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flaq_sc2_humanclean.nf
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flaq_sc2_humanclean.nf
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#!/usr/bin/env nextflow
/*
Note:
Before running the script, please set the parameters in the config file params.yaml
*/
//Step1:input data files
nextflow.enable.dsl=2
def L001R1Lst = []
def sampleNames = []
myDir = file("$params.input")
myDir.eachFileMatch ~/.*_1.fastq.gz/, {L001R1Lst << it.name}
L001R1Lst.sort()
L001R1Lst.each{
def x = it.minus("_1.fastq.gz")
//println x
sampleNames.add(x)
}
//println L001R1Lst
//println sampleNames
//Step2: process the inputed data
A = Channel.fromList(sampleNames)
//A.view()
process quality {
input:
val x
output:
//stdout
//path 'xfile.txt', emit: aLook
val "${params.output}/${x}", emit: outputpath1
//path "${params.output}/${x}_trim_2.fastq", emit: trimR2
"""
#echo ${params.input}/${x}_1.fastq.gz >> xfile.txt
mkdir -p ${params.output}/assemblies
mkdir -p ${params.output}/variants
mkdir -p ${params.output}/vadr_error_reports
mkdir -p ${params.output}/${x}
cp ${params.input}/${x}_*.fastq.gz ${params.output}/${x}
#Run fastqc on original reads
singularity exec --cleanenv /apps/staphb-toolkit/containers/fastqc_0.11.9.sif fastqc ${params.output}/${x}/${x}_1.fastq.gz ${params.output}/${x}/${x}_2.fastq.gz
mv ${params.output}/${x}/${x}_1_fastqc.html ${params.output}/${x}/${x}_1_original_fastqc.html
mv ${params.output}/${x}/${x}_1_fastqc.zip ${params.output}/${x}/${x}_1_original_fastqc.zip
mv ${params.output}/${x}/${x}_2_fastqc.html ${params.output}/${x}/${x}_2_original_fastqc.html
mv ${params.output}/${x}/${x}_2_fastqc.zip ${params.output}/${x}/${x}_2_original_fastqc.zip
# Run sra-human-scrubber to remove human reads
gzip -d ${params.output}/${x}/${x}_1.fastq.gz
gzip -d ${params.output}/${x}/${x}_2.fastq.gz
singularity exec -B ${params.output}/${x}:/data /apps/staphb-toolkit/containers/sra-human-scrubber_1.1.2021-05-05.sif /opt/scrubber/scripts/scrub.sh -r -i /data/${x}_1.fastq -o /data/${x}_1_humanclean.fastq
singularity exec -B ${params.output}/${x}:/data /apps/staphb-toolkit/containers/sra-human-scrubber_1.1.2021-05-05.sif /opt/scrubber/scripts/scrub.sh -r -i /data/${x}_2.fastq -o /data/${x}_2_humanclean.fastq
gzip ${params.output}/${x}/${x}_1_humanclean.fastq
gzip ${params.output}/${x}/${x}_2_humanclean.fastq
#Run trimmomatic
singularity exec --cleanenv /apps/staphb-toolkit/containers/trimmomatic_0.39.sif trimmomatic PE -phred33 -trimlog ${params.output}/${x}/${x}.log ${params.output}/${x}/${x}_1_humanclean.fastq.gz ${params.output}/${x}/${x}_2_humanclean.fastq.gz ${params.output}/${x}/${x}_trim_1.fastq.gz ${params.output}/${x}/${x}_unpaired_trim_1.fastq.gz ${params.output}/${x}/${x}_trim_2.fastq.gz ${params.output}/${x}/${x}_unpaired_trim_2.fastq.gz SLIDINGWINDOW:4:30 MINLEN:75 TRAILING:20 > ${params.output}/${x}/${x}_trimstats.txt
rm ${params.output}/${x}/${x}_unpaired_trim_*.fastq.gz
rm ${params.output}/${x}/${x}_1.fastq ${params.output}/${x}/${x}_2.fastq
#Run bbduk to remove Illumina adapter sequences and any PhiX contamination
singularity exec --cleanenv /apps/staphb-toolkit/containers/bbtools_38.76.sif bbduk.sh in1=${params.output}/${x}/${x}_trim_1.fastq.gz in2=${params.output}/${x}/${x}_trim_2.fastq.gz out1=${params.output}/${x}/${x}_1.rmadpt.fq.gz out2=${params.output}/${x}/${x}_2.rmadpt.fq.gz ref=/bbmap/resources/adapters.fa stats=${params.output}/${x}/${x}.adapters.stats.txt ktrim=r k=23 mink=11 hdist=1 tpe tbo
singularity exec --cleanenv /apps/staphb-toolkit/containers/bbtools_38.76.sif bbduk.sh in1=${params.output}/${x}/${x}_1.rmadpt.fq.gz in2=${params.output}/${x}/${x}_2.rmadpt.fq.gz out1=${params.output}/${x}/${x}_1.fq.gz out2=${params.output}/${x}/${x}_2.fq.gz outm=${params.output}/${x}/${x}_matchedphix.fq ref=/bbmap/resources/phix174_ill.ref.fa.gz k=31 hdist=1 stats=${params.output}/${x}/${x}_phixstats.txt
rm ${params.output}/${x}/${x}_trim*.fastq.gz
rm ${params.output}/${x}/${x}*rmadpt.fq.gz
#Run fastqc on clean forward and reverse reads
singularity exec --cleanenv /apps/staphb-toolkit/containers/fastqc_0.11.9.sif fastqc ${params.output}/${x}/${x}_1.fq.gz ${params.output}/${x}/${x}_2.fq.gz
#Rename fastqc output files
mv ${params.output}/${x}/${x}_1_fastqc.html ${params.output}/${x}/${x}_1_clean_fastqc.html
mv ${params.output}/${x}/${x}_1_fastqc.zip ${params.output}/${x}/${x}_1_clean_fastqc.zip
mv ${params.output}/${x}/${x}_2_fastqc.html ${params.output}/${x}/${x}_2_clean_fastqc.html
mv ${params.output}/${x}/${x}_2_fastqc.zip ${params.output}/${x}/${x}_2_clean_fastqc.zip
#Run multiqc
singularity exec --cleanenv /apps/staphb-toolkit/containers/multiqc_1.8.sif multiqc ${params.output}/${x}/${x}_*_fastqc.zip -o ${params.output}/${x}
#Map reads to reference
mkdir ${params.output}/${x}/alignment
"""
}
process nofrag {
input:
val mypath
output:
//stdout
val mypath
//path "pyoutputs.txt", emit: pyoutputs
"""
samplename=\$(echo ${mypath} | rev | cut -d "/" -f 1 | rev)
singularity exec /apps/staphb-toolkit/containers/bwa_0.7.17.sif bwa mem ${params.reference}/nCoV-2019.reference.fasta ${mypath}/\${samplename}_1.fq.gz ${mypath}/\${samplename}_2.fq.gz | singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools view - -F 4 -u -h | singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools sort > ${mypath}/alignment/\${samplename}.sorted.bam
#Index final sorted bam
#singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools index ${mypath}/alignment/\${samplename}.sorted.bam
"""
}
process frag {
input:
val mypath
output:
//stdout
val mypath
//path "pyoutputs.txt", emit: pyoutputs
"""
samplename=\$(echo ${mypath} | rev | cut -d "/" -f 1 | rev)
#singularity exec /apps/staphb-toolkit/containers/bwa_0.7.17.sif bwa mem ${params.reference}/nCoV-2019.reference.fasta ${mypath}/\${samplename}_1.fq.gz ${mypath}/\${samplename}_2.fq.gz | singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools view - -F 4 -u -h | singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools sort > ${mypath}/alignment/\${samplename}.sorted.bam
singularity exec /apps/staphb-toolkit/containers/bwa_0.7.17.sif bwa mem ${params.reference}/nCoV-2019.reference.fasta ${mypath}/\${samplename}_1.fq.gz ${mypath}/\${samplename}_2.fq.gz | singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools view - -F 4 -u -h | singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools sort -n > ${mypath}/alignment/\${samplename}.namesorted.bam
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools fixmate -m ${mypath}/alignment/\${samplename}.namesorted.bam ${mypath}/alignment/\${samplename}.fixmate.bam
#Create positional sorted bam from fixmate.bam
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools sort -o ${mypath}/alignment/\${samplename}.positionsort.bam ${mypath}/alignment/\${samplename}.fixmate.bam
#Mark duplicate reads
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools markdup ${mypath}/alignment/\${samplename}.positionsort.bam ${mypath}/alignment/\${samplename}.markdup.bam
#Remove duplicate reads
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools markdup -r ${mypath}/alignment/\${samplename}.positionsort.bam ${mypath}/alignment/\${samplename}.dedup.bam
#Sort dedup.bam and rename to .sorted.bam
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools sort -o ${mypath}/alignment/\${samplename}.sorted.bam ${mypath}/alignment/\${samplename}.dedup.bam
#Index final sorted bam
#singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools index ${mypath}/alignment/\${samplename}.sorted.bam
"""
}
process primer {
input:
val mypath
output:
//stdout
val mypath
//path "pyoutputs.txt", emit: pyoutputs
"""
samplename=\$(echo ${mypath} | rev | cut -d "/" -f 1 | rev)
#Index final sorted bam
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools index ${mypath}/alignment/\${samplename}.sorted.bam
#Trim primers with iVar
ivar trim -i ${mypath}/alignment/\${samplename}.sorted.bam -b ${params.primer}/ARTIC-V4.1.bed -p ${mypath}/alignment/\${samplename}.primertrim -e
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools sort ${mypath}/alignment/\${samplename}.primertrim.bam -o ${mypath}/alignment/\${samplename}.primertrim.sorted.bam
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools index ${mypath}/alignment/\${samplename}.primertrim.sorted.bam
singularity exec /apps/staphb-toolkit/containers/samtools_1.12.sif samtools coverage ${mypath}/alignment/\${samplename}.primertrim.sorted.bam -o ${mypath}/alignment/\${samplename}.coverage.txt
"""
}
process assembly {
input:
val mypath
output:
//stdout
val mypath
//path "pyoutputs.txt", emit: pyoutputs
"""
samplename=\$(echo ${mypath} | rev | cut -d "/" -f 1 | rev)
#Call variants
mkdir ${mypath}/variants
samtools mpileup -A -d 8000 --reference ${params.reference}/nCoV-2019.reference.fasta -B -Q 0 ${mypath}/alignment/\${samplename}.primertrim.sorted.bam | ivar variants -r ${params.reference}/nCoV-2019.reference.fasta -m 10 -p ${mypath}/variants/\${samplename}.variants -q 20 -t 0.25 -g ${params.reference}/GCF_009858895.2_ASM985889v3_genomic.gff
#Generate consensus assembly
mkdir ${mypath}/assembly
samtools mpileup -A -B -d 8000 --reference ${params.reference}/nCoV-2019.reference.fasta -Q 0 ${mypath}/alignment/\${samplename}.primertrim.sorted.bam | ivar consensus -t 0 -m 10 -n N -p ${mypath}/assembly/\${samplename}.consensus
"""
}
process pystats {
input:
val mypath
output:
stdout
//val mypath
//path "pyoutputs.txt", emit: pyoutputs
$/
#!/usr/bin/env python3
import subprocess
items = "${mypath}".strip().split("/")
#print(items[-1])
filepath1 = "${mypath}"+"/alignment/"+items[-1]+".coverage.txt"
#print(filepath1)
with open(filepath1, 'r') as cov_report:
header = cov_report.readline()
header = header.rstrip()
stats = cov_report.readline()
stats = stats.rstrip()
stats = stats.split()
ref_name = stats[0]
#print(ref_name)
start = stats[1]
end = stats[2]
reads_mapped = stats[3]
cov_bases = stats[4]
cov = stats[5]
depth = stats[6]
baseq = stats[7]
#print(reads_mapped)
mapq = stats[8]
#Get number of raw reads
proc_1 = subprocess.run('zcat ' + "${mypath}/" + items[-1] + '_1_humanclean.fastq.gz | wc -l', shell=True, capture_output=True, text=True, check=True)
wc_out_1 = proc_1.stdout.rstrip()
reads_1 = int(wc_out_1) / 4
proc_2 = subprocess.run('zcat ' + "${mypath}/" + items[-1] + '_2_humanclean.fastq.gz | wc -l', shell=True, capture_output=True, text=True, check=True)
wc_out_2 = proc_2.stdout.rstrip()
reads_2 = int(wc_out_2) / 4
raw_reads = reads_1 + reads_2
raw_reads = int(raw_reads)
#Get number of clean reads
proc_c1x = subprocess.run('zcat ' + "${mypath}/" + items[-1] + '_1.fq.gz | wc -l', shell=True, capture_output=True, text=True, check=True)
wc_out_c1x = proc_c1x.stdout.rstrip()
reads_c1x = int(wc_out_c1x) / 4
proc_c2x = subprocess.run('zcat ' + "${mypath}/" + items[-1] + '_2.fq.gz | wc -l', shell=True, capture_output=True, text=True, check=True)
wc_out_c2x = proc_c2x.stdout.rstrip()
reads_c2x = int(wc_out_c2x) / 4
clean_reads = reads_c1x + reads_c2x
clean_reads = int(clean_reads)
#print(clean_reads)
#Get percentage of mapped reads/clean reads
percent_map = "%0.4f"%((int(reads_mapped)/int(clean_reads))*100)
#print(percent_map)
#Gather QC metrics for consensus assembly
filepath2 = "${mypath}"+"/assembly/"+items[-1]+".consensus.fa"
with open(filepath2, 'r') as assem:
header = assem.readline()
header = header.rstrip()
bases = assem.readline()
bases = bases.rstrip()
num_bases = len(bases)
ns = bases.count('N')
called = num_bases - ns
pg = "%0.4f"%((called/int(end))*100)
#print(called)
#print(end)
#print(pg)
#Rename header in fasta to just sample name
subprocess.run("sed -i \'s/^>.*/>"+items[-1]+"/\' "+filepath2, shell=True, check=True)
#print("sed -i \'s/^>.*/>"+items[-1]+"/\' "+filepath2)
#QC flag
pg_flag = ''
dp_flag = ''
qc_flag = ''
if float(pg) < 79.5:
pg_flag = 'FAIL: Percent genome < 80%'
qc_flag = qc_flag + pg_flag
else:
if float(depth) < 100:
dp_flag = 'FAIL: Mean read depth < 100x'
qc_flag = qc_flag + dp_flag
if qc_flag == '':
qc_flag = qc_flag + 'PASS'
#print(qc_flag)
if qc_flag == 'PASS':
subprocess.run("cp "+filepath2+" "+"${params.output}"+"/assemblies/", shell=True, check=True)
subprocess.run('cp ' + "${mypath}" + '/variants/' + items[-1] + '.variants.tsv ' + "${params.output}"+'/variants/', shell=True, check=True)
#Run VADR
out_log = open("${mypath}/"+items[-1]+'.out', 'w')
err_log = open("${mypath}/"+items[-1]+'.err', 'w')
subprocess.run("singularity exec -B "+"${mypath}"+"/assembly"+":/data /apps/staphb-toolkit/containers/vadr_1.3.sif /opt/vadr/vadr/miniscripts/fasta-trim-terminal-ambigs.pl --minlen 50 --maxlen 30000 /data/" + items[-1]+".consensus.fa > " + "${mypath}"+"/assembly/"+items[-1]+".trimmed.fasta", shell=True, stdout=out_log, stderr=err_log, check=True)
subprocess.run("singularity exec -B "+"${mypath}"+"/assembly:/data /apps/staphb-toolkit/containers/vadr_1.3.sif v-annotate.pl --split --cpu 8 --glsearch -s -r --nomisc --mkey sarscov2 --lowsim5seq 6 --lowsim3seq 6 --alt_fail lowscore,insertnn,deletinn --mdir /opt/vadr/vadr-models/ /data/"+items[-1]+".trimmed.fasta -f /data/"+"vadr_results", shell=True, stdout=out_log, stderr=err_log, check=True)
#Parse through VADR outputs to get PASS or REVIEW flag
vadr_flag = ''
with open("${mypath}"+"/assembly/vadr_results/vadr_results.vadr.pass.list", 'r') as p_list:
result = p_list.readline()
result = result.rstrip()
if result == items[-1]:
vadr_flag = 'PASS'
with open("${mypath}"+"/assembly/vadr_results/vadr_results.vadr.fail.list", 'r') as f_list:
result = f_list.readline()
result = result.rstrip()
if result == items[-1]:
vadr_flag = 'REVIEW'
#Copy VADR error report to main analysis folder for easier review
if vadr_flag == 'REVIEW':
subprocess.run("cp " + "${mypath}"+"/assembly/vadr_results/vadr_results.vadr.alt.list "+"${params.output}"+"/vadr_error_reports/"+items[-1]+".vadr.alt.list", shell=True, check=True)
#subprocess.run('mv vadr_error_reports/vadr_results.vadr.alt.list vadr_error_reports/' + s + '.vadr.alt.list', shell=True, check=True)
#print("cp " + "${mypath}"+"/assembly/vadr_results/vadr_results.vadr.alt.list "+"${params.output}"+"/vadr_error_reports/"+items[-1]+".vadr.alt.list")
#Run pangolin
###--pango_path /apps/staphb-toolkit/containers/pangolin_4.1.2-pdata-1.13.sif --pangolin v4.1.2 --pangolin_data v1.13
pangolin = "v4.1.2_pdata-v1.13"
subprocess.run('singularity exec -B '+"${mypath}"+"/assembly:/data /apps/staphb-toolkit/containers/pangolin_4.1.2-pdata-1.13.sif" + ' pangolin -o /data /data/'+items[-1]+".consensus.fa", shell=True, check=True)
#Get lineage
proc = subprocess.run("tail -n 1 "+"${mypath}"+"/assembly/lineage_report.csv | cut -d \',\' -f 2", shell=True, check=True, capture_output=True, text=True)
lineage = proc.stdout.rstrip()
#print(lineage)
#Run nextclade
subprocess.run("singularity exec -B "+"${mypath}"+"/assembly:/data /apps/staphb-toolkit/containers/nextclade_2021-03-15.sif nextclade --input-fasta /data/"+items[-1]+".consensus.fa --output-csv /data/nextclade_report.csv", shell=True, check=True)
#Parse nextclade output and screen for sotc
sotc_v = "${params.sotc}".split(',')
with open("${mypath}"+"/assembly/nextclade_report.csv", 'r') as nc:
header = nc.readline()
c_results = nc.readline()
c_results = c_results.rstrip()
data = c_results.split(',')
#print(data)
sotc = []
for v in sotc_v:
print(v)
if v in data:
sotc.append(v)
sotc_out = (',').join(sotc)
out_log.close()
err_log.close()
else:
vadr_flag = 'NA'
lineage = 'NA'
sotc_out = 'NA'
with open("${mypath}"+"/report.txt", 'w') as report:
header = ['sampleID', 'reference', 'start', 'end', 'num_raw_reads', 'num_clean_reads', 'num_mapped_reads', 'percent_mapped_clean_reads', 'cov_bases_mapped', 'percent_genome_cov_map', 'mean_depth', 'mean_base_qual', 'mean_map_qual', 'assembly_length', 'numN', 'percent_ref_genome_cov', 'VADR_flag', 'QC_flag', 'pangolin_version', 'lineage', 'SOTC']
report.write('\t'.join(map(str,header)) + '\n')
results = [items[-1], ref_name, start, end, raw_reads, clean_reads, reads_mapped, percent_map, cov_bases, cov, depth, baseq, mapq, num_bases, ns, pg, vadr_flag, qc_flag, pangolin, lineage, sotc_out]
report.write('\t'.join(map(str,results)) + '\n')
/$
}
workflow {
if("${params.frag}" == "frag"){
quality(A) | frag | primer | assembly | pystats | view
}
else{
quality(A) | nofrag | primer | assembly | pystats | view
}
}