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#! /usr/bin/env python
# -*- coding: utf-8 -*-
"""
Read qc utilities
Created: Jul 24 2013
sulsj
"""
import os
import subprocess
import matplotlib
import numpy as np
from common import checkpoint_step
from os_utility import make_dir, change_mod, run_sh_command, rm_dir
from readqc_constants import RQCReadQcConfig, RQCContamDb, RQCReadQcReferenceDatabases, RQCReadQc
from rqc_utility import safe_basename, get_cat_cmd, localize_file, safe_dirname
from rqc_constants import RQCExitCodes
matplotlib.use("Agg") ## This needs to skip the DISPLAY env var checking
import matplotlib.pyplot as plt
from matplotlib.font_manager import FontProperties
import mpld3
from matplotlib.ticker import MultipleLocator, FormatStrFormatter
""" STEP1 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
fast_subsample_fastq_sequences
Title : fast_subsample_fastq_sequences
Function : This function subsamples the data from a specified fastq file.
Usage : fast_subsample_fastq_sequences( $seq_unit, subsampledFile, $subsamplingRate, $max_count, \$totalBaseCount, $totalReadNum, log)
Args : 1) The source fastq file.
2) The destination fastq file.
3) The percentage of read subsampling.
4) The maximum number of reads at which to stop subsampling.
5) A reference to the variable that will store the
basecount.
6) A reference to the variable that will store the
number of reads.
7) A reference to a JGI_Log object.
Returns : JGI_SUCCESS: The fastq data was successfully sampled.
JGI_FAILURE: The fastq data could not be sampled.
Comments : Pass as parameters both the subsample_rate and the read_count
in order to stop subsampling at the read_count.
The read_count can also be null, in which case the number
of reads corresponding to the percentage subsample_rate will be subsampled.
@param fastq: source fastq file (full path)
@param outputFileName: subsampled output file name (basename)
@param subsamplingRate: sample rate < 1.0
@param isStatGenOnly: boolean -> generate stats output or not
@param log
@return retCode: success or failure
@return subsampledFile: subsampled output file name (full path)
@return totalBaseCount: total #bases (to be added to readqc_stats.txt)
@return totalReadNum: total #reads (to be added to readqc_stats.txt)
@return subsampledReadNum: total #reads sampled (to be added to readqc_stats.txt)
"""
def fast_subsample_fastq_sequences(sourceFastq, outputFileName, subsamplingRate, isStatGenOnly, log):
## Tools
cdir = os.path.dirname(__file__)
bbtoolsReformatShCmd = os.path.join(cdir, '../../reformat.sh') #RQCReadQcCommands.BBTOOLS_REFORMAT_CMD
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
log.info("Sampling %s at %.2f rate", sourceFastq, subsamplingRate)
retCode = None
totalBaseCount = 0
totalReadNum = 0
subsampledReadNum = 0
bIsPaired = False
readLength = 0
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
make_dir(subsamplePath)
change_mod(subsamplePath, "0755")
subsampledFile = os.path.join(subsamplePath, outputFileName)
fileSize = os.path.getsize(sourceFastq)
log.info("Source fastq file size = %s", fileSize)
## Replaced subsampler with reformat.sh
## subsample with bbtoolsReformatShCmd:
## $ reformat.sh in=7348.8.68143.fastq out=subsample.fastq samplerate=0.01 qout=33
## - 21G == 180.399 seconds ~ 6x faster than subsample_fastq_pl
## new subampler from BBTOOLS
## without qin=33 then it uses auto detect, Illumina is phread64 but we need to convert to phred33
##reformat.sh in=7257.1.64419.CACATTGTGAG.s1.0.fastq out=temp.out samplerate=0.02 qin=33 qout=33 overwrite
## 20140820
## bhist=<file> Write a base composition histogram to file. ## Cycle Nucleotide Composition
## gchist=<file> Write a gc content histogram to file. ## Read GC, mean, std
## qhist=<file> Write a quality histogram to file. ## Average Base Position Quality
## bqhist=<file> Write a quality histogram designed for box plots. ## Average Base Position Quality Boxplot
## obqhist=<file> Write a base quality histogram to file. ## Base quality histogram; *.base_qual.stats
reformatPrefix = os.path.basename(subsampledFile).replace(".fastq", "")
reformatLogFile = os.path.join(subsamplePath, reformatPrefix + ".reformat.log")
reformatGchistFile = os.path.join(subsamplePath, reformatPrefix + ".reformat.gchist.txt") ## Read GC
reformatBhistFile = os.path.join(subsamplePath, reformatPrefix + ".reformat.bhist.txt") ## Cycle Nucleotide Composition
reformatQhistFile = os.path.join(subsamplePath, reformatPrefix + ".reformat.qhist.txt") ## Average Base Position Quality
reformatBqhistFile = os.path.join(subsamplePath, reformatPrefix + ".reformat.bqhist.txt") ## Average Base Position Quality Boxplot
reformatObqhistFile = os.path.join(subsamplePath, reformatPrefix + ".reformat.obqhist.txt") ## Base quality histogram
if not isStatGenOnly: ## if subsampling for blast, do not generate the stat files
subsampleCmd = "%s in=%s out=%s samplerate=%s qin=33 qout=33 ow=t > %s 2>&1 " % \
(bbtoolsReformatShCmd, sourceFastq, subsampledFile, subsamplingRate, reformatLogFile)
else:
subsampleCmd = "%s in=%s out=%s samplerate=%s qin=33 qout=33 ow=t gcplot=t bhist=%s qhist=%s gchist=%s gcbins=auto bqhist=%s obqhist=%s > %s 2>&1 " % \
(bbtoolsReformatShCmd, sourceFastq, subsampledFile, subsamplingRate, reformatBhistFile,
reformatQhistFile, reformatGchistFile, reformatBqhistFile, reformatObqhistFile, reformatLogFile)
_, _, exitCode = run_sh_command(subsampleCmd, True, log, True)
if exitCode == 0:
##java -ea -Xmx200m -cp /usr/common/jgi/utilities/bbtools/prod-33.18/lib/BBTools.jar jgi.ReformatReads in=7257.1.64419.CACATTGTGAG.s1.0.fastq out=temp.out samplerate=0.02 qin=33 qout=33 overwrite
##Executing jgi.ReformatReads [in=7257.1.64419.CACATTGTGAG.s1.0.fastq, out=temp.out, samplerate=0.02, qin=33, qout=33, overwrite]
##
##Unspecified format for output temp.out; defaulting to fastq.
##Input is being processed as paired
##Writing interleaved.
##Input: 6661 reads 1671911 bases
##Processed: 278 reads 69778 bases
##Output: 278 reads (4.17%) 69778 bases (4.17%)
##
##Time: 0.181 seconds.
##Reads Processed: 278 1.54k reads/sec
##Bases Processed: 69778 0.39m bases/sec
## NEW
if os.path.isfile(reformatLogFile):
with open(reformatLogFile) as STAT_FH:
for l in STAT_FH.readlines():
if l.startswith("Input:"):
toks = l.split()
totalBaseCount = int(toks[3])
totalReadNum = int(toks[1])
# elif l.startswith("Processed:") or l.startswith("Output:"):
elif l.startswith("Output:"):
toks = l.split()
subsampledReadNum = int(toks[1])
elif l.startswith("Input is being processed as"):
toks = l.split()
if toks[-1].strip() == "paired":
bIsPaired = True
log.info("Total base count of input fastq = %s", totalBaseCount)
log.info("Total num reads of input fastq = %s", totalReadNum)
log.info("Total num reads of sampled = %s", subsampledReadNum)
readLength = int(totalBaseCount / totalReadNum)
log.info("Read length = %d", readLength)
log.info("Paired = %s", bIsPaired)
if totalReadNum > 0 and subsampledReadNum > 0:
##
## TODO: deal with sequnits with small number of reads
## How to record the status in readqc.log
##
retCode = RQCExitCodes.JGI_SUCCESS
log.info("illumina_readqc_subsampling complete: output file = %s", subsampledFile)
elif totalReadNum > 0 and subsampledReadNum <= 0:
retCode = RQCExitCodes.JGI_FAILURE
log.error("illumina_readqc_subsampling failure. subsampledReadNum <= 0.")
else:
retCode = RQCExitCodes.JGI_FAILURE
log.error("illumina_readqc_subsampling failure. totalReadNum <= 0 and subsampledReadNum <= 0.")
else:
retCode = RQCExitCodes.JGI_FAILURE
log.error("illumina_readqc_subsampling failure. Can't find stat file from subsampling.")
else:
retCode = RQCExitCodes.JGI_FAILURE
log.error("illumina_readqc_subsampling failure. Failed to run bbtoolsReformatShCmd. Exit code != 0")
with open(reformatLogFile, 'r') as f:
log.error(f.read())
retCode = -2
return retCode, subsampledFile, totalBaseCount, totalReadNum, subsampledReadNum, bIsPaired, readLength
""" STEP2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
write_unique_20_mers
Title: write_unique_k_mers (k=20 or 25)
Function: Given a fastq file, finds unique 20/25 mers from the start
of the read and along a random position of the read
Usage: write_unique_20_mers(\@seq_files, $log)
Args: 1) ref to an array containing bz2 zipped fastq file path(s)
2) output directory
3) log file object
Returns: exitCode, merSamplerOutFile, pngPlotFile, htmlPlotFile
Comments: Using bbcountunique.sh's output file named merSampler.<fastq_name>.m20.e25000
create a plot png file, merSampler.<fastq_name>.m20.e25000.png
bbcountunique.sh: Generates a kmer uniqueness histogram, binned by file position.
There are 3 columns for single reads, 6 columns for paired:
count number of reads or pairs processed
r1_first percent unique 1st kmer of read 1
r1_rand percent unique random kmer of read 1
r2_first percent unique 1st kmer of read 2
r2_rand percent unique random kmer of read 2
pair percent unique concatenated kmer from read 1 and 2
@param fastq: source fastq file (full path)
@param log
@return retCode: success or failure
@return mersampler_out_file: plot data (to be added to readqc_files.txt)
@return pngPlotFile: output plot (to be added to readqc_files.txt)
@return htmlPlotFile: output d3 interactive plot (to be added to readqc_files.txt)
"""
def write_unique_20_mers(fastq, log):
## Tools
cdir = os.path.dirname(__file__)
bbcountuniqueShCmd = os.path.join(cdir, '../../bbcountunique.sh') #RQCReadQcCommands.BBCOUNTUNIQUE_SH_CMD
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
uniqMerDir = "uniqueness"
uniqMerPath = os.path.join(READ_OUTPUT_PATH, uniqMerDir)
make_dir(uniqMerPath)
change_mod(uniqMerPath, "0755")
## cmd line for merSampler
uniqMerSize = RQCReadQc.ILLUMINA_MER_SAMPLE_MER_SIZE ## 20 ==> 25 RQC-823 08102016
reportFreq = RQCReadQc.ILLUMINA_MER_SAMPLE_REPORT_FRQ ## 25000
sequnitFileName, exitCode = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
##~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
## bbcountunique.sh, a new version of sampler from bbtools
## bbcountunique.sh in=$FILENAME out=out.txt percent=t count=t cumulative=t
## ex) bbcountunique.sh in=$SEQDIR/dna/$SEQFILE.fastq.gz out=7378.1.69281.CGATG-2.txt percent=t count=t cumulative=f int=f
## ex2)
## cmd: bbcountunique.sh k=20 interval=25000 in=7601.1.77813.CTTGTA.fastq.gz out=7601.1.77813.CTTGTA.merSampler.m20.e25000_2 percent=t count=t cumulative=f int=f
log.info("bbcountunique.sh started.")
merSamplerOutFile = os.path.join(uniqMerPath, sequnitFileNamePrefix + ".merSampler.m" + str(uniqMerSize) + ".e" + str(reportFreq) + "_2")
## RQC-823
## Adding shuffling before bbcountunique
## 08302016 Reverted to no shuffling
##
## shuffle.sh in=input.fastq.gz out=stdout.fq -Xmx40g | bbcountunique.sh in=stdin.fq -Xmx40g ==> not working
# shuffledFastqFile = os.path.join(uniqMerPath, sequnitFileNamePrefix + ".shuffled.fq")
# suffleCmd = "%s in=%s out=%s" % (shuffleShCmd, fastq, shuffledFastqFile)
# stdOut, stdErr, exitCode = run_sh_command(suffleCmd, True, log, True)
# if exitCode != 0:
# log.error("failed to suffle fastq for unique mer analysis.")
# return RQCExitCodes.JGI_FAILURE, None, None, None
bbcountuniqCmd = "%s in=%s out=%s k=%s interval=%s percent=t count=t cumulative=f int=f ow=t" \
% (bbcountuniqueShCmd, fastq, merSamplerOutFile, uniqMerSize, reportFreq)
_, _, exitCode = run_sh_command(bbcountuniqCmd, True, log, True)
if exitCode != 0:
log.error("Failed to sample unique %s mers by bbcountunique.sh.", uniqMerSize)
return RQCExitCodes.JGI_FAILURE, None, None, None
log.info("bbcountunique.sh completed.")
## Old plotting data
# nSeq nStartUniMer fracStartUniMer nRandUniMer fracRandUniMer
## 0 1 2 3 4
##25000 2500 0.1 9704 0.3882
## New plotting data from bbcountunique
# count first rand first_cnt rand_cnt
# 0 1 2 3 4
# 25000 66.400 76.088 16600 19022
# 50000 52.148 59.480 13037 14870
# 75000 46.592 53.444 11648 13361
# 100000 43.072 49.184 10768 12296 ...
pngPlotFile = None
htmlPlotFile = None
if os.path.isfile(merSamplerOutFile):
## sanity check
## OLD
## #nSeq nStartUniMer fracStartUniMer nRandUniMer fracRandUniMer
## ex) 25000 16594 0.6638 18986 0.7594
## 50000 29622 0.5211 33822 0.5934
## 75000 41263 0.4656 47228 0.5362
## 100000 52026 0.4305 59545 0.4927 ...
"""
2016-09-07
#count first rand first_cnt rand_cnt avg_quality perfect_prob
25000 96.480 98.636 24120 24659 30.36 80.94
50000 96.204 97.996 24051 24499 30.41 81.17
75000 95.512 97.568 23878 24392 29.99 80.06
100000 95.408 97.588 23852 24397 30.24 80.78
125000 95.176 97.240 23794 24310 30.23 80.86
"""
line = None
numData = 0
with open(merSamplerOutFile, "r") as FH:
lines = FH.readlines()
line = lines[-1] ## get the last line
numData = sum(1 for l in lines)
toks = line.split()
assert len(toks) == 7, "ERROR: merSamplerOutFile format error: %s " % (merSamplerOutFile)
if numData < 3:
log.error("Not enough data in merSamplerOutFile: %s", merSamplerOutFile)
return RQCExitCodes.JGI_SUCCESS, None, None, None
## Generating plots
rawDataMatrix = np.loadtxt(merSamplerOutFile, delimiter='\t', comments='#')
# Bryce: 2016-09-07, its 7 now. Its failed 622 pipelines ...
# assert len(rawDataMatrix[1][:]) == 5
fig, ax = plt.subplots()
markerSize = 5.0
lineWidth = 1.5
## Note: no need to show all the data points
## If the number of data points > 5k, get only 5k data.
jump = 1
if len(rawDataMatrix[:, 0]) > 10000:
jump = int(len(rawDataMatrix[:, 0]) / 5000)
xData = rawDataMatrix[:, 0][0::jump]
yData = rawDataMatrix[:, 1][0::jump]
yData2 = rawDataMatrix[:, 2][0::jump]
totalReadNum = rawDataMatrix[-1, 0] ## sampled read num from the last line of the data file
assert int(totalReadNum) > 0
maxX = int(totalReadNum) * 3
p1 = ax.plot(xData, yData, 'g', marker='x', markersize=markerSize, linewidth=lineWidth, label="Starting %s Mer Uniqueness" % (str(uniqMerSize)), alpha=0.5)
p2 = ax.plot(xData, yData2, 'b', marker='x', markersize=markerSize, linewidth=lineWidth, label="Random %s Mer Uniqueness" % (str(uniqMerSize)), alpha=0.5)
## Curve-fitting
from scipy.optimize import curve_fit
## fit function: f(x)=a*log(x)+b
def fit_func(x, a, b):
return a * np.log(x) + b
fitpars, _ = curve_fit(fit_func, rawDataMatrix[:, 0], rawDataMatrix[:, 1])
fix_x = [i for i in range(25000, maxX, 25000)]
ax.plot(fix_x, fit_func(fix_x, *fitpars), 'r', linewidth=lineWidth, label="fit", alpha=0.5)
ax.set_xlabel("Read Sampled", fontsize=12, alpha=0.5)
ax.set_ylabel("Percentage Unique", fontsize=12, alpha=0.5)
ax.spines["top"].set_visible(False)
ax.spines["right"].set_visible(False)
ax.get_xaxis().tick_bottom()
ax.get_yaxis().tick_left()
fontProp = FontProperties()
fontProp.set_size("small")
fontProp.set_family("Bitstream Vera Sans")
ax.legend(loc=1, prop=fontProp)
ax.set_xlim([0, maxX])
ax.set_ylim([0, 100])
ax.grid(color="gray", linestyle=':')
## Add tooltip
labels = ["%.2f" % i for i in rawDataMatrix[:, 1]]
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=labels))
labels = ["%.2f" % i for i in rawDataMatrix[:, 2]]
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p2[0], labels=labels))
## Create both dynamic and static plots
pngPlotFile = merSamplerOutFile + "_mer_sampler_plot.png"
plt.savefig(pngPlotFile, dpi=fig.dpi)
htmlPlotFile = merSamplerOutFile + "_mer_sampler_plot_d3.html"
mpld3.save_html(fig, htmlPlotFile)
log.info("New data file from bbcountunique: %s", merSamplerOutFile)
log.info("New png plot: %s", pngPlotFile)
log.info("New D3 plot: %s", htmlPlotFile)
else:
log.error("Cannot find merSamplerOutFile by bbcountunique.sh, %s", merSamplerOutFile)
return RQCExitCodes.JGI_FAILURE, None, None, None
##~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
# if os.path.isfile(merSamplerOutFile):
# line = None
# numData = 0
#
# with open(merSamplerOutFile, "r") as FH:
# line = FH.readlines()[-1] ## get the last line
# numData = len(line)
#
# ## #nSeq nStartUniMer fracStartUniMer nRandUniMer fracRandUniMer
# ## ex) 25000 16594 0.6638 18986 0.7594
# ## 50000 29622 0.5211 33822 0.5934
# ## 75000 41263 0.4656 47228 0.5362
# ## 100000 52026 0.4305 59545 0.4927 ...
# """
# 2016-09-07
# #count first rand first_cnt rand_cnt avg_quality perfect_prob
# 25000 96.480 98.636 24120 24659 30.36 80.94
# 50000 96.204 97.996 24051 24499 30.41 81.17
# 75000 95.512 97.568 23878 24392 29.99 80.06
# 100000 95.408 97.588 23852 24397 30.24 80.78
# 125000 95.176 97.240 23794 24310 30.23 80.86
# """
#
# toks = line.split()
# assert len(toks)==7, "ERROR: merSamplerOutFile format error: %s " % (merSamplerOutFile)
#
# # totalReadNum = int(toks[0])
# # log.info("Total number of reads = %s." % (totalReadNum))
# # assert totalReadNum > 0
#
# else:
# log.error("cannot find mersampler_out_file, %s" % (merSamplerOutFile))
# return RQCExitCodes.JGI_FAILURE, None, None, None
#
# if numData < 3:
# log.error("not enough data in %s" % (merSamplerOutFile))
# return RQCExitCodes.JGI_FAILURE, None, None, None
## verify that the mer sampler output file was created
# if not os.path.isfile(merSamplerOutFile):
# log.error("failed to find output file for %s Mer Uniqueness: %s" % (str(uniqMerSize), merSamplerOutFile))
# else:
# log.info("MerSampler output file successfully generated (%s)." % (merSamplerOutFile))
## verify that the mer sampler plot png file was created
if not os.path.isfile(pngPlotFile):
log.warning("Failed to find output plot png file for %s Mer Uniqueness", str(uniqMerSize))
else:
log.info("MerSampler output png file successfully generated (%s)", pngPlotFile)
if not os.path.isfile(htmlPlotFile):
log.warning("Failed to find output d3 plot html file for %s Mer Uniqueness", str(uniqMerSize))
else:
log.info("MerSampler output png file successfully generated (%s)", htmlPlotFile)
return RQCExitCodes.JGI_SUCCESS, merSamplerOutFile, pngPlotFile, htmlPlotFile
""" STEP3 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_read_gc
Title: illumina_read_gc
Function: Takes path to fastq file and generates
read gc histograms (txt and png)
Usage: illumina_read_gc($fastq_path, $log)
Args: 1) path to subsampled fastq file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: None.
@param fastq: source fastq file (full path)
@param log
@return reformatGchistFile: hist text data (to be added to readqc_files.txt)
@return pngFile: output plot (to be added to readqc_files.txt)
@return htmlFile: output d3 interactive plot (to be added to readqc_files.txt)
@return meanVal: gc mean (to be added to readqc_stats.txt)
@return stdevVal: gc stdev (to be added to readqc_stats.txt)
"""
def illumina_read_gc(fastq, log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, _ = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
reformatGchistFile = os.path.join(subsamplePath, sequnitFileNamePrefix + ".reformat.gchist.txt") ## gc hist
log.debug("gchist file: %s", reformatGchistFile)
## Gen Average Base Position Quality plot
if not os.path.isfile(reformatGchistFile):
log.error("Gchist file not found: %s", reformatGchistFile)
return None, None, None, None, None, None, None
## File format
## #Mean 41.647
## #Median 42.000
## #Mode 42.000
## #STDev 4.541
## #GC Count
## 0.0 0
## 1.0 0
## 2.0 0
## 3.0 0
## 4.0 0
meanVal = None
medVal = None
modeVal = None
stdevVal = None
with open(reformatGchistFile, "r") as STAT_FH:
for l in STAT_FH.readlines():
if l.startswith("#Mean"):
meanVal = l.strip().split('\t')[1]
elif l.startswith("#Median"):
medVal = l.strip().split('\t')[1]
elif l.startswith("#Mode"):
modeVal = l.strip().split('\t')[1]
elif l.startswith("#STDev"):
stdevVal = l.strip().split('\t')[1]
rawDataMatrix = np.loadtxt(reformatGchistFile, comments='#', usecols=(0, 1, 2)) ## only use 3 colums: GC, Count, Cumulative
## In addition to the %GC and # reads, the cumulative read % is added.
assert len(rawDataMatrix[1][:]) == 3
fig, ax = plt.subplots()
markerSize = 5.0
lineWidth = 1.5
p1 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 1], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, alpha=0.5)
ax.set_xlabel("%GC", fontsize=12, alpha=0.5)
ax.set_ylabel("Read count", fontsize=12, alpha=0.5)
ax.grid(color="gray", linestyle=':')
## Add tooltip
toolTipStrReadCnt = ["Read count=%d" % i for i in rawDataMatrix[:, 1]]
toolTipStrGcPerc = ["GC percent=%.1f" % i for i in rawDataMatrix[:, 0]]
toolTipStrReadPerc = ["Read percent=%.1f" % (i * 100.0) for i in rawDataMatrix[:, 2]]
toolTipStr = ["%s, %s, %s" % (i, j, k) for (i, j, k) in
zip(toolTipStrGcPerc, toolTipStrReadCnt, toolTipStrReadPerc)]
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=toolTipStr))
pngFile = os.path.join(qualPath, sequnitFileNamePrefix + ".gchist.png")
htmlFile = os.path.join(qualPath, sequnitFileNamePrefix + ".gchist.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFile)
## Save Matplotlib plot in png format
plt.savefig(pngFile, dpi=fig.dpi)
return reformatGchistFile, pngFile, htmlFile, meanVal, stdevVal, medVal, modeVal
""" STEP5 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
write_avg_base_quality_stats
Title: write_base_quality_stats
Function: Takes path to fastq file and generates
quality plots for each read
Usage: write_base_quality_stats($fastq_path, $analysis, $log)
Args: 1) path to subsampled fastq file
2) log file object
Returns: None.
Comments: None.
@param fastq: source fastq file (full path)
@param log
@return reformatObqhistFile: output data file (to be added to readqc_files.txt)
"""
def write_avg_base_quality_stats(fastq, log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, _ = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
reformatObqhistFile = os.path.join(subsamplePath, sequnitFileNamePrefix + ".reformat.obqhist.txt") ## base composition histogram
log.debug("obqhist file: %s", reformatObqhistFile)
## Gen base composition histogram
if not os.path.isfile(reformatObqhistFile):
log.error("Obqhist file not found: %s", reformatObqhistFile)
return None
else:
return reformatObqhistFile
""" STEP6 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_count_q_score
Title: count_q_score
Function: Given a fastq (bz2 zipped or unzipped)
file path, creates a histogram of
the quality scores in the file
Usage: count_q_score($fastq, $log)
Args: 1) path to subsampled fastq file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: Generates a file named <fastq_path>.qhist that has
the quality score histogram.
@param fastq: source fastq file (full path)
@param log
@return reformatObqhistFile: output data file (to be added to readqc_files.txt)
@return pngFile: output plot file (to be added to readqc_files.txt)
@return htmlFile: output d3 interactive plot file (to be added to readqc_files.txt)
"""
def illumina_count_q_score(fastq, log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, _ = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
reformatObqhistFile = os.path.join(subsamplePath, sequnitFileNamePrefix + ".reformat.obqhist.txt") ## base composition histogram
log.debug("obqhist file: %s", reformatObqhistFile)
rawDataMatrix = np.loadtxt(reformatObqhistFile, delimiter='\t', comments='#')
assert len(rawDataMatrix[1][:]) == 3
## Qavg nrd percent
## 0 1 2
fig, ax = plt.subplots()
markerSize = 5.0
lineWidth = 1.5
p1 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 2], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, alpha=0.5)
ax.set_xlabel("Average Read Quality", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction of Reads", fontsize=12, alpha=0.5)
ax.grid(color="gray", linestyle=':')
## Add tooltip
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=list(rawDataMatrix[:, 2])))
pngFile = os.path.join(qualPath, sequnitFileNamePrefix + ".avg_read_quality_histogram.png")
htmlFile = os.path.join(qualPath, sequnitFileNamePrefix + ".avg_read_quality_histogram.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFile)
## Save Matplotlib plot in png format
plt.savefig(pngFile, dpi=fig.dpi)
return reformatObqhistFile, pngFile, htmlFile
""" STEP7 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_calculate_average_quality
Title: illumina_calculate_average_quality
Function: Given a fastq (subsampled) file, calculates average quality in 21 mer windows.
Usage: illumina_calculate_average_quality($fastq_path, $log)
Args: 1) path to subsampled fastq file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: Several output files are generated in the directory that
the script is run.
1) Text output file with 21mer start position, number of mers read,
total mers, and average accuracy of the bin
2) A gnuplot png file named <fastq_name>.21mer.qual.png
The function assumes that the fastq file exists.
The 21mer qual script was writtten by mli
@return retCode: success or failure
@return stat_file: plot data (to be added to readqc_files.txt)
@return pngFile: output plot (to be added to readqc_files.txt)
"""
## Removed!
##def illumina_calculate_average_quality(fastq, log):
""" STEP8 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_find_common_motifs
Title: illumina_find_common_motifs
Function: Given a fastq (subsampled) file, finds most common N-string motifs.
Usage: illumina_find_common_motifs($fastq_path, $log)
Args: 1) path to subsampled fastq file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: An output file is generated in the directory
1) Text output summary file most common motifs and perecent total motifs.
The function assumes that the fastq file exists.
The nstutter script was writtten by jel
@param fastq: source fastq file (full path)
@param log
@return retCode: success or failure
@return nstutterStatFile: output stutter data file (to be added to readqc_files.txt)
"""
def illumina_find_common_motifs(fastq, log):
## Tools
cdir = os.path.dirname(__file__)
patterNFastqPlCmd = os.path.join(cdir, '../tools/patterN_fastq.pl') #RQCReadQcCommands.PATTERN_FASTQ_PL
sequnitFileName, exitCode = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
stutterDir = "stutter"
stutterPath = os.path.join(READ_OUTPUT_PATH, stutterDir)
make_dir(stutterPath)
change_mod(stutterPath, "0755")
nstutterStatFile = os.path.join(stutterPath, sequnitFileNamePrefix + ".nstutter.stat")
## ex) patterN_fastq.pl -analog -PCT 0.1 -in 7601.1.77813.CTTGTA.s0.01.fastq > 7601.1.77813.CTTGTA.s0.01.nstutter.stat ; wait;
makeStatFileCmd = "%s -analog -PCT 0.1 -in %s > %s " % (patterNFastqPlCmd, fastq, nstutterStatFile)
combinedCmd = "%s; wait; " % (makeStatFileCmd)
_, _, exitCode = run_sh_command(combinedCmd, True, log, True)
if exitCode != 0:
log.error("failed to run patterNFastqPlCmd. Exit code != 0.")
return RQCExitCodes.JGI_FAILURE, None
if os.path.isfile(nstutterStatFile):
log.info("N stutter stat file successfully created (%s)", nstutterStatFile)
else:
log.warning("Could not locate N stutter stat file %s", nstutterStatFile)
nstutterStatFile = "failed to generate"
return RQCExitCodes.JGI_SUCCESS, nstutterStatFile
""" STEP9 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_run_bwa
Title: illumina_run_bwa
Function: Given a fastq (subsampled) file path, runs bwa aligner
Reads are aligned to each other.
Usage: illumina_run_bwa($fastq_file_path, $log)
Args: 1) path to subsampled fastq file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: None.
@param fastq: source fastq file (full path)
@param log
@return retCode: success or failure
@return summary_file: output bwa summary file (to be added to readqc_files.txt)
"""
## REMOVED!
##def illumina_run_bwa(fastq, log):
def illumina_run_dedupe(fastq, log):
## Tools
cdir = os.path.dirname(__file__)
bbdedupeShCmd = os.path.join(cdir, '../../bbdedupe.sh') #RQCReadQcCommands.BBDEDUPE_SH
sequnitFileName, exitCode = safe_basename(fastq, log)
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
dupDir = "dupes"
dupPath = os.path.join(READ_OUTPUT_PATH, dupDir)
make_dir(dupPath)
change_mod(dupPath, "0755")
dedupeSummaryFile = os.path.join(dupPath, sequnitFileName + ".bwa.summary")
## dedupe.sh in=reads.fq s=0 ftr=49 ac=f int=f
xmx = "-Xmx23G"
bbdedupeShCmd = "%s %s in=%s out=null qin=33 ow=t s=0 ftr=49 ac=f int=f> %s 2>&1 " % (bbdedupeShCmd, xmx, fastq, dedupeSummaryFile)
_, _, exitCode = run_sh_command(bbdedupeShCmd, True, log, True)
if exitCode != 0:
log.error("Failed to run bbdedupeShCmd.sh")
return RQCExitCodes.JGI_FAILURE, None
return RQCExitCodes.JGI_SUCCESS, dedupeSummaryFile
""" STEP10 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_run_tagdust
Title: illumina_run_tagdust
Function: Given a fastq (subsampled) file path, runs tag dust to
find common illumina artifacts.
Usage: illumina_run_tagdust($fastq_file_path, $log)
Args: 1) path to subsampled fastq file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: None.
@param fastq: source fastq file (full path)
@param log
@return retCode: success or failure
@return tagdust_out: output tagdust file (to be added to readqc_files.txt)
"""
## No longer needed!!
##def illumina_run_tagdust(fastq, log):
""" STEP11 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_detect_read_contam
@param fastq: source fastq file (full path)
@param firstBp: first bp length to cut for read contam detection
@param log
@return retCode: success or failure
@return outFileList: output duk stat file list (to be added to readqc_files.txt)
@return ratioResultDict: output stat value dict (to be added to readqc_stats.txt)
"""
##def illumina_detect_read_contam(fastq, log):
## REMOVED!
# # def illumina_detect_read_contam2(fastq, firstBp, log):
## REMOVED! 08302016
"""
Contam removal by seal.sh
"""
def illumina_detect_read_contam3(fastq, firstBp, log):
## Tools
cdir = os.path.dirname(__file__)
sealShCmd = os.path.join(cdir, '../../seal.sh') #RQCReadQcCommands.SEAL_SH_CMD
sequnitFileName, exitCode = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
numBadFiles = 0
ratio = 0
outFileDict = {}
ratioResultDict = {}
contamStatDict = {}
catCmd, exitCode = get_cat_cmd(fastq, log)
## TODO: remove JGI Contaminants from CONTAM_DBS
# CONTAM_DBS['artifact'] = ARTIFACT_FILE_NO_SPIKEIN
# CONTAM_DBS['artifact_50bp'] = ARTIFACT_FILE_NO_SPIKEIN ## 20131203 Added for 50bp contam
# CONTAM_DBS['DNA_spikein'] = ARTIFACT_FILE_DNA_SPIKEIN
# CONTAM_DBS['RNA_spikein'] = ARTIFACT_FILE_RNA_SPIKEIN
# CONTAM_DBS['contaminants'] = CONTAMINANTS
# CONTAM_DBS['fosmid'] = FOSMID_VECTOR
# CONTAM_DBS['mitochondrion'] = MITOCHONDRION_NCBI_REFSEQ
# CONTAM_DBS['phix'] = PHIX
# CONTAM_DBS['plastid'] = CHLOROPLAST_NCBI_REFSEQ
# CONTAM_DBS['rrna'] = GENERAL_RRNA_FILE
# CONTAM_DBS['microbes'] = MICROBES ## non-synthetic
# CONTAM_DBS['synthetic'] = SYNTHETIC
# CONTAM_DBS['adapters'] = ADAPTERS
for db in RQCContamDb.CONTAM_DBS.iterkeys():
if db == "artifact_50bp" and int(firstBp) == 20:
sealStatsFile = os.path.join(qualPath, sequnitFileNamePrefix + ".artifact_20bp.seal.stats")
else:
sealStatsFile = os.path.join(qualPath, sequnitFileNamePrefix + "." + db + ".seal.stats")
## Localization file to /scratch/rqc
# log.info("Contam DB localization started for %s", db)
# localizedDb = localize_file(RQCContamDb.CONTAM_DBS[db], log)
## 04262017 Skip localization temporarily until /scratch can be mounted
## in shifter container
# if os.environ['NERSC_HOST'] == "genepool":
# localizedDb = localize_file(RQCContamDb.CONTAM_DBS[db], log)
# else:
# localizedDb = None
#
# if localizedDb is None:
# localizedDb = RQCContamDb.CONTAM_DBS[db] ## use the orig location
# else:
# log.info("Use the localized file, %s", localizedDb)
localizedDb = RQCContamDb.CONTAM_DBS[db]
## 09112017 Manually add -Xmx23G
xmx = "-Xmx23G"
if db == "artifact_50bp":
cutCmd = "cut -c 1-%s | " % (firstBp)
cmd = "set -e; %s %s | %s %s in=stdin.fq out=null ref=%s k=22 hdist=0 stats=%s ow=t statscolumns=3 %s " % \
(catCmd, fastq, cutCmd, sealShCmd, localizedDb, sealStatsFile, xmx)
elif db == "microbes":
cmd = "%s in=%s out=null ref=%s hdist=0 mm=f mkf=0.5 ambig=random minlen=120 qtrim=rl trimq=10 stats=%s ow=t statscolumns=3 %s " % \
(sealShCmd, fastq, localizedDb, sealStatsFile, xmx)
else:
cmd = "%s in=%s out=null ref=%s k=22 hdist=0 stats=%s ow=t statscolumns=3 %s " % \
(sealShCmd, fastq, localizedDb, sealStatsFile, xmx)
_, _, exitCode = run_sh_command(cmd, True, log, True)
if exitCode != 0:
log.error("Failed to run seal.sh cmd")
return RQCExitCodes.JGI_FAILURE, None, None, None
## Parsing seal output
if not os.path.isfile(sealStatsFile):
log.warning("Cannot open contam output file %s", sealStatsFile)
numBadFiles += 1
continue ## continue to next contam db
maxStatCount = 0
with open(sealStatsFile, "r") as sealFH:
for line in sealFH:
line.strip()
if line.find("#Matched") != -1:
## ex) #Matched 1123123 77.31231%
toks = line.split()
assert len(toks) == 3
ratio = toks[-1].replace('%', '')
## contamintaion stat
if not line.startswith('#'):
t = line.rstrip().split('\t')
# contamStatDict["%s:%s" % (db, t[0])] = t[2].replace('%', '')
if maxStatCount < 10 and t[0].startswith("gi|"):
# contamStatDict["contam:%s:%s" % (db, t[0])] = t[2].replace('%', '')
contamStatDict["contam:%s:%s" % (db, "|".join(t[0].split('|')[:2]))] = t[2].replace('%', '') ## save only gi part (RQC-906)
maxStatCount += 1
## RQC-743
if db == "artifact_50bp" and int(firstBp) == 20:
db = "artifact_20bp"
outFileDict[db + ".seal.stats"] = sealStatsFile
log.debug("Contam db and matched ratio: %s = %f", RQCContamDb.CONTAM_KEYS[db], float(ratio))
ratioResultDict[RQCContamDb.CONTAM_KEYS[db]] = float(ratio)
if numBadFiles:
log.info("Number of bad I/O cases = %s", numBadFiles)
return RQCExitCodes.JGI_FAILURE, None, None, None
return RQCExitCodes.JGI_SUCCESS, outFileDict, ratioResultDict, contamStatDict
""" STEP12 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_sciclone_analysis
Title: illumina_sciclone_analysis
Function: Takes path to fastq file and determines
if it is from a multiplexed run or not
Usage: illumina_sciclone_analysis($subfastq, $log)
Args: 1) fastq file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: None.
@param origFastq: source fastq file (full path)
@param isPairedEnd: pair- or single-ended
@param log
@return retCode: success or failure
@return ratioResultDict: output stat value dict (to be added to readqc_stats.txt)
@return dnaCountFile: output sam stat file (to be added to readqc_files.txt)
@return rnaCountFile: output sam stat file (to be added to readqc_files.txt)
"""
## Removed!
##def illumina_sciclone_analysis(origFastq, isPairedEnd, log, libName=None, isRna=None):
def illumina_sciclone_analysis2(origFastq, isPairedEnd, log, libName=None, isRna=None):
## detect lib is rna or not
sequnitFileName, exitCode = safe_basename(origFastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
# if libName is None and isRna is None:
# _, _, libName, isRna = get_lib_info(sequnitFileNamePrefix, log) ## seqProjectId, ncbiOrganismName not used
#
# if isRna == "N/A":
# log.error("Failed to get lib info for %s", sequnitFileNamePrefix)
# return RQCExitCodes.JGI_FAILURE, -1, -1
if isRna == '1':
isRna = True
elif isRna == '0':
isRna = False
if isRna:
log.debug("The lib is RNA (%s)", libName)
else:
log.debug("The lib is DNA (%s)", libName)
if isPairedEnd:
log.debug("It's pair-ended.")
else:
log.debug("It's single-ended.")
## output dir
sciclone_dir = "sciclone_analysis"
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sciclone_path = os.path.join(READ_OUTPUT_PATH, sciclone_dir)
## Define the subdirectory. If it exists already, remove it
if os.path.isdir(sciclone_path):
rm_dir(sciclone_path)
make_dir(sciclone_path)
change_mod(sciclone_path, "0755")
## NOTE: Save count file in analysis object
dnaCountFile = None
rnaCountFile = None
if isRna:
rnaCountFile = os.path.join(sciclone_path, sequnitFileNamePrefix + "_bbduk_sciclone_rna_count.txt")
else:
dnaCountFile = os.path.join(sciclone_path, sequnitFileNamePrefix + "_bbduk_sciclone_dna_count.txt")
cdir = os.path.dirname(__file__)
bbdukShCmd = os.path.join(cdir, '../../bbduk.sh') #RQCReadQcCommands.BBDUK_SH_CMD
bbdukRnaDb = RQCReadQcReferenceDatabases.SCICLONE_RNA2
# bbdukDnaDb = RQCReadQcReferenceDatabases.SCICLONE_DNA2
cmd = None
if isRna:
## Localization file to /scratch/rqc
# log.info("Sciclone RNA ref DB localization started for %s", bbdukRnaDb)
# localizedDb = localize_file(bbdukRnaDb, log)
## 04262017 Skip localization temporarily until /scratch can be mounted
## in shifter container
# if os.environ['NERSC_HOST'] == "genepool":
# localizedDb = localize_file(bbdukRnaDb, log)
# else:
# localizedDb = None
#
# if localizedDb is None:
# localizedDb = bbdukRnaDb ## use the orig location
# else:
# log.info("Use the localized file, %s", localizedDb)
localizedDb = bbdukRnaDb ## use the orig location
## bbduk.sh in=7365.2.69553.AGTTCC.fastq.gz ref=/global/projectb/sandbox/gaag/bbtools/data/sciclone_rna.fa out=null fbm=t k=31 mbk=0 stats=sciclone2.txt
cmd = "%s in=%s ref=%s out=null fbm=t k=31 mbk=0 stats=%s statscolumns=3 " % (bbdukShCmd, origFastq, localizedDb, rnaCountFile)
else:
## Localization file to /scratch/rqc
# log.info("Sciclone DNA ref DB localization started for %s", bbdukDnaDb)
# localizedDb = localize_file(bbdukDnaDb, log)
## 04262017 Skip localization temporarily until /scratch can be mounted
## in shifter container
# if os.environ['NERSC_HOST'] == "genepool":
# localizedDb = localize_file(bbdukRnaDb, log)
# else:
# localizedDb = None
#
# if localizedDb is None:
# localizedDb = bbdukRnaDb ## use the orig location
# else:
# log.info("Use the localized file, %s", localizedDb)
localizedDb = bbdukRnaDb ## use the orig location
## bbduk.sh in=7257.1.64419.CACATTGTGAG.fastq.gz ref=/global/projectb/sandbox/gaag/bbtools/data/sciclone_dna.fa out=null fbm=t k=31 mbk=0 stats=sciclone1.txt
cmd = "%s in=%s ref=%s out=null fbm=t k=31 mbk=0 stats=%s statscolumns=3 " % (bbdukShCmd, origFastq, localizedDb, dnaCountFile)
_, _, exitCode = run_sh_command(cmd, True, log, True)
if exitCode != 0:
log.error("Failed to run bbduk.sh cmd")
return RQCExitCodes.JGI_FAILURE, None, None
log.debug("rnaCountFile = %s", rnaCountFile)
log.debug("dnaCountFile = %s", dnaCountFile)
return RQCExitCodes.JGI_SUCCESS, dnaCountFile, rnaCountFile
""" STEP13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_read_megablast
Title: illumina_read_megablast
Function: Takes path(s) to bz2 zipped or gzipped fastq file
and runs megablast against the reads.
Usage: illumina_read_megablast(\@seq_files, $subsampledFile, $read_length, $log)
Args: 1) reference to an array containing bz2 zipped or gzipped fastq file path(s);
the files should all be compressed the same way
2) subsampled fastq file
3) read length
4) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: None.
@param subsampledFile: source fastq file (full path)
@param log
@return retCode: success or failure
"""
## No longer needed. Removed.
##def illumina_read_megablast(subsampledFile, read_num_to_pass, log, blastDbPath=None):
""" STEP14 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
"""
##def illumina_read_blastn_refseq_microbial(subsampledFile, log, blastDbPath=None):
## 12212015 sulsj REMOVED!
""" STEP14 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
"""
def illumina_read_blastn_refseq_archaea(subsampledFile, log):
## Tools
cdir = os.path.dirname(__file__)
bbtoolsReformatShCmd = os.path.join(cdir, '../../reformat.sh') #RQCReadQcCommands.BBTOOLS_REFORMAT_CMD
## verify the fastq file
if not os.path.isfile(subsampledFile):
log.error("Failed to find fastq file")
return RQCExitCodes.JGI_FAILURE
log.info("Read level contamination analysis using blastn")
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
## output dir
megablastDir = "megablast"
megablastPath = os.path.join(READ_OUTPUT_PATH, megablastDir)
make_dir(megablastPath)
change_mod(megablastPath, "0755")
## 20140929 Replaced with reformat.sh
queryFastaFileName = "reads.fa"
## reformat.sh for converting fastq to fasta
cmd = "%s in=%s out=%s qin=33 qout=33 ow=t " % (bbtoolsReformatShCmd, subsampledFile, os.path.join(megablastPath, queryFastaFileName))
_, _, exitCode = run_sh_command(cmd, True, log, True)
if exitCode != 0:
log.error("Failed to run reformat.sh to convert fastq to fasta: %s", cmd)
return RQCExitCodes.JGI_FAILURE
megablastOutputFile = None
db = "refseq.archaea"
log.info("---------------------------------------------")
log.info("Start blastn search against %s", db)
log.info("---------------------------------------------")
## final output ==> READ_OUTPUT_PATH/megablast
retCode, megablastOutputFile = run_blastplus_py(os.path.join(megablastPath, queryFastaFileName), db, log)
if retCode != RQCExitCodes.JGI_SUCCESS:
if megablastOutputFile is None:
log.error("Failed to run blastn against %s. Ret = %s", db, retCode)
retCode = RQCExitCodes.JGI_FAILURE
elif megablastOutputFile == -143:
log.warning("Blast overtime. Skip the search against %s.", db)
retCode = -143 ## blast overtime
else:
log.info("Successfully ran blastn of reads against %s", db)
retCode = RQCExitCodes.JGI_SUCCESS
return retCode
""" STEP15 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
"""
def illumina_read_blastn_refseq_bacteria(subsampledFile, log):
## Tools
cdir = os.path.dirname(__file__)
bbtoolsReformatShCmd = os.path.join(cdir, '../../reformat.sh') #RQCReadQcCommands.BBTOOLS_REFORMAT_CMD
## verify the fastq file
if not os.path.isfile(subsampledFile):
log.error("Failed to find fastq file for blastn")
return RQCExitCodes.JGI_FAILURE
log.info("Read level contamination analysis using blastn")
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
## output dir
megablastDir = "megablast"
megablastPath = os.path.join(READ_OUTPUT_PATH, megablastDir)
make_dir(megablastPath)
change_mod(megablastPath, "0755")
## 20140929 Replaced with reformat.sh
queryFastaFileName = "reads.fa"
## reformat.sh for converting fastq to fasta
cmd = "%s in=%s out=%s qin=33 qout=33 ow=t " % (bbtoolsReformatShCmd, subsampledFile, os.path.join(megablastPath, queryFastaFileName))
_, _, exitCode = run_sh_command(cmd, True, log, True)
if exitCode != 0:
log.error("Failed to run reformat.sh to convert fastq to fasta: %s", cmd)
return RQCExitCodes.JGI_FAILURE
megablastOutputFile = None
db = "refseq.bacteria"
log.info("---------------------------------------------")
log.info("Start blastn search against %s", db)
log.info("---------------------------------------------")
## final output ==> READ_OUTPUT_PATH/megablast
retCode, megablastOutputFile = run_blastplus_py(os.path.join(megablastPath, queryFastaFileName), db, log)
if retCode != RQCExitCodes.JGI_SUCCESS:
if megablastOutputFile is None:
log.error("Failed to run blastn against %s. Ret = %s", db, retCode)
retCode = RQCExitCodes.JGI_FAILURE
elif megablastOutputFile == -143:
log.warning("Blast overtime. Skip the search against %s.", db)
retCode = -143 ## blast overtime
else:
log.info("Successfully ran blastn of reads against %s", db)
retCode = RQCExitCodes.JGI_SUCCESS
return retCode
""" STEP16 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
"""
def illumina_read_blastn_nt(subsampledFile, log):
## Tools
cdir = os.path.dirname(__file__)
bbtoolsReformatShCmd = os.path.join(cdir, '../../reformat.sh') #RQCReadQcCommands.BBTOOLS_REFORMAT_CMD
## verify the fastq file
if not os.path.isfile(subsampledFile):
log.error("Failed to find fastq file for blastn")
return RQCExitCodes.JGI_FAILURE
log.info("Read level contamination analysis using blastn")
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
## output dir
megablastDir = "megablast"
megablastPath = os.path.join(READ_OUTPUT_PATH, megablastDir)
make_dir(megablastPath)
change_mod(megablastPath, "0755")
## 20140929 Replaced with reformat.sh
queryFastaFileName = "reads.fa"
## reformat.sh
cmd = "%s in=%s out=%s qin=33 qout=33 ow=t " % (bbtoolsReformatShCmd, subsampledFile, os.path.join(megablastPath, queryFastaFileName))
_, _, exitCode = run_sh_command(cmd, True, log, True)
if exitCode != 0:
log.error("Failed to run reformat.sh to convert fastq to fasta: %s", cmd)
return RQCExitCodes.JGI_FAILURE
megablastOutputFile = None
db = "nt"
log.info("----------------------------------")
log.info("Start blastn search against %s", db)
log.info("----------------------------------")
## final output ==> READ_OUTPUT_PATH/megablast
retCode, megablastOutputFile = run_blastplus_py(os.path.join(megablastPath, queryFastaFileName), db, log)
if retCode != RQCExitCodes.JGI_SUCCESS:
if megablastOutputFile is None:
log.error("Failed to run blastn against %s. Ret = %s", db, retCode)
retCode = RQCExitCodes.JGI_FAILURE
elif megablastOutputFile == -143:
log.warning("Blast overtime. Skip the search against %s.", db)
retCode = -143 ## blast overtime
else:
log.info("Successfully ran blastn of reads against %s", db)
retCode = RQCExitCodes.JGI_SUCCESS
return retCode
""" STEP17 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
illumina_generate_index_sequence_detection_plot
"""
def illumina_generate_index_sequence_detection_plot(fastq, log, isMultiplexed=None): ## TO BE REMOVED!
isMultiplexed = 0
if not os.path.isfile(fastq):
log.error("Failed to find the input fastq file, %s", fastq)
return RQCExitCodes.JGI_FAILURE, None, None, None
else:
log.info("fastq file for index sequence analysis: %s", fastq)
sequnitFileName, exitCode = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
# if isMultiplexed is None:
# isMultiplexed = get_multiplex_info(sequnitFileNamePrefix, log)
#retCode = None
demultiplexStatsFile = None
demultiplexPlotDataFile = None
detectionPlotPngFile = None
storedDemultiplexStatsFile = None
if int(isMultiplexed) == 1:
log.info("Multiplexed - start analyzing...")
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
demul_dir = "demul"
demulPath = os.path.join(READ_OUTPUT_PATH, demul_dir)
make_dir(demulPath)
change_mod(demulPath, "0755")
## This version is sorted by percent for readability of stats
demultiplexStatsFile = os.path.join(demulPath, sequnitFileNamePrefix + ".demultiplex_stats")
## This version has index column and sort by index for plot
demultiplexPlotDataFile = os.path.join(demulPath, sequnitFileNamePrefix + ".demultiplex_stats.tmp")
## This path is relative to final qual location to be stored in analysis obj.
detectionPlotPngFile = os.path.join(demulPath, sequnitFileNamePrefix + ".index_sequence_detection.png")
storedDemultiplexStatsFile = os.path.join(demulPath, sequnitFileNamePrefix + ".demultiplex_stats")
if not os.path.isfile(demultiplexStatsFile):
indexSeq = None
line = None
header = None
indexSeqCounter = {}
catCmd, exitCode = get_cat_cmd(fastq, log)
if fastq.endswith(".gz"):
catCmd = "zcat" ## pigz does not work with subprocess
seqCount = 0
try:
proc = subprocess.Popen([catCmd, fastq], bufsize=2 ** 16, stdout=subprocess.PIPE, stderr=subprocess.STDOUT)
while 1:
line = proc.stdout.readline()
if not line:
break
## First line is header
line.strip()
header = line
## Get second line of record - sequence
line = proc.stdout.readline()
## Get third line - junk
line = proc.stdout.readline()
## Get the final line (4th line) - quality
line = proc.stdout.readline()
## Parse the header
headerFields = header.split(":")
## The last index is the index
indexSeq = headerFields[-1].strip()
assert indexSeq
## Increment the counts and store the index
if indexSeq in indexSeqCounter:
indexSeqCounter[indexSeq] += 1
else:
indexSeqCounter[indexSeq] = 1
seqCount += 1
except Exception as e:
if log:
log.error("Exception in file reading: %s", e)
log.error("Failed to read the given fastq file [%s]", fastq)
log.error("Fastq header doesn't have the index sequence: %s", header)
log.error("Index sequence analysis is skipped!")
return RQCExitCodes.JGI_SUCCESS, None, None, None
## Open the output file handles for writing
log.info("demultiplexPlotDataFile = %s", demultiplexPlotDataFile)
log.info("detectionPlotPngFile = %s", detectionPlotPngFile)
log.info("demultiplexStatsFile = %s", demultiplexStatsFile)
log.info("storedDemultiplexStatsFile = %s", storedDemultiplexStatsFile)
plotDataFH = open(demultiplexPlotDataFile, "w")
statsFH = open(demultiplexStatsFile, "w")
## Count the total number of indexes found
numIndexesFound = len(indexSeqCounter)
## Store the data header information for printing
reportHeader = """# Demultiplexing Summary
#
# Seq unit name: %s
# Total sequences: %s
# Total indexes found: %s
# 1=indexSeq 2=index_sequence_count 3=percent_of_total
#
""" % (sequnitFileName, seqCount, numIndexesFound)
statsFH.write(reportHeader)
## Sort by value, descending
log.debug("Sort by value of indexSeqCounter")
for indexSeq in sorted(indexSeqCounter, key=indexSeqCounter.get, reverse=True):
perc = float(indexSeqCounter[indexSeq]) / float(seqCount) * 100
l = "%s\t%s\t%.6f\n" % (indexSeq, indexSeqCounter[indexSeq], perc)
statsFH.write(l)
## Sort by index and add id column for plotting
log.debug("Sort by index of indexSeqCounter")
i = 1
for indexSeq in sorted(indexSeqCounter.iterkeys()):
perc = float(indexSeqCounter[indexSeq]) / float(seqCount) * 100
l = "%s\t%s\t%s\t%.6f\n" % (i, indexSeq, indexSeqCounter[indexSeq], perc)
plotDataFH.write(l)
i += 1
plotDataFH.close()
statsFH.close()
log.debug("demultiplex plotting...")
## matplotlib plotting
# data
# Index_seq_id Index_seq indexSeqCounter percent
# 1 AAAAAAAAAAAA 320 0.000549
# 2 AAAAAAAAAAAC 16 0.000027
# 3 AAAAAAAAAAAG 8 0.000014
# 4 AAAAAAAAAACA 4 0.000007
# 5 AAAAAAAAAACG 2 0.000003
# 6 AAAAAAAAAAGA 6 0.000010
# 7 AAAAAAAAAATA 6 0.000010
# rawDataMatrix = np.loadtxt(demultiplexPlotDataFile, delimiter='\t', comments='#')
# assert len(rawDataMatrix[1][:]) == 4
## For a textfile with 4000x4000 words this is about 10 times faster than loadtxt.
## http://stackoverflow.com/questions/14985233/load-text-file-as-strings-using-numpy-loadtxt
def load_data_file(fname):
data = []
with open(fname, 'r') as FH:
lineCnt = 0
for line in FH:
if lineCnt > 10000: ## experienced out of mem with a stat file with 9860976 index sequences
log.warning("Too many index sequences. Only 10000 index sequences will be used for plotting.")
break
data.append(line.replace('\n', '').split('\t'))
lineCnt += 1
return data
def column(matrix, i, opt):
if opt == "int":
return [int(row[i]) for row in matrix]
elif opt == "float":
return [float(row[i]) for row in matrix]
else:
return [row[i] for row in matrix]
rawDataMatrix = load_data_file(demultiplexPlotDataFile)
fig, ax = plt.subplots()
markerSize = 6.0
lineWidth = 1.5
p1 = ax.plot(column(rawDataMatrix, 0, "int"), column(rawDataMatrix, 3, "float"), 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='N', alpha=0.5)
ax.set_xlabel("Index Sequence ID", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction", fontsize=12, alpha=0.5)
ax.grid(color="gray", linestyle=':')
## Add tooltip
labels = ["%s" % i for i in column(rawDataMatrix, 1, "str")]
## Show index_seq in the plot
for i in rawDataMatrix:
ax.text(i[0], float(i[3]) + .2, "%s" % i[1])
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=labels))
detectionPlotHtml = os.path.join(demulPath, sequnitFileNamePrefix + ".index_sequence_detection.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, detectionPlotHtml)
## Save Matplotlib plot in png format
plt.savefig(detectionPlotPngFile, dpi=fig.dpi)
if exitCode != 0:
log.error("Failed to create demulplex plot")
return RQCExitCodes.JGI_FAILURE, None, None, None
log.info("demulplex stats and plot generation completed!")
else:
log.info("Not multiplexed - skip this analysis.")
return RQCExitCodes.JGI_SUCCESS, None, None, None
if detectionPlotPngFile is not None and storedDemultiplexStatsFile is not None and os.path.isfile(
detectionPlotPngFile) and os.path.isfile(storedDemultiplexStatsFile):
return RQCExitCodes.JGI_SUCCESS, demultiplexStatsFile, detectionPlotPngFile, detectionPlotHtml
else:
return RQCExitCodes.JGI_FAILURE, None, None, None
""" STEP18 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
end_of_read_illumina_adapter_check
usage: kmercount_pos.py [-h] [-k <int>] [-c <int>] [-t <int>] [-p <file>]
fastaFile fastqFile [fastqFile ...]
Count occurance of database kmers in reads
positional arguments:
fastaFile Input FASTA file(s). Text or gzip
fastqFile Input FASTQ file(s). Text or gzip
optional arguments:
-h, --help show this help message and exit
-k <int> kmer length (default: 16)
-c <int> minimum allowed coverage (default: 2)
-t <int> target coverage (default: 30)
-p <file>, --plot <file> plot data and save as png to <file> (default: None)
* NOTE
- RQC-383 04082014: Updated to the newest version of kmercount_pos.py (readqc ver 5.0.4)
"""
def end_of_read_illumina_adapter_check(firstSubsampledFastqFile, log):
cdir = os.path.dirname(__file__)
kmercountPosCmd = os.path.join(cdir, 'kmercount_pos.py') #RQCReadQcCommands.KMERCOUNT_POS_CMD
adapterDbName = RQCReadQcReferenceDatabases.END_OF_READ_ILLUMINA_ADAPTER_CHECK_DB
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, exitCode = safe_basename(firstSubsampledFastqFile, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
plotFile = ""
dataFile = ""
adapterCheckDir = "adapter"
adapterCheckPath = os.path.join(READ_OUTPUT_PATH, adapterCheckDir)
make_dir(adapterCheckPath)
change_mod(adapterCheckPath, "0755")
plotFile = os.path.join(adapterCheckPath, sequnitFileNamePrefix + ".end_of_read_adapter_check.png") ## ignored
dataFile = os.path.join(adapterCheckPath, sequnitFileNamePrefix + ".end_of_read_adapter_check.txt")
## Localization file to /scratch/rqc
log.info("illumina_adapter_check DB localization started for %s", adapterDbName)
# if os.environ['NERSC_HOST'] == "genepool":
# localizedDb = localize_file(adapterDbName, log)
# else:
# localizedDb = None
#
# if localizedDb is None:
# localizedDb = adapterDbName ## use the orig location
# else:
# log.info("Use the localized file, %s", localizedDb)
localizedDb = adapterDbName ## use the orig location
## ex) kmercount_pos.py --plot plot.png Artifacts.adapters_primers_only.fa subsample.fastq > counts_by_pos.txt
cmd = "%s --plot %s %s %s > %s " % (kmercountPosCmd, plotFile, localizedDb, firstSubsampledFastqFile, dataFile)
## Run cmd
_, _, exitCode = run_sh_command(cmd, True, log, True)
assert exitCode == 0
## mpld3 plots gen
rawDataMatrix = np.loadtxt(dataFile, delimiter='\t', comments='#', skiprows=0)
assert len(rawDataMatrix[1][:]) == 3 or len(rawDataMatrix[1][:]) == 5
##pos read1 read2
## 0 1 2
## This output file format is changed on 2013.06.26 (RQC-442)
##
## pos read1_count read1_perc read2_count read2_perc
##
fig, ax = plt.subplots()
markerSize = 3.5
lineWidth = 1.0
if len(rawDataMatrix[1][:]) != 5: ## support for old file
p1 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 1], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label="read1", alpha=0.5)
p2 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 2], 'g', marker='d', markersize=markerSize, linewidth=lineWidth, label="read2", alpha=0.5)
ax.set_ylabel("Read Count with Database K-mer", fontsize=12, alpha=0.5)
else:
p1 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 2], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label="read1", alpha=0.5)
p2 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 4], 'g', marker='d', markersize=markerSize, linewidth=lineWidth, label="read2", alpha=0.5)
ax.set_ylim([0, 100])
ax.set_ylabel("Percent Reads with Database K-mer", fontsize=12, alpha=0.5)
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.yaxis.set_label_coords(-0.095, 0.75)
fontProp = FontProperties()
fontProp.set_size("small")
fontProp.set_family("Bitstream Vera Sans")
ax.legend(loc=1, prop=fontProp)
ax.grid(color="gray", linestyle=':')
## Add tooltip
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=list(rawDataMatrix[:, 1])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p2[0], labels=list(rawDataMatrix[:, 2])))
pngFile = os.path.join(adapterCheckPath, sequnitFileNamePrefix + ".end_of_read_adapter_check.png")
htmlFile = os.path.join(adapterCheckPath, sequnitFileNamePrefix + ".end_of_read_adapter_check.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFile)
## Save Matplotlib plot in png format
plt.savefig(pngFile, dpi=fig.dpi)
if exitCode != 0:
log.error("Failed to run kmercountPosCmd")
return RQCExitCodes.JGI_FAILURE, None, None, None
if os.path.isfile(plotFile) and os.path.isfile(dataFile):
log.info("kmercount_pos completed.")
return RQCExitCodes.JGI_SUCCESS, dataFile, pngFile, htmlFile
else:
log.error("cannot find the output files from kmercount_pos. kmercount_pos failed.")
return RQCExitCodes.JGI_FAILURE, None, None, None
""" STEP19 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
insert_size_analysis
Using bbmerge.sh from bbtools, create insert size histogram (static/interactive) plots using D3 and data file
e.q.
java -ea -Xmx200m -cp /usr/common/jgi/utilities/bbtools/prod-v32.28/lib/BBTools.jar jgi.BBMerge
in=/global/projectb/scratch/brycef/rqc-dev/staging/00/00/66/26/6626.2.48981.TTCTCC.fastq ihist=ihist.txt
Executing jgi.BBMerge [in=/global/projectb/scratch/brycef/rqc-dev/staging/00/00/66/26/6626.2.48981.TTCTCC.fastq, ihist=ihist.txt]
e.g.
bbmerge.sh in=[path-to-fastq] hist=hist.txt
- you should use the whole fastq, not just the subsampled fastq
"""
def insert_size_analysis(fastq, log):
## Tools
cdir = os.path.dirname(__file__)
bbmergeShCmd = os.path.join(cdir, '../../bbmerge.sh') #RQCReadQcCommands.BBMERGE_SH_CMD
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, exitCode = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
plotFile = ""
dataFile = ""
insertSizeOutDir = "insert_size_analysis"
insertSizeOutPath = os.path.join(READ_OUTPUT_PATH, insertSizeOutDir)
make_dir(insertSizeOutPath)
change_mod(insertSizeOutPath, "0755")
plotFile = os.path.join(insertSizeOutPath, sequnitFileNamePrefix + ".insert_size_histo.png")
htmlFile = os.path.join(insertSizeOutPath, sequnitFileNamePrefix + ".insert_size_histo.html")
dataFile = os.path.join(insertSizeOutPath, sequnitFileNamePrefix + ".insert_size_histo.txt")
## TODO
## if it's single ended
## 1. rqcfilter.sh for adapter trim
## 2. reformat.sh for getting lhist
## 3. analyze lhist.txt
## ex) bbmerge.sh in=7601.1.77813.CTTGTA.fastq.gz hist=.../insert_size_analysis/7601.1.77813.CTTGTA.insert_size_histo.txt
## reads=1000000 --> 1M reads are enough for insert size analysis
cmd = "%s in=%s hist=%s reads=1000000 " % (bbmergeShCmd, fastq, dataFile)
## Run cmd
_, stdErr, exitCode = run_sh_command(cmd, True, log, True)
if exitCode != 0:
log.error("Failed to run bbmerge_sh_cmd.")
return RQCExitCodes.JGI_FAILURE, None, None, None, None
retCode = {}
## File format
## BBMerge version 5.0
## Finished reading
## Total time: 8.410 seconds.
##
## Pairs: 1000000
## Joined: 556805 55.681%
## Ambiguous: 9665 0.967%
## No Solution: 433474 43.347%
## Too Short: 56 0.006%
## Avg Insert: 234.6
## Standard Deviation: 33.9
## Mode: 250
##
## Insert range: 26 - 290
## 90th percentile: 277
## 75th percentile: 262
## 50th percentile: 238
## 25th percentile: 211
## 10th percentile: 188
for l in stdErr.split('\n'):
toks = l.split()
if l.startswith("Total time"):
retCode["total_time"] = toks[2]
elif l.startswith("Reads"):
retCode["num_reads"] = toks[1]
elif l.startswith("Pairs"):
retCode["num_reads"] = toks[1]
elif l.startswith("Joined"):
retCode["joined_num"] = toks[1]
retCode["joined_perc"] = toks[2]
elif l.startswith("Ambiguous"):
retCode["ambiguous_num"] = toks[1]
retCode["ambiguous_perc"] = toks[2]
elif l.startswith("No Solution"):
retCode["no_solution_num"] = toks[2]
retCode["no_solution_perc"] = toks[3]
elif l.startswith("Too Short"):
retCode["too_short_num"] = toks[2]
retCode["too_short_perc"] = toks[3]
elif l.startswith("Avg Insert"):
retCode["avg_insert"] = toks[2]
elif l.startswith("Standard Deviation"):
retCode["std_insert"] = toks[2]
elif l.startswith("Mode"):
retCode["mode_insert"] = toks[1]
elif l.startswith("Insert range"):
retCode["insert_range_start"] = toks[2]
retCode["insert_range_end"] = toks[4]
elif l.startswith("90th"):
retCode["perc_90th"] = toks[2]
elif l.startswith("50th"):
retCode["perc_50th"] = toks[2]
elif l.startswith("10th"):
retCode["perc_10th"] = toks[2]
elif l.startswith("75th"):
retCode["perc_75th"] = toks[2]
elif l.startswith("25th"):
retCode["perc_25th"] = toks[2]
log.debug("Insert size stats: %s", str(retCode))
## -------------------------------------------------------------------------
## plotting
rawDataMatrix = np.loadtxt(dataFile, delimiter='\t', comments='#')
## File format
## loc val
## 1 11
try:
rawDataX = rawDataMatrix[:, 0]
rawDataY = rawDataMatrix[:, 1]
except IndexError:
log.info("No solution from bbmerge.")
return RQCExitCodes.JGI_SUCCESS, dataFile, None, None, retCode
fig, ax = plt.subplots()
markerSize = 5.0
lineWidth = 1.5
p1 = ax.plot(rawDataX, rawDataY, 'r', marker='o', markersize=markerSize, linewidth=lineWidth, alpha=0.5)
ax.set_xlabel("Insert Size", fontsize=12, alpha=0.5)
ax.set_ylabel("Count", fontsize=12, alpha=0.5)
ax.grid(color="gray", linestyle=':')
## Add tooltip
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=list(rawDataY)))
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFile)
## Save Matplotlib plot in png format
plt.savefig(plotFile, dpi=fig.dpi)
## Checking outputs
if os.path.isfile(plotFile) and os.path.isfile(dataFile) and os.path.isfile(htmlFile):
log.info("insert_size_analysis completed.")
return RQCExitCodes.JGI_SUCCESS, dataFile, plotFile, htmlFile, retCode
else:
log.error("cannot find the output files. insert_size_analysis failed.")
return RQCExitCodes.JGI_FAILURE, None, None, None, None
""" STEP20 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
gc_divergence_analysis
"""
def gc_divergence_analysis(fastq, bIsPaired, srcDir, log):
## Tools
# rscriptCmd = RQCReadQcCommands.RSCRIPT_CMD
READ_FILES_FILE = RQCReadQcConfig.CFG["files_file"]
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, exitCode = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
## get the bhist.txt produced by reformat.sh
reformatBhistFile = None
with open(READ_FILES_FILE, "r") as FFH:
for l in FFH.readlines():
if l.startswith("read base count text 1"):
reformatBhistFile = l.split("=")[1].strip()
assert os.path.isfile(reformatBhistFile), "reformatBhistFile does not exist: %s" % (l)
break
assert reformatBhistFile, "ERROR: reformatBhistFile cannot be found in %s." % (READ_FILES_FILE)
log.debug("gc_divergence_analysis(): bhist file = %s", reformatBhistFile)
gcDivergenceTransformedFile = os.path.join(qualPath, sequnitFileNamePrefix + ".gc.divergence.transformed.csv") ## gc divergence transformed csv
gcDivergenceTransformedPlot = os.path.join(qualPath, sequnitFileNamePrefix + ".gc.divergence.transformed.png") ## gc divergence transformed plot
gcDivergenceCoefficientsFile = os.path.join(qualPath, sequnitFileNamePrefix + ".gc.divergence.coefficients.csv") ## gc divergence coefficients csv
## Check base composition histogram
if not os.path.isfile(reformatBhistFile):
log.error("Bhist file not found: %s", reformatBhistFile)
return RQCExitCodes.JGI_FAILURE, None, None, None, None
##
## Need R/3.1.2 b/c of library(dplyr) and library(tidyr) are only installed for the R/3.1.2 version
##
## Transform bhist output from reformat.sh to csv
if bIsPaired:
# transformCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; format_signal_data --input %s --output %s --read both --type composition " %\
transformCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; format_signal_data --input %s --output %s --read both --type composition " %\
(reformatBhistFile, gcDivergenceTransformedFile)
# cmd = " ".join(["module load R; Rscript", "--vanilla", os.path.join(SRCDIR, "tools", "jgi-fastq-signal-processing", "format_signal_data"), ])
# transformCmd = "module unload R; module load R; module load R/3.3.2; Rscript --vanilla %s --input %s --output %s --read both --type composition" %\
if os.environ['NERSC_HOST'] in ("denovo", "cori"):
transformCmd = "Rscript --vanilla %s --input %s --output %s --read both --type composition" %\
(os.path.join(srcDir, "tools/jgi-fastq-signal-processing/bin", "format_signal_data"), reformatBhistFile, gcDivergenceTransformedFile)
else:
# transformCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; format_signal_data --input %s --output %s --read 1 --type composition " %\
transformCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; format_signal_data --input %s --output %s --read 1 --type composition " %\
(reformatBhistFile, gcDivergenceTransformedFile)
# transformCmd = "module unload R; module load R; module load R/3.3.2; Rscript --vanilla %s --input %s --output %s --read 1 --type composition" %\
if os.environ['NERSC_HOST'] in ("denovo", "cori"):
transformCmd = "Rscript --vanilla %s --input %s --output %s --read 1 --type composition" %\
(os.path.join(srcDir, "tools/jgi-fastq-signal-processing/bin", "format_signal_data"), reformatBhistFile, gcDivergenceTransformedFile)
log.debug("Transform cmd = %s", transformCmd)
_, _, exitCode = run_sh_command(transformCmd, True, log, True)
if exitCode != 0:
log.info("Failed to run GC_DIVERGENCE_TRANSFORM.")
return -1, None, None, None, None
## Compute divergence value
coeff = []
# modelCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; model_read_signal --input %s --output %s " %\
modelCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; model_read_signal --input %s --output %s " %\
(gcDivergenceTransformedFile, gcDivergenceCoefficientsFile)
# modelCmd = "module unload python; module unload R; module load R/3.3.2; Rscript --vanilla %s --input %s --output %s" %\
if os.environ['NERSC_HOST'] in ("denovo", "cori"):
modelCmd = "Rscript --vanilla %s --input %s --output %s" %\
(os.path.join(srcDir, "tools/jgi-fastq-signal-processing/bin", "model_read_signal"), gcDivergenceTransformedFile, gcDivergenceCoefficientsFile)
log.debug("Model cmd = %s", modelCmd)
_, _, exitCode = run_sh_command(modelCmd, True, log, True)
if exitCode != 0:
log.info("Failed to run GC_DIVERGENCE_MODEL.")
return -1, None, None, None, None
## Parsing coefficients.csv
## ex)
## "read","variable","coefficient"
## "Read 1","AT",2
## "Read 1","AT+CG",2.6
## "Read 1","CG",0.6
## "Read 2","AT",1.7
## "Read 2","AT+CG",2.3
## "Read 2","CG",0.7
assert os.path.isfile(gcDivergenceCoefficientsFile), "GC divergence coefficient file not found."
with open(gcDivergenceCoefficientsFile) as COEFF_FH:
for l in COEFF_FH.readlines():
if l.startswith("\"read\""):
continue ## skip header line
toks = l.strip().split(',')
assert len(toks) == 3, "Unexpected GC divergence coefficient file format."
coeff.append({"read": toks[0], "variable": toks[1], "coefficient": toks[2]})
## Plotting
# plotCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; plot_read_signal --input %s --output %s --type composition " %\
plotCmd = "module unload R; module load R/3.2.4; module load jgi-fastq-signal-processing/2.x; plot_read_signal --input %s --output %s --type composition " %\
(gcDivergenceTransformedFile, gcDivergenceTransformedPlot)
# plotCmd = "module unload python; module unload R; module load R/3.3.2; Rscript --vanilla %s --input %s --output %s --type composition" %\
if os.environ['NERSC_HOST'] in ("denovo", "cori"):
plotCmd = "Rscript --vanilla %s --input %s --output %s --type composition" %\
(os.path.join(srcDir, "tools/jgi-fastq-signal-processing/bin", "plot_read_signal"), gcDivergenceTransformedFile, gcDivergenceTransformedPlot)
log.debug("Plot cmd = %s", plotCmd)
_, _, exitCode = run_sh_command(plotCmd, True, log, True)
if exitCode != 0:
log.info("Failed to run GC_DIVERGENCE_PLOT.")
return -1, None, None, None, None
return RQCExitCodes.JGI_SUCCESS, gcDivergenceTransformedFile, gcDivergenceTransformedPlot, gcDivergenceCoefficientsFile, coeff
""" STEP22 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
cleanup_readqc
Cleaning up the ReadQC analysis directory with unwanted files.
@param log
@return retCode: always return success
"""
def cleanup_readqc(log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
## Purge FASTQs
cmd = "rm -f %s/%s/*.fastq " % (READ_OUTPUT_PATH, "subsample")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
cmd = "rm -f %s/%s/*.fastq " % (READ_OUTPUT_PATH, "qual")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
## Purge FASTA qual and Fasta file
cmd = "rm -f %s/%s/reads.fa " % (READ_OUTPUT_PATH, "megablast")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
cmd = "rm -f %s/%s/reads.qual " % (READ_OUTPUT_PATH, "megablast")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
## Delete Sciclone files
cmd = "rm -f %s/%s/*.fastq " % (READ_OUTPUT_PATH, "sciclone_analysis")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
cmd = "rm -f %s/%s/*.fq " % (READ_OUTPUT_PATH, "sciclone_analysis")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
cmd = "rm -f %s/%s/*.sam " % (READ_OUTPUT_PATH, "sciclone_analysis")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
cmd = "rm -f %s/%s/*.sai " % (READ_OUTPUT_PATH, "sciclone_analysis")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
## Purge Blast files
cmd = "rm -f %s/%s/megablast*v*JFfTIT " % (READ_OUTPUT_PATH, "megablast")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
## purge megablast.reads.fa.v.nt.FmLD2a10p90E30JFfTITW45; megablast.reads.fa.v.refseq.microbial.FmLD2a10p90E30JFfTITW45
cmd = "rm -f %s/%s/megablast*v*FfTITW45 " % (READ_OUTPUT_PATH, "megablast")
_, _, exitCode = run_sh_command(cmd, True, log)
if exitCode != 0:
log.error("Failed to execute %s; may be already purged.", cmd)
return RQCExitCodes.JGI_SUCCESS
## ===========================================================================================================================
""" For NEW STEP4
QC plot generation using the outputs from reformat.sh
"""
def gen_average_base_position_quality_plot(fastq, bIsPaired, log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, _ = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
reformatQhistFile = os.path.join(subsamplePath, sequnitFileNamePrefix + ".reformat.qhist.txt") ## Average Base Position Quality
log.debug("qhist file: %s", reformatQhistFile)
## Gen Average Base Position Quality Plot
if not os.path.isfile(reformatQhistFile):
log.error("Qhist file not found: %s", reformatQhistFile)
return None, None, None
## New data format
## Load data from txt
rawDataMatrix = np.loadtxt(reformatQhistFile, delimiter='\t', comments='#', skiprows=0)
assert len(rawDataMatrix[1][:]) == 5 or len(rawDataMatrix[1][:]) == 3
## New data (paired)
# BaseNum Read1_linear Read1_log Read2_linear Read2_log
# 1 33.469 30.347 32.459 29.127
# 2 33.600 32.236 32.663 29.532
# 3 33.377 30.759 32.768 29.719
fig, ax = plt.subplots()
markerSize = 3.5
lineWidth = 1.0
p1 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 1], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='read1')
if bIsPaired:
p2 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 3], 'g', marker='d', markersize=markerSize, linewidth=lineWidth, label='read2')
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.set_ylabel("Average Quality Score", fontsize=12, alpha=0.5)
ax.set_ylim([0, 45])
fontProp = FontProperties()
fontProp.set_size("small")
fontProp.set_family("Bitstream Vera Sans")
ax.legend(loc=1, prop=fontProp)
ax.grid(color="gray", linestyle=':')
## Add tooltip
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=list(rawDataMatrix[:, 1])))
if bIsPaired:
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p2[0], labels=list(rawDataMatrix[:, 3])))
pngFile = os.path.join(qualPath, sequnitFileNamePrefix + ".r1_r2_baseposqual.png")
htmlFile = os.path.join(qualPath, sequnitFileNamePrefix + ".r1_r2_baseposqual.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFile)
## Save Matplotlib plot in png format
plt.savefig(pngFile, dpi=fig.dpi)
return reformatQhistFile, pngFile, htmlFile
def gen_average_base_position_quality_boxplot(fastq, log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, _ = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
reformatBqhistFile = os.path.join(subsamplePath, sequnitFileNamePrefix + ".reformat.bqhist.txt") ## Average Base Position Quality Boxplot
log.debug("qhist file: %s", reformatBqhistFile)
## Gen Average Base Position Quality Boxplot
if not os.path.isfile(reformatBqhistFile):
log.error("Bqhist file not found: %s", reformatBqhistFile)
return None, None, None, None, None
## New data format (paired)
# 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
##BaseNum count_1 min_1 max_1 mean_1 Q1_1 med_1 Q3_1 LW_1 RW_1 count_2 min_2 max_2 mean_2 Q1_2 med_2 Q3_2 LW_2 RW_2
# 0 6900 0 36 33.48 33 34 34 29 36 6900 0 36 33.48 33 34 34 29 36
rawDataMatrix = np.loadtxt(reformatBqhistFile, delimiter='\t', comments='#', skiprows=0)
assert len(rawDataMatrix[1][:]) == 19 or len(rawDataMatrix[1][:]) == 10
bIsPaired = True
if len(rawDataMatrix[1][:]) == 10:
bIsPaired = False
## create data for boxplot
boxplot_data_r1 = []
boxplot_data_r2 = []
for i in rawDataMatrix:
idx = int(i[0]) - 1 ## read base loc
spread = [rawDataMatrix[idx, 5], rawDataMatrix[idx, 7]] # Q1 ~ Q3
center = [rawDataMatrix[idx, 6]] # median
flier_high = [rawDataMatrix[idx, 8]] # whisker lW 2%
flier_low = [rawDataMatrix[idx, 9]] # whisker rW 98%
boxplot_data_r1.append(np.concatenate((spread, center, flier_high, flier_low), 0))
if bIsPaired:
spread = [rawDataMatrix[idx, 14], rawDataMatrix[idx, 16]] # Q1 ~ Q3
center = [rawDataMatrix[idx, 15]] # median
flier_high = [rawDataMatrix[idx, 17]] # whisker lW 2%
flier_low = [rawDataMatrix[idx, 18]] # whisker rW 98%
boxplot_data_r2.append(np.concatenate((spread, center, flier_high, flier_low), 0))
fig, ax = plt.subplots()
ax.boxplot(boxplot_data_r1)
plt.subplots_adjust(left=0.06, right=0.9, top=0.9, bottom=0.1)
F = plt.gcf()
## How to get the current size?
## DPI = F.get_dpi() ## = 80
## DefaultSize = F.get_size_inches() ## = (8, 6)
F.set_size_inches(18, 6) ## plot size (w, h)
ax.set_xlabel("Read Position", fontsize=11, alpha=0.5)
ax.set_ylabel("Quality Score (Solexa Scale: 40=Highest, -15=Lowest)", fontsize=11, alpha=0.5)
ax.set_ylim([-20, 45])
ax.yaxis.grid(True, linestyle=':', which="major")
majorLocator_x = MultipleLocator(5)
majorFormatter = FormatStrFormatter("%d")
minorLocator = MultipleLocator(1)
ax.xaxis.set_major_locator(majorLocator_x)
ax.xaxis.set_major_formatter(majorFormatter)
ax.xaxis.set_minor_locator(minorLocator)
majorLocator_y = MultipleLocator(5)
minorLocator = MultipleLocator(1)
ax.yaxis.set_major_locator(majorLocator_y)
ax.yaxis.set_minor_locator(minorLocator)
pngFileR1 = os.path.join(qualPath, sequnitFileNamePrefix + ".r1_average_base_position_quality_boxplot.png")
htmlFileR1 = os.path.join(qualPath, sequnitFileNamePrefix + ".r1_average_base_position_quality_boxplot.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFileR1)
## Save Matplotlib plot in png format
plt.savefig(pngFileR1, dpi=fig.dpi)
plotPngPlotFileR2 = None
plotHtmlPlotFileR2 = None
if bIsPaired:
fig, ax = plt.subplots()
ax.boxplot(boxplot_data_r2)
plt.subplots_adjust(left=0.06, right=0.9, top=0.9, bottom=0.1)
F = plt.gcf()
## How to get the current size?
## DPI = F.get_dpi() ## = 80
## DefaultSize = F.get_size_inches() ## = (8, 6)
F.set_size_inches(18, 6) ## plot size (w, h)
ax.set_xlabel("Read Position", fontsize=11, alpha=0.5)
ax.set_ylabel("Quality Score (Solexa Scale: 40=Highest, -15=Lowest)", fontsize=11, alpha=0.5)
ax.set_ylim([-20, 45])
ax.yaxis.grid(True, linestyle=':', which='major')
majorLocator_x = MultipleLocator(5)
majorFormatter = FormatStrFormatter('%d')
minorLocator = MultipleLocator(1)
ax.xaxis.set_major_locator(majorLocator_x)
ax.xaxis.set_major_formatter(majorFormatter)
ax.xaxis.set_minor_locator(minorLocator)
majorLocator_y = MultipleLocator(5)
minorLocator = MultipleLocator(1)
ax.yaxis.set_major_locator(majorLocator_y)
ax.yaxis.set_minor_locator(minorLocator)
plotPngPlotFileR2 = os.path.join(qualPath, sequnitFileNamePrefix + ".r2_average_base_position_quality_boxplot.png")
plotHtmlPlotFileR2 = os.path.join(qualPath, sequnitFileNamePrefix + ".r2_average_base_position_quality_boxplot.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, plotHtmlPlotFileR2)
## Save Matplotlib plot in png format
plt.savefig(plotPngPlotFileR2, dpi=fig.dpi)
return reformatBqhistFile, pngFileR1, plotPngPlotFileR2, htmlFileR1, plotHtmlPlotFileR2
def gen_cycle_nucleotide_composition_plot(fastq, readLength, isPairedEnd, log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, _ = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
reformatBhistFile = os.path.join(subsamplePath, sequnitFileNamePrefix + ".reformat.bhist.txt") ## base composition histogram
log.debug("gen_cycle_nucleotide_composition_plot(): bhist file = %s", reformatBhistFile)
## Genbase composition histogram
if not os.path.isfile(reformatBhistFile):
log.error("Bhist file not found: %s", reformatBhistFile)
return None, None, None, None, None
## data
# 0 1 2 3 4 5
##Pos A C G T N
# 0 0.15111 0.26714 0.51707 0.06412 0.00056
# 1 0.20822 0.20773 0.25543 0.32795 0.00068
rawDataMatrix = np.loadtxt(reformatBhistFile, delimiter='\t', comments='#')
assert len(rawDataMatrix[1][:]) == 6
if isPairedEnd:
rawDataR1 = rawDataMatrix[:readLength - 1][:]
rawDataR2 = rawDataMatrix[readLength:][:]
## r1 ------------------------------------------------------------------
fig, ax = plt.subplots()
markerSize = 2.5
lineWidth = 1.0
p2 = ax.plot(rawDataR1[:, 0], rawDataR1[:, 1], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='A', alpha=0.5)
p3 = ax.plot(rawDataR1[:, 0], rawDataR1[:, 4], 'g', marker='s', markersize=markerSize, linewidth=lineWidth, label='T', alpha=0.5)
p4 = ax.plot(rawDataR1[:, 0], rawDataR1[:, 3], 'b', marker='*', markersize=markerSize, linewidth=lineWidth, label='G', alpha=0.5)
p5 = ax.plot(rawDataR1[:, 0], rawDataR1[:, 2], 'm', marker='d', markersize=markerSize, linewidth=lineWidth, label='C', alpha=0.5)
p6 = ax.plot(rawDataR1[:, 0], rawDataR1[:, 5], 'c', marker='v', markersize=markerSize, linewidth=lineWidth, label='N', alpha=0.5)
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction", fontsize=12, alpha=0.5)
fontProp = FontProperties()
fontProp.set_size("small")
fontProp.set_family("Bitstream Vera Sans")
ax.legend(loc=1, prop=fontProp)
ax.grid(color="gray", linestyle=':')
## Add tooltip
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p2[0], labels=list(rawDataR1[:, 1])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p3[0], labels=list(rawDataR1[:, 4])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p4[0], labels=list(rawDataR1[:, 3])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p5[0], labels=list(rawDataR1[:, 2])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p6[0], labels=list(rawDataR1[:, 5])))
pngFileR1 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_nucl_composition_r1.png")
htmlFileR1 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_nucl_composition_r1.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFileR1)
## Save Matplotlib plot in png format
plt.savefig(pngFileR1, dpi=fig.dpi)
## r2 ------------------------------------------------------------------
fig, ax = plt.subplots()
markerSize = 2.5
lineWidth = 1.0
p2 = ax.plot([(x - readLength) for x in rawDataR2[:, 0]], rawDataR2[:, 1], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='A', alpha=0.5)
p3 = ax.plot([(x - readLength) for x in rawDataR2[:, 0]], rawDataR2[:, 4], 'g', marker='s', markersize=markerSize, linewidth=lineWidth, label='T', alpha=0.5)
p4 = ax.plot([(x - readLength) for x in rawDataR2[:, 0]], rawDataR2[:, 3], 'b', marker='*', markersize=markerSize, linewidth=lineWidth, label='G', alpha=0.5)
p5 = ax.plot([(x - readLength) for x in rawDataR2[:, 0]], rawDataR2[:, 2], 'm', marker='d', markersize=markerSize, linewidth=lineWidth, label='C', alpha=0.5)
p6 = ax.plot([(x - readLength) for x in rawDataR2[:, 0]], rawDataR2[:, 5], 'c', marker='v', markersize=markerSize, linewidth=lineWidth, label='N', alpha=0.5)
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction", fontsize=12, alpha=0.5)
fontProp = FontProperties()
fontProp.set_size("small")
fontProp.set_family("Bitstream Vera Sans")
ax.legend(loc=1, prop=fontProp)
ax.grid(color="gray", linestyle=':')
## Add tooltip
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p2[0], labels=list(rawDataR2[:, 1])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p3[0], labels=list(rawDataR2[:, 4])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p4[0], labels=list(rawDataR2[:, 3])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p5[0], labels=list(rawDataR2[:, 2])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p6[0], labels=list(rawDataR2[:, 5])))
plotPngPlotFileR2 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_nucl_composition_r2.png")
plotHtmlPlotFileR2 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_nucl_composition_r2.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, plotHtmlPlotFileR2)
## Save Matplotlib plot in png format
plt.savefig(plotPngPlotFileR2, dpi=fig.dpi)
return reformatBhistFile, pngFileR1, htmlFileR1, plotPngPlotFileR2, plotHtmlPlotFileR2
else:
fig, ax = plt.subplots()
markerSize = 2.5
lineWidth = 1.0
p2 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 1], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='A', alpha=0.5)
p3 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 4], 'g', marker='s', markersize=markerSize, linewidth=lineWidth, label='T', alpha=0.5)
p4 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 3], 'b', marker='*', markersize=markerSize, linewidth=lineWidth, label='G', alpha=0.5)
p5 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 2], 'm', marker='d', markersize=markerSize, linewidth=lineWidth, label='C', alpha=0.5)
p6 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 5], 'c', marker='v', markersize=markerSize, linewidth=lineWidth, label='N', alpha=0.5)
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction", fontsize=12, alpha=0.5)
fontProp = FontProperties()
fontProp.set_size("small")
fontProp.set_family("Bitstream Vera Sans")
ax.legend(loc=1, prop=fontProp)
ax.grid(color="gray", linestyle=':')
## Add tooltip
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p2[0], labels=list(rawDataMatrix[:, 1])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p3[0], labels=list(rawDataMatrix[:, 4])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p4[0], labels=list(rawDataMatrix[:, 3])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p5[0], labels=list(rawDataMatrix[:, 2])))
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p6[0], labels=list(rawDataMatrix[:, 5])))
pngFile = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_nucl_composition.png")
htmlFile = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_nucl_composition.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFile)
## Save Matplotlib plot in png format
plt.savefig(pngFile, dpi=fig.dpi)
return reformatBhistFile, pngFile, htmlFile, None, None
def gen_cycle_n_base_percent_plot(fastq, readLength, isPairedEnd, log):
READ_OUTPUT_PATH = RQCReadQcConfig.CFG["output_path"]
sequnitFileName, _ = safe_basename(fastq, log)
sequnitFileNamePrefix = sequnitFileName.replace(".fastq", "").replace(".gz", "")
subsampleDir = "subsample"
subsamplePath = os.path.join(READ_OUTPUT_PATH, subsampleDir)
qualDir = "qual"
qualPath = os.path.join(READ_OUTPUT_PATH, qualDir)
make_dir(qualPath)
change_mod(qualPath, "0755")
reformatBhistFile = os.path.join(subsamplePath, sequnitFileNamePrefix + ".reformat.bhist.txt") ## base composition histogram
log.debug("gen_cycle_n_base_percent_plot(): bhist file = %s", reformatBhistFile)
## Genbase composition histogram
if not os.path.isfile(reformatBhistFile):
log.error("Bhist file not found: %s", reformatBhistFile)
return None, None, None, None, None
## data
# 0 1 2 3 4 5
##Pos A C G T N
# 0 0.15111 0.26714 0.51707 0.06412 0.00056
# 1 0.20822 0.20773 0.25543 0.32795 0.00068
rawDataMatrix = np.loadtxt(reformatBhistFile, delimiter='\t', comments='#')
assert len(rawDataMatrix[1][:]) == 6
if isPairedEnd:
rawDataR1 = rawDataMatrix[:readLength - 1][:]
rawDataR2 = rawDataMatrix[readLength:][:]
## r1 ------------------------------------------------------------------
fig, ax = plt.subplots()
markerSize = 5.0
lineWidth = 1.5
p1 = ax.plot(rawDataR1[:, 0], rawDataR1[:, 5], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='N', alpha=0.5)
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction", fontsize=12, alpha=0.5)
ax.grid(color="gray", linestyle=':')
ax.set_xlim([0, readLength])
## Add tooltip
labels = ["%.5f" % i for i in rawDataR1[:, 5]]
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=labels))
pngFileR1 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_n_base_percent_r1.png")
htmlFileR1 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_n_base_percent_r1.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFileR1)
## Save Matplotlib plot in png format
plt.savefig(pngFileR1, dpi=fig.dpi)
## r2 ------------------------------------------------------------------
fig, ax = plt.subplots()
markerSize = 5.0
lineWidth = 1.5
p1 = ax.plot([(x - readLength) for x in rawDataR2[:, 0]], rawDataR2[:, 5], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='N', alpha=0.5)
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction", fontsize=12, alpha=0.5)
ax.grid(color="gray", linestyle=':')
ax.set_xlim([0, readLength])
## Add tooltip
labels = ["%.5f" % i for i in rawDataR2[:, 5]]
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=labels))
plotPngPlotFileR2 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_n_base_percent_r2.png")
plotHtmlPlotFileR2 = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_n_base_percent_r2.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, plotHtmlPlotFileR2)
## Save Matplotlib plot in png format
plt.savefig(plotPngPlotFileR2, dpi=fig.dpi)
return reformatBhistFile, pngFileR1, htmlFileR1, plotPngPlotFileR2, plotHtmlPlotFileR2
else:
fig, ax = plt.subplots()
markerSize = 5.0
lineWidth = 1.5
p1 = ax.plot(rawDataMatrix[:, 0], rawDataMatrix[:, 5], 'r', marker='o', markersize=markerSize, linewidth=lineWidth, label='N', alpha=0.5)
ax.set_xlabel("Read Position", fontsize=12, alpha=0.5)
ax.set_ylabel("Fraction", fontsize=12, alpha=0.5)
ax.grid(color="gray", linestyle=':')
## Add tooltip
labels = ["%.5f" % i for i in rawDataMatrix[:, 5]]
mpld3.plugins.connect(fig, mpld3.plugins.PointLabelTooltip(p1[0], labels=labels))
pngFile = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_n_base_percent.png")
htmlFile = os.path.join(qualPath, sequnitFileNamePrefix + ".cycle_n_base_percent.html")
## Save D3 interactive plot in html format
mpld3.save_html(fig, htmlFile)
## Save Matplotlib plot in png format
plt.savefig(pngFile, dpi=fig.dpi)
return reformatBhistFile, pngFile, htmlFile, None, None
""" For STEP12
sciclone_sam2summary
@param sam_file: input sam file
@param count_file: stat file for writing
@return retCode: success or failure
@return count_file: output count file
"""
## Removed!
##def sciclone_sam2summary(sam_file, log):
""" For STEP12
run_rna_strandedness
Title: run_rna_strandedness
Function: Takes sam file generated from rna data set and determines
mapping to sense and antisense strand
Usage: run_rna_strandedness($sam_file, $log)
Args: 1) sam file
2) log file object
Returns: JGI_SUCCESS
JGI_FAILURE
Comments: None.
@param sam_file
@return retCode
@return outputLogFile: log file
@return outResultDict: stat value dict (to be added to readqc_stats.txt)
"""
## Removed!
""" For STEP12
separate_paired_end_fq
Title : separate_paired_end_fq
Function : Given a fastq file, this function splits the fastq into
read1 and read2 fastq file.
Usage : sequence_lengths( $fastq, $read1_fq, $read2_fq, $log )
Args : 1) The name of a fastq sequence file.
2) The name of the read1 fastq output file
3) The name of the read2 fastq output file
4) A JGI_Log object.
Returns : JGI_SUCCESS: The fastq file was successfully separated.
JGI_FAILURE: The fastq file could not be separated.
Comments : For paired end fastq files only.
sulsj
- Added gzip'd fastq support
## TODO: need to write a fastq IO class
@param fastq
@param read1_outfile
@param read2_outfile
@param log
@return retCode
"""
## Removed!
""" For STEP12
NOTE: Borrowed from alignment.py and updated to get lib name and isRna
get_lib_info
Look up the seqProjectId, bio_name from the library_info table
- to look up the references
@param sequnitFileName: name of the seq unit in the seq_units.sequnitFileName field
@return seqProjectId
@return ncbiOrganismName
@return libraryName
@return isRna
"""
""" For STEP14 & STEP15
run_blastplus_py
Call jgi-rqc-pipeline/tools/run_blastplus.py
"""
def run_blastplus_py(queryFastaFile, db, log):
timeoutCmd = 'timeout' #RQCReadQcCommands.TIMEOUT_CMD
blastOutFileNamePrefix = None
# retCode = None
outDir, exitCode = safe_dirname(queryFastaFile, log)
queryFastaFileBaseName, exitCode = safe_basename(queryFastaFile, log)
dbFileBaseName, exitCode = safe_basename(db, log)
# runBlastnCmd = "/global/homes/s/sulsj/work/bitbucket-repo/jgi-rqc-pipeline/tools/run_blastplus.py" ## debug
# runBlastnCmd = "/global/dna/projectdirs/PI/rqc/prod/jgi-rqc-pipeline/tools/run_blastplus.py"
# runBlastnCmd = "/global/homes/s/sulsj/work/bitbucket-repo/jgi-rqc-pipeline/tools/run_blastplus_taxserver.py" ## debug
runBlastnCmd = "/global/dna/projectdirs/PI/rqc/prod/jgi-rqc-pipeline/tools/run_blastplus_taxserver.py"
blastOutFileNamePrefix = outDir + "/megablast." + queryFastaFileBaseName + ".vs." + dbFileBaseName
## Should use jigsaw/2.6.0 for not checking database reference fasta file
## 07212016 Added -s to add lineage to the subject field
cmd = "%s 21600s %s -d %s -o %s -q %s -s > %s.log 2>&1 " % (timeoutCmd, runBlastnCmd, db, outDir, queryFastaFile, blastOutFileNamePrefix)
_, _, exitCode = run_sh_command(cmd, True, log, True)
## Added timeout to terminate blast run manually after 6hrs
## If exitCode == 124 or exitCode = 143, this means the process exits with timeout.
## Timeout exits with 128 plus the signal number. 143 = 128 + 15 (SGITERM)
## Ref) http://stackoverflow.com/questions/4189136/waiting-for-a-command-to-return-in-a-bash-script
## timeout man page ==> If the command times out, and --preserve-status is not set, then exit with status 124.
##
if exitCode in (124, 143):
## BLAST timeout
## Exit with success so that the blast step can be skipped.
log.warning("##################################")
log.warning("BLAST TIMEOUT. JUST SKIP THE STEP.")
log.warning("##################################")
return RQCExitCodes.JGI_FAILURE, -143
elif exitCode != 0:
log.error("Failed to run_blastplus_py. Exit code != 0")
return RQCExitCodes.JGI_FAILURE, None
else:
log.info("run_blastplus_py complete.")
return RQCExitCodes.JGI_SUCCESS, blastOutFileNamePrefix
"""===========================================================================
checkpoint_step_wrapper
"""
def checkpoint_step_wrapper(status):
assert RQCReadQcConfig.CFG["status_file"]
checkpoint_step(RQCReadQcConfig.CFG["status_file"], status)
"""===========================================================================
get the file content
"""
def get_analysis_file_contents(fullPath):
retCode = ""
if os.path.isfile(fullPath):
with open(fullPath, "r") as FH:
retCode = FH.readlines()
return retCode
else:
return "file not found"
'''===========================================================================
file_name_trim
'''
def file_name_trim(fname):
return fname.replace(".gz", "").replace(".fastq", "").replace(".fasta", "")
## EOF
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