimated average fragment length. Default: 149")
parser.add_argument('-sd', type=int, metavar="read_length_sd", default=30,
help="Only used for single-end mode of kallisto/rsem. "
"Use '-sd' along with '-rl. "
"Estimated standard deviation of fragment length. Default: 30")
parser.add_argument('-mapper', type=str, metavar="align_tool", default='bowtie2',
help='RSEM argument. Only bowtie, bowtie2 and star are supported. '
'Default: bowtie2')
parser.add_argument('-mapper_path', type=str, metavar="mapper_path",
default='/mnt/ilustre/users/sanger-dev/app/bioinfo/align/bowtie2-2.2.9/',
help='RSEM argument. Absolute path of alignment tool/mapper')
args = parser.parse_args()
toolbox = QuantExpToolbox(transcriptome=args.t,
fastq=args.fq,
transcript2gene=args.t2g,
method=args.m,
output=args.o,
pool=args.pool,
threads=args.thread,
libtype=args.strand,
salmon=args.salmon,
kallisto=args.kallisto,
rsem=args.rsem,
read_len=args.rl,
read_len_sd=args.sd,
map_tool=args.mapper,
map_tool_path=args.mapper_path,
samtools=None,
)
toolbox.build_index()
toolbox.run_quant()
toolbox.run_sort_bam()