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main.nf
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#!/usr/bin/env nextflow
nextflow.enable.dsl = 2
/*
VirReport workflow
Roberto Barrero, 14/03/2019
Desmond Schmidt, 2/7/2019
Converted to Nextflow by Craig Windell, 11/2020
Modified by Maely Gauthier, 2021-2023
*/
import java.util.*;
import java.util.stream.IntStream;
import java.util.stream.Collectors;
def helpMessage () {
log.info """
VirReport workflow
Roberto Barrero, 14/03/2019
Desmond Schmidt, 2/7/2019
Converted to Nextflow by Craig Windell, 11/2020
Modified by Maely Gauthier, 2021-2023
Usage:
Run the command
nextflow run eresearchqut/virreport -profile ...
Mandatory arguments:
-profile '[docker, singularity]' Profile to use. Choose docker or singularity
Optional arguments:
--indexfile '[path/to/file]' Path to the csv file that contains the list of
samples to be analysed by this pipeline.
'index.csv'
Contents of indexfile csv:
sampleid,samplepath
MT019,/user/folder/MT019_sRNA.fastq
--blast_db_dir '[path/to/files]' Path to the blast NT and/or NR database file base name
[none]
--blast_viral_db Run blastn and megablast homology search on cap3 de novo assembly against a virus and viroid database
[False]
--blast_viral_nt_db '[path/to/file]' Path to the viral nucleotide database file base name. Required if --blast_viral_db option is specified
[none]
--blastn_evalue '[value]' Blastn evalue.
'0.0001'
--blastn_method ['blastn/megablast'] Specify blastn homology search on cap3 de novo assembly againts NCBI NT
[default megablast]
--blastx [True/False] Run blastX againts NCBI NR
[False]
--bowtie_db_dir Path to the bowtie indices (for RNA source step and filtering of non-informative reads)
--cap3_len '[value]' Trim value used in the CAP3 step.
'40'
--contamination_detection [True/False] Run false positive prediction due to cross-sample contamination for detections
obtained via blastn search against NT
[False]
--contamination_detection_viral_db Run false positive prediction due to cross-sample contamination for detections
obtained via blastn search against a viral database
[False]
--contamination_flag '[value]' Threshold value to predict false positives due to cross-sample contamination.
Required if --contamination_detection option is specified
'0.01'
--dedup Use UMI-tools dedup to remove duplicate reads
--maxlen '[value]' Maximum read length to extract
['22']
--merge_lane Specify this option if sequencing was peformed on several flow cells and 2 or more fastq files were generated for one sample and require to be merged
--minlen '[value]' Minimum read length to extract
['21']
--orf_circ_minsize '[value]' The value of minsize for getorf -circular
'75'
--orf_minsize '[value]' The value of minsize for getorf
'75'
--qualityfilter [True/False] Perform adapter and quality filtering of fastq files
[False]
--rna_source_profile Evaluates the sRNA library content
[False]
--spadesmem '[value]' Memory usage for SPAdes de novo assembler
[60]
--targets [True/False] Filter the blastn results to viruses/viroids of interest
[False]
--targets_file '[path/to/folder]' File specifying the name of the viruses/viroids of interest to filter from the blast results output
['Targetted_Viruses_Viroids.txt']
--tblastn_evalue tblastn evalue. Required if --tblatsn option is specified
'0.0001'
--virusdetect [True/False] Run VirusDetect
[False]
--virusdetect_db_path '[path/to/filebasename]' Path to the virusdetect blast virus database base name
[none]
####
Internal SSG usage only
--diagno Additional information will be added to each viral detection to facilitate interpretation
[False]
--synthetic_oligos Reads will be aligned to specific synthetic oligos
[False]
--sampleinfo Appends additional sample information to final summary to facilitate diagnostics reporting
[False]
--sampleinfo_path Path_to_sample_info to be appended
[none]
--samplesheet_path Path_to_sample_sheet to be appended
[none]
""".stripIndent()
}
// Show help message
if (params.help) {
helpMessage()
exit 0
}
if (params.blast_db_dir != null) {
blastn_db_name = "${params.blast_db_dir}/nt"
blastp_db_name = "${params.blast_db_dir}/nr"
}
if (params.blast_viral_db_path != null) {
blast_viral_db_name = file(params.blast_viral_db_path).name
blast_viral_db_dir = file(params.blast_viral_db_path).parent
}
if (params.virusdetect_db_path != null) {
virusdetect_db_dir = file(params.virusdetect_db_path).parent
}
size_range = "${params.minlen}-${params.maxlen}nt"
if (params.sampleinfo_path != null) {
sampleinfo_dir = file(params.sampleinfo_path).parent
sampleinfo_name = file(params.sampleinfo_path).name
}
if (params.samplesheet_path != null) {
samplesheet_dir = file(params.samplesheet_path).parent
samplesheet_name = file(params.samplesheet_path).name
}
switch (workflow.containerEngine) {
case "docker":
bindbuild = "";
if (params.blast_viral_db_path != null) {
bindbuild = "-v ${blast_viral_db_dir}:${blast_viral_db_dir} "
}
if (params.blast_db_dir != null) {
bindbuild = (bindbuild + "-v ${params.blast_db_dir}:${params.blast_db_dir} ")
}
if (params.bowtie_db_dir != null) {
bindbuild = (bindbuild + "-v ${params.bowtie_db_dir}:${params.bowtie_db_dir} ")
}
if (params.virusdetect_db_path != null) {
bindbuild = (bindbuild + "-v ${virusdetect_db_dir}:${virusdetect_db_dir} ")
}
if (params.sampleinfo_path != null) {
bindbuild = (bindbuild + "-v ${sampleinfo_dir}:${sampleinfo_dir} ")
}
if (params.samplesheet_path != null) {
bindbuild = (bindbuild + "-v ${samplesheet_dir}:${samplesheet_dir} ")
}
bindOptions = bindbuild;
break;
case "singularity":
bindbuild = "";
if (params.blast_viral_db_path != null) {
bindbuild = "-B ${blast_viral_db_dir} "
}
if (params.blast_db_dir != null) {
bindbuild = (bindbuild + "-B ${params.blast_db_dir} ")
}
if (params.bowtie_db_dir != null) {
bindbuild = (bindbuild + "-B ${params.bowtie_db_dir} ")
}
if (params.virusdetect_db_path != null) {
bindbuild = (bindbuild + "-B ${virusdetect_db_dir} ")
}
if (params.sampleinfo_path != null) {
bindbuild = (bindbuild + "-B ${sampleinfo_dir} ")
}
if (params.samplesheet_path != null) {
bindbuild = (bindbuild + "-B ${samplesheet_dir} ")
}
bindOptions = bindbuild;
break;
default:
bindOptions = "";
}
process FASTQC_RAW {
tag "$sampleid"
publishDir "${params.outdir}/00_quality_filtering/${sampleid}", mode: 'copy'
input:
tuple val(sampleid), file(fastqfile_path)
output:
path("*_fastqc.{zip,html}")
script:
"""
fastqc --quiet --threads ${task.cpus} ${fastqfile_path}
"""
}
process MERGE_LANES {
tag "$sampleid"
input:
tuple val(sampleid), path(samplepath)
output:
tuple val(sampleid), file("${sampleid}_R1.merged.fastq.gz"), emit: merged
script:
if (params.merge_lane) {
samplepathList = samplepath.collect{it.toString()}
if (samplepathList.size > 1 ) {
"""
cat ${samplepath} > ${sampleid}_R1.merged.fastq.gz
"""
}
} else {
"""
ln ${samplepath} ${sampleid}_R1.merged.fastq.gz
"""
}
}
//This step takes > 1h to run for the large flow cells
process ADAPTER_TRIMMING {
label "setting_6"
tag "$sampleid"
publishDir "${params.outdir}/00_quality_filtering/${sampleid}", mode: 'link', overwrite: true, pattern: "*{log,json,html,trimmed.fastq.gz,zip,html,pdf,txt}"
input:
tuple val(sampleid), path(fastqfile)
output:
path("${sampleid}_umi_tools.log")
path("${sampleid}_truseq_adapter_cutadapt.log")
path("${sampleid}_umi_tools.log"), emit: umi_tools_results
tuple val(sampleid), path(fastqfile), path("${sampleid}_umi_cleaned.fastq.gz"), emit: adapter_trimmed
tuple val(sampleid), path("${sampleid}_umi_cleaned.fastq.gz"), emit: adapter_trimmed2
script:
"""
#Checks Illumina seq adapters have been removed
cutadapt -j ${task.cpus} \
--no-indels \
-a "AGATCGGAAGAGCACACGTCTGAACTCCAGTCA;min_overlap=12" \
-g "ACACTCTTTCCCTACACGACGCTCTTCCGATCT;min_overlap=9" \
--times 2 \
-o ${sampleid}_trimmed.fastq.gz \
${fastqfile} > ${sampleid}_truseq_adapter_cutadapt.log
umi_tools extract --extract-method=regex \
--bc-pattern=".+(?P<discard_1>AACTGTAGGCACCATCAAT){s<=2}(?P<umi_1>.{12})\$" \
-I ${sampleid}_trimmed.fastq.gz \
-S ${sampleid}_umi_cleaned.fastq.gz > ${sampleid}_umi_tools.log
rm ${sampleid}_trimmed.fastq.gz
"""
}
process QUAL_TRIMMING_AND_QC {
label "setting_3"
tag "$sampleid"
publishDir "${params.outdir}/00_quality_filtering/${sampleid}", mode: 'link', overwrite: true, pattern: "*{log,json,html,trimmed.fastq.gz,zip,html,png,pdf,txt}"
input:
tuple val(sampleid), path(fastqfile), path(fastq_filt_by_size)
output:
file "*_fastqc.{zip,html}"
file "${sampleid}_fastp.json"
file "${sampleid}_fastp.html"
file "${sampleid}_read_length_dist.pdf"
file "${sampleid}_read_length_dist.png"
file "${sampleid}_read_length_dist.txt"
file "${sampleid}_quality_trimmed.fastq.gz"
file "${sampleid}_qual_filtering_cutadapt.log"
path("${sampleid}_qual_filtering_cutadapt.log"), emit: cutadapt_qual_filt_results
tuple val(sampleid), file(fastqfile), path("${sampleid}_quality_trimmed.fastq"), emit: qual_trimmed
path("${sampleid}_fastp.json"), emit: fastp_results
path("${sampleid}_read_length_dist.txt"), emit: read_length_dist_results
script:
"""
cutadapt -j ${task.cpus} \
--trim-n --max-n 0 -m 18 -q 30 \
-o ${sampleid}_quality_trimmed.fastq \
${sampleid}_umi_cleaned.fastq.gz > ${sampleid}_qual_filtering_cutadapt.log
pigz --best --force -p ${task.cpus} -r ${sampleid}_quality_trimmed.fastq -c > ${sampleid}_quality_trimmed.fastq.gz
fastqc --quiet --threads ${task.cpus} ${sampleid}_quality_trimmed.fastq.gz
fastp --in1=${sampleid}_quality_trimmed.fastq.gz --out1=${sampleid}_fastp_trimmed.fastq.gz \
--disable_adapter_trimming \
--disable_quality_filtering \
--disable_length_filtering \
--json=${sampleid}_fastp.json \
--html=${sampleid}_fastp.html \
--thread=${task.cpus}
#derive distribution for quality filtered reads > 5 bp long
cutadapt -j ${task.cpus} \
--trim-n --max-n 0 -m 5 -q 30 \
-o ${sampleid}_quality_trimmed_temp.fastq \
${sampleid}_umi_cleaned.fastq.gz
fastq2fasta.pl ${sampleid}_quality_trimmed_temp.fastq > ${sampleid}_quality_trimmed.fasta
read_length_dist.py --input ${sampleid}_quality_trimmed.fasta
mv ${sampleid}_quality_trimmed.fasta_read_length_dist.txt ${sampleid}_read_length_dist.txt
mv ${sampleid}_quality_trimmed.fasta_read_length_dist.png ${sampleid}_read_length_dist.png
mv ${sampleid}_quality_trimmed.fasta_read_length_dist.pdf ${sampleid}_read_length_dist.pdf
rm ${sampleid}_quality_trimmed_temp.fastq
"""
}
process RNA_SOURCE_PROFILE {
label "setting_2"
tag "$sampleid"
publishDir "${params.outdir}/00_quality_filtering/${sampleid}", mode: 'link', overwrite: true, pattern: "*{log}"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(fastqfile)
output:
path("${sampleid}_bowtie.log")
path("${sampleid}_bowtie.log"), emit: rna_source_bowtie_results
script:
"""
cutadapt -j ${task.cpus} \
--trim-n --max-n 0 -m 15 -q 30 \
-o ${sampleid}_quality_trimmed_temp2.fastq \
${sampleid}_umi_cleaned.fastq.gz
#derive distribution for quality filtered reads > 15 bp bp long
echo ${sampleid} > ${sampleid}_bowtie.log;
count=1
for rnatype in rRNA miRNA plant_tRNA plant_pt_mt_other_genes plant_noncoding artefacts plant_virus_viroid; do
if [[ \${count} == 1 ]]; then
fastqfile=${sampleid}_quality_trimmed_temp2.fastq
fi
echo \${rnatype} alignment: >> ${sampleid}_bowtie.log;
bowtie -q -v 1 -k 1 -p ${task.cpus} \
--un ${sampleid}_\${rnatype}_cleaned_sRNA.fq \
-x ${params.bowtie_db_dir}/\${rnatype} \
\${fastqfile} \
${sampleid}_\${rnatype}_match 2>>${sampleid}_bowtie.log
count=\$((count+1))
if [[ \${count} > 1 ]]; then
fastqfile=${sampleid}_\${rnatype}_cleaned_sRNA.fq
fi
rm ${sampleid}_\${rnatype}_match;
done
rm *cleaned_sRNA.fq
"""
}
process RNA_SOURCE_PROFILE_REPORT {
publishDir "${params.outdir}/00_quality_filtering/qc_report", mode: 'link'
containerOptions "${bindOptions}"
input:
path("*bowtie.log")
output:
path("read_origin_pc_summary*.txt")
path("read_origin_counts*.txt")
path("read_RNA_source*.pdf")
path("read_RNA_source*.png")
path("read_origin_detailed_pc*.txt")
script:
"""
rna_source_summary.py
"""
}
process DERIVE_USABLE_READS {
label "setting_4"
tag "$sampleid"
publishDir "${params.outdir}/00_quality_filtering/${sampleid}", mode: 'link', overwrite: true, pattern: "*{.log,.fastq.gz}"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(fastqfile), file(qual_trimmed_fastqfile)
output:
path("${sampleid}*_cutadapt.log")
path("${sampleid}_blacklist_filter.log")
path("${sampleid}_${params.minlen}-${params.maxlen}nt.fastq.gz")
tuple val(sampleid),
path(fastqfile),
path("${sampleid}_${params.minlen}-${params.maxlen}nt.fastq"),
emit: usable_reads
path("*_18-25nt_cutadapt.log"), emit: cutadapt_18_25nt_results
path("*_21-22nt_cutadapt.log"), emit: cutadapt_21_22nt_results
path("*_24nt_cutadapt.log"), emit: cutadapt_24nt_results
path("*_blacklist_filter.log"), emit: bowtie_usable_read_results
script:
"""
bowtie -q -v 1 \
-k 1 --un ${sampleid}_cleaned.fastq -p ${task.cpus} \
-x ${params.bowtie_db_dir}/blacklist \
${qual_trimmed_fastqfile} \
${sampleid}_blacklist_match 2>${sampleid}_blacklist_filter.log
cutadapt -j ${task.cpus} -m 18 -M 25 -o ${sampleid}_18-25nt.fastq.gz ${sampleid}_cleaned.fastq > ${sampleid}_18-25nt_cutadapt.log
cutadapt -j ${task.cpus} -m 21 -M 22 -o ${sampleid}_21-22nt.fastq ${sampleid}_cleaned.fastq > ${sampleid}_21-22nt_cutadapt.log
cutadapt -j ${task.cpus} -m 24 -M 24 -o ${sampleid}_24nt.fastq.gz ${sampleid}_cleaned.fastq > ${sampleid}_24nt_cutadapt.log
if [[ ${params.minlen} != 21 ]] || [[ ${params.maxlen} != 22 ]]; then
cutadapt -j ${task.cpus} -m ${params.minlen} -M ${params.maxlen} -o ${sampleid}_${params.minlen}-${params.maxlen}nt.fastq ${sampleid}_cleaned.fastq > ${sampleid}_${params.minlen}-${params.maxlen}nt_cutadapt.log
fi
rm ${sampleid}_24nt.fastq.gz ${sampleid}_18-25nt.fastq.gz
pigz --best --force -p ${task.cpus} -r ${sampleid}_${params.minlen}-${params.maxlen}nt.fastq -c > ${sampleid}_${params.minlen}-${params.maxlen}nt.fastq.gz
"""
}
/*
file("*qual_filtering_cutadapt.log")
file("*fastp.json")
file("*_read_length_dist.txt")
file("*_18-25nt_cutadapt.log")
file("*_21-22nt_cutadapt.log")
file("*_24nt_cutadapt.log")
file("*_blacklist_filter.log")
file("*_umi_tools.log")
*/
process QCREPORT {
publishDir "${params.outdir}/00_quality_filtering/qc_report", mode: 'link', overwrite: true
containerOptions "${bindOptions}"
input:
path multiqc_files
output:
path("run_qc_report*.txt")
path("run_read_size_distribution*.pdf")
path("run_read_size_distribution*.png")
script:
"""
if [[ ${params.sampleinfo} == true ]]; then
seq_run_qc_report.py --sampleinfopath ${params.sampleinfo_path} --samplesheetpath ${params.samplesheet_path}
else
seq_run_qc_report.py
fi
grouped_bar_chart.py
"""
}
process READPROCESSING {
tag "$sampleid"
publishDir "${params.outdir}/01_VirReport/${sampleid}/assembly", mode: 'link'
input:
tuple val(sampleid), file(fastqfile)
output:
path("${sampleid}_${size_range}_cutadapt.log")
path("${sampleid}_${size_range}.fastq")
tuple val(sampleid),
path("unzipped.fastq"),
path("${sampleid}_${size_range}.fastq"),
emit: fastq
script:
"""
if [[ ${fastqfile} == *.gz ]];
then
gunzip -c ${fastqfile} > unzipped.fastq
else
ln ${fastqfile} unzipped.fastq
fi
cutadapt -j ${task.cpus} -m ${params.minlen} -M ${params.maxlen} -o ${sampleid}_${size_range}.fastq unzipped.fastq > ${sampleid}_${size_range}_cutadapt.log
"""
}
// This process performs separate velvet and SPAdes de novo assemblies
// After merging the assemblies, the contigs are collapsed using cap3
process DENOVO_ASSEMBLY {
publishDir "${params.outdir}/01_VirReport/${sampleid}/assembly", mode: 'link', overwrite: true, pattern: "*{fasta,log}"
tag "$sampleid"
label "setting_1"
input:
tuple val(sampleid), file(fastqfile), file(fastq_filt_by_size)
output:
file "${sampleid}_velvet_assembly_${size_range}.fasta"
file "${sampleid}_velvet_log"
file "${sampleid}_spades_assembly_${size_range}.fasta"
file "${sampleid}_spades_log"
file "${sampleid}_cap3_${size_range}.fasta"
tuple val(sampleid),
file(fastqfile),
file(fastq_filt_by_size),
file("${sampleid}_cap3_${size_range}.fasta"),
emit: assembly_for_blastn
tuple val(sampleid),
file("${sampleid}_cap3_${size_range}.fasta"),
emit: assembly_for_tblastn
script:
"""
#run velvet de novo assembler
export OMP_NUM_THREADS=2
echo 'Starting velvet de novo assembly';
velveth ${sampleid}_velvet_${size_range}_k15 15 -short -fastq ${fastq_filt_by_size}
velvetg ${sampleid}_velvet_${size_range}_k15 -exp_cov 2
#edit contigs name and rename velvet assembly
sed 's/>/>velvet_/' ${sampleid}_velvet_${size_range}_k15/contigs.fa > ${sampleid}_velvet_assembly_${size_range}.fasta
cp ${sampleid}_velvet_${size_range}_k15/Log ${sampleid}_velvet_log
#run spades de novo assembler
spades.py --rna -t ${task.cpus} -k 19,21 -m ${params.spadesmem} -s ${fastq_filt_by_size} -o ${sampleid}_spades_k19_21
#edit contigs name and rename spades assembly
if [[ ! -s ${sampleid}_spades_k19_21/transcripts.fasta ]]
then
touch ${sampleid}_spades_assembly_${size_range}.fasta
else
sed 's/>/>spades_/' ${sampleid}_spades_k19_21/transcripts.fasta > ${sampleid}_spades_assembly_${size_range}.fasta
fi
cp ${sampleid}_spades_k19_21/spades.log ${sampleid}_spades_log
#merge velvet and spades assemblies
cat ${sampleid}_velvet_assembly_${size_range}.fasta ${sampleid}_spades_assembly_${size_range}.fasta > ${sampleid}_merged_spades_velvet_assembly_${size_range}.fasta
#collapse derived contigs
cap3 ${sampleid}_merged_spades_velvet_assembly_${size_range}.fasta -s 300 -j 31 -i 30 -p 90 -o 16
cat ${sampleid}_merged_spades_velvet_assembly_${size_range}.fasta.cap.singlets ${sampleid}_merged_spades_velvet_assembly_${size_range}.fasta.cap.contigs > ${sampleid}_cap3_${size_range}_temp.fasta
#retain only contigs > 30 bp long
extract_seqs_rename.py ${sampleid}_cap3_${size_range}_temp.fasta ${params.cap3_len} \
| sed "s/CONTIG/${sampleid}_${params.minlen}-${params.maxlen}_/" \
| sed 's/|>/ |/' | awk '{print \$1}' \
> ${sampleid}_cap3_${size_range}.fasta
"""
}
process BLASTN_VIRAL_DB_CAP3 {
label "setting_4"
publishDir "${params.outdir}/01_VirReport/${sampleid}/blastn/viral_db", mode: 'link', overwrite: true, pattern: "*{vs_viral_db.bls,.txt}"
tag "$sampleid"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(fastqfile), file(fastq_filt_by_size), file("${sampleid}_cap3_${size_range}.fasta")
output:
file "${sampleid}_cap3_${size_range}_blastn_vs_viral_db.bls"
file "${sampleid}_cap3_${size_range}_megablast_vs_viral_db.bls"
tuple val(sampleid),
file(fastqfile),
file(fastq_filt_by_size),
file("${sampleid}_cap3_${size_range}.fasta"),
file("${sampleid}_cap3_${size_range}_blastn_vs_viral_db.bls"),
file("${sampleid}_cap3_${size_range}_megablast_vs_viral_db.bls"),
emit: blast_results
script:
"""
#1. blastn search
blastn -task blastn \
-query ${sampleid}_cap3_${size_range}.fasta \
-db ${blast_viral_db_dir}/${blast_viral_db_name} \
-out ${sampleid}_cap3_${size_range}_blastn_vs_viral_db.bls \
-evalue ${params.blastn_evalue} \
-num_threads ${task.cpus} \
-outfmt '6 qseqid sgi sacc length pident mismatch gapopen qstart qend qlen sstart send slen sstrand evalue bitscore qcovhsp stitle staxids qseq sseq sseqid qcovs qframe sframe' \
-max_target_seqs 50
#2. megablast search
blastn -query ${sampleid}_cap3_${size_range}.fasta \
-db ${blast_viral_db_dir}/${blast_viral_db_name} \
-out ${sampleid}_cap3_${size_range}_megablast_vs_viral_db.bls \
-evalue ${params.blastn_evalue} \
-num_threads ${task.cpus} \
-outfmt '6 qseqid sgi sacc length pident mismatch gapopen qstart qend qlen sstart send slen sstrand evalue bitscore qcovhsp stitle staxids qseq sseq sseqid qcovs qframe sframe' \
-max_target_seqs 50
"""
}
process FILTER_BLASTN_VIRAL_DB_CAP3 {
publishDir "${params.outdir}/01_VirReport/${sampleid}/blastn/viral_db", mode: 'link', overwrite: true, pattern: "*{.txt}"
tag "$sampleid"
input:
tuple val(sampleid), \
file(fastqfile), \
file(fastq_filt_by_size), \
file("${sampleid}_cap3_${size_range}.fasta"), \
file("${sampleid}_cap3_${size_range}_blastn_vs_viral_db.bls"), \
file("${sampleid}_cap3_${size_range}_megablast_vs_viral_db.bls")
output:
file "summary_${sampleid}_cap3_${size_range}_*_vs_viral_db.bls_viruses_viroids*.txt"
file "summary_${sampleid}_cap3_${size_range}_*_vs_viral_db.bls_filtered.txt"
tuple val(sampleid),
file(fastqfile),
file(fastq_filt_by_size),
file("summary_${sampleid}_cap3_${size_range}_megablast_vs_viral_db.bls_viruses_viroids.txt"),
emit: viral_db_blast_results
script:
"""
c1grep() { grep "\$@" || test \$? = 1; }
#retain 1st blast hit
for var in ${sampleid}_cap3_${size_range}_megablast_vs_viral_db.bls ${sampleid}_cap3_${size_range}_blastn_vs_viral_db.bls;
do
cat \${var} | awk '{print \$1}' | sort | uniq > \${var}.top1.ids
for i in `cat \${var}.top1.ids`; do echo "fetching top hits..." \$i; grep \$i \${var} | head -1 >> \${var}.top1Hits.txt ; done
cat \${var}.top1Hits.txt | sed 's/ /_/g' > \${var}.txt
#summarise the blast files
java -jar ${projectDir}/bin/BlastTools.jar -t blastn \${var}.txt
sequence_length.py --virus_list summary_\${var}.txt --contig_fasta ${sampleid}_cap3_${size_range}.fasta --out summary_\${var}_with_contig_lengths.txt
#only retain hits to plant viruses
c1grep "virus\\|viroid\\|Endogenous" summary_\${var}_with_contig_lengths.txt > summary_\${var}_filtered.txt
sed -i 's/Elephantopus_scaber_closterovirus/Citrus_tristeza_virus/' summary_\${var}_filtered.txt
sed -i 's/Hop_stunt_viroid_-_cucumber/Hop_stunt_viroid/' summary_\${var}_filtered.txt
if [[ ! -s summary_\${var}_filtered.txt ]]
then
echo -e "Species\tsacc\tnaccs\tlength\tslen\tcov\tav-pident\tstitle\tqseqids\tcontig_ind_lengths\tcumulative_contig_len\tcontig_lenth_min\tcontig_lenth_max" > summary_\${var}_viruses_viroids.txt;
exit 0
else
#fetch unique virus/viroid species name from Blast summary reports
cat summary_\${var}_filtered.txt | awk '{print \$7}' | awk -F "|" '{print \$2}'| sort | uniq | sed 's/Species://' > \${var}_uniq.ids
#retrieve the best hit for each unique virus/viroid species name by selecting longest alignment (column 3) and highest genome coverage (column 5)
touch \${var}_filtered.txt
for id in `cat \${var}_uniq.ids`;
do
grep \${id} summary_\${var}_filtered.txt | sort -k3,3nr -k5,5nr | head -1 >> \${var}_filtered.txt
done
#print the header of the inital summary_blastn file
cat summary_\${var}_with_contig_lengths.txt | head -1 > header
#report 1
cat header \${var}_filtered.txt > summary_\${var}_viruses_viroids.txt
#fetch genus names of identified hits
awk '{print \$7}' summary_\${var}_viruses_viroids.txt | awk -F "|" '{print \$2}' | sed 's/Species://' | sed 1d > wanted.names
#add species to report
paste wanted.names \${var}_filtered.txt | sort | awk '\$4>=40' > summary_\${var}_viruses_viroids.tmp.txt
#report 2
awk '{print "Species" "\\t" \$0 }' header > header2
cat header2 summary_\${var}_viruses_viroids.tmp.txt | awk -F"\\t" '\$1!=""&&\$2!=""&&\$3!=""' > summary_\${var}_viruses_viroids.txt
fi
done
"""
}
process COVSTATS_VIRAL_DB {
tag "$sampleid"
label "setting_2"
publishDir "${params.outdir}/01_VirReport/${sampleid}/alignments/viral_db", mode: 'link', overwrite: true, pattern: "*{.fa*,.fasta,metrics.txt,scores.txt,targets.txt,stats.txt,log.txt,.bcf*,.vcf.gz*,.bam*}"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(fastqfile), path(fastq_filt_by_size), path(samplefile)
output:
path("${sampleid}_${size_range}*")
path("${sampleid}_${size_range}_top_scoring_targets_with_cov_stats_viral_db.txt"), emit: viral_db_detections_summary
script:
"""
if [[ ${fastqfile} == *.gz ]];
then
gunzip -c ${fastqfile} > qfilt.fastq
else
ln ${fastqfile} qfilt.fastq
fi
filter_and_derive_stats.py --sample ${sampleid} --rawfastq qfilt.fastq --fastqfiltbysize ${fastq_filt_by_size} --results ${samplefile} --read_size ${size_range} --blastdbpath ${blast_viral_db_dir}/${blast_viral_db_name} --dedup ${params.dedup} --mode viral_db --cpu ${task.cpus}
"""
}
process DETECTION_REPORT_VIRAL_DB {
label "local"
publishDir "${params.outdir}/01_VirReport/Summary", mode: 'copy', overwrite: true
containerOptions "${bindOptions}"
input:
file ('*')
output:
path("VirReport_detection_summary*viral_db*.txt")
script:
"""
if ${params.sampleinfo}; then
detection_report.py --read_size ${size_range} --threshold ${params.contamination_flag} --viral_db true --diagno ${params.diagno} --dedup ${params.dedup} --sampleinfo ${params.sampleinfo_path}
else
detection_report.py --read_size ${size_range} --threshold ${params.contamination_flag} --viral_db true --diagno ${params.diagno} --dedup ${params.dedup}
fi
"""
}
process TBLASTN_VIRAL_DB {
label "setting_4"
publishDir "${params.outdir}/01_VirReport/${sampleid}/tblastn/viral_db", mode: 'link', overwrite: true
tag "$sampleid"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(cap3_fasta)
output:
path("${sampleid}_cap3_${size_range}_getorf.all.fasta")
path("${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_out.bls")
path("${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids_final.txt")
script:
"""
getorf -sequence ${cap3_fasta} -outseq ${sampleid}_cap3_${size_range}_getorf.fasta -minsize ${params.orf_minsize}
getorf -sequence ${cap3_fasta} -circular True -outseq ${sampleid}_cap3_${size_range}_getorf.circular.fasta -minsize ${params.orf_circ_minsize}
cat ${sampleid}_cap3_${size_range}_getorf.fasta ${sampleid}_cap3_${size_range}_getorf.circular.fasta > ${sampleid}_cap3_${size_range}_getorf.all.fasta
#cat ${sampleid}_cap3_${size_range}_getorf.all.fasta | grep ">" | sed 's/>//' | awk '{print \$1}' > ${sampleid}_cap3_${size_range}_getorf.all.fasta.ids
tblastn -query ${sampleid}_cap3_${size_range}_getorf.all.fasta \
-db ${blast_viral_db_dir}/${blast_viral_db_name} \
-evalue ${params.tblastn_evalue} \
-out ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_out.bls \
-num_threads ${task.cpus} \
-max_target_seqs 10 \
-outfmt '6 qseqid sseqid pident nident length mismatch gapopen gaps qstart qend qlen qframe sstart send slen evalue bitscore qcovhsp sallseqid stitle'
grep ">" ${sampleid}_cap3_${size_range}_getorf.all.fasta | sed 's/>//' | cut -f1 -d ' ' | sort | uniq > ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_out.wanted.ids
for i in `cat ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_out.wanted.ids`; do
grep \$i ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_out.bls | head -n5 >> ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits.txt;
done
grep -i "Virus" ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits.txt > ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids.txt || [[ \$? == 1 ]]
grep -i "Viroid" ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits.txt >> ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids.txt || [[ \$? == 1 ]]
#modify accordingly depending on version of viral_db
cut -f2 ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids.txt | cut -f2 -d '|' > seq_ids.txt
cut -f20 ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids.txt | cut -f2 -d '|' | sed 's/Species://' > species_name_extraction.txt
paste seq_ids.txt ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids.txt species_name_extraction.txt > ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids_mod.txt
awk -v OFS='\\t' '{ print \$1,\$2,\$4,\$5,\$6,\$7,\$8,\$9,\$10,\$11,\$12,\$13,\$14,\$15,\$16,\$17,\$18,\$19,\$20,\$22}' ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids_mod.txt | sed 's/ /_/g' > ${sampleid}_cap3_${size_range}_getorf.all_tblastn_vs_viral_db_top5Hits_virus_viroids_final.txt
"""
}
process BLASTN_NT_CAP3 {
label "setting_2"
publishDir "${params.outdir}/01_VirReport/${sampleid}/blastn/NT", mode: 'link', overwrite: true, pattern: "*{vs_NT.bls,_top5Hits.txt,_final.txt,taxonomy.txt}"
tag "$sampleid"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(fastqfile), file(fastq_filt_by_size), file(cap3_fasta)
output:
path("${cap3_fasta.baseName}_blastn_vs_NT.bls")
path("${cap3_fasta.baseName}_blastn_vs_NT_top5Hits.txt")
path("${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids.txt")
path("summary_${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_final.txt")
path("summary_${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids.txt")
tuple val(sampleid),
file(fastqfile),
file(fastq_filt_by_size),
file("summary_${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_final.txt"),
file("${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_seq_ids_taxonomy.txt"),
emit: viral_ncbi_blast_results
tuple val(sampleid),
file(cap3_fasta),
file("${cap3_fasta.baseName}_blastn_vs_NT_top5Hits.txt"),
emit: viral_ncbi_blast_results_for_blastx
script:
def blast_task_param = (params.blastn_method == "blastn") ? "-task blastn" : ''
"""
#To extract the taxonomy, copy the taxonomy databases associated with your blast NT database
if [[ ! -f ${params.blast_db_dir}/taxdb.btd || ! -f ${params.blast_db_dir}/taxdb.bti ]]; then
update_blastdb.pl taxdb
tar -xzf taxdb.tar.gz
else
cp ${params.blast_db_dir}/taxdb.btd .
cp ${params.blast_db_dir}/taxdb.bti .
fi
blastn ${blast_task_param} \
-query ${cap3_fasta} \
-db ${blastn_db_name} \
-negative_seqidlist ${params.negative_seqid_list} \
-out ${cap3_fasta.baseName}_blastn_vs_NT.bls \
-evalue ${params.blastn_evalue} \
-num_threads ${task.cpus} \
-outfmt '6 qseqid sgi sacc length pident mismatch gapopen qstart qend qlen sstart send slen sstrand evalue bitscore qcovhsp stitle staxids qseq sseq sseqid qcovs qframe sframe sscinames' \
-max_target_seqs 50 \
-word_size 24
grep ">" ${cap3_fasta.baseName}.fasta | sed 's/>//' > ${cap3_fasta.baseName}.ids
#fetch top blastn hits
for i in `cat ${cap3_fasta.baseName}.ids`; do
grep \$i ${cap3_fasta.baseName}_blastn_vs_NT.bls | head -n5 >> ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits.txt;
done
grep -i "Virus" ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits.txt > ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_tmp.txt || [[ \$? == 1 ]]
grep -i "Viroid" ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits.txt >> ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_tmp.txt || [[ \$? == 1 ]]
cat ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_tmp.txt | sed 's/ /_/g' > ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids.txt
cut -f3,26 ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids.txt | sort | uniq > ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_seq_ids_taxonomy.txt
java -jar ${projectDir}/bin/BlastTools.jar -t blastn ${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids.txt
rm taxdb.btd
rm taxdb.bti
sequence_length.py --virus_list summary_${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids.txt --contig_fasta ${cap3_fasta.baseName}.fasta --out summary_${cap3_fasta.baseName}_blastn_vs_NT_top5Hits_virus_viroids_final.txt
"""
}
process COVSTATS_NT {
tag "$sampleid"
label "setting_2"
publishDir "${params.outdir}/01_VirReport/${sampleid}/alignments/NT", mode: 'link', overwrite: true, pattern: "*{.fa*,.fasta,metrics.txt,scores.txt,targets.txt,stats.txt,log.txt,.bcf*,.vcf.gz*,.bam*}"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(fastqfile), file(fastq_filt_by_size), file(samplefile), file(taxonomy)
output:
path("${sampleid}_${size_range}*")
path("${sampleid}_${size_range}_top_scoring_targets_*with_cov_stats.txt"), emit: viral_ncbi_detections_summary
script:
"""
if [[ ${fastqfile} == *.gz ]];
then
gunzip -c ${fastqfile} > qfilt.fastq
else
ln ${fastqfile} qfilt.fastq
fi
filter_and_derive_stats.py --sample ${sampleid} --rawfastq qfilt.fastq --fastqfiltbysize ${fastq_filt_by_size} --results ${samplefile} --read_size ${size_range} --taxonomy ${taxonomy} --blastdbpath ${blastn_db_name} --dedup ${params.dedup} --cpu ${task.cpus} --mode ncbi
"""
}
process DETECTION_REPORT_NT {
label "local"
publishDir "${params.outdir}/01_VirReport/Summary", mode: 'copy', overwrite: true
containerOptions "${bindOptions}"
input:
path('*')
output:
file "VirReport_detection_summary*.txt"
script:
"""
if [[ ${params.sampleinfo} == true ]]; then
detection_report.py --read_size ${size_range} --threshold ${params.contamination_flag} --dedup ${params.dedup} --diagno ${params.diagno} --targets ${params.targets_file} --sampleinfopath ${params.sampleinfo_path}
else
detection_report.py --read_size ${size_range} --threshold ${params.contamination_flag} --dedup ${params.dedup} --diagno ${params.diagno} --targets ${params.targets_file}
fi
"""
}
//blastx jobs runs out of memory if only given 64Gb
process BLASTX {
label "setting_8"
publishDir "${params.outdir}/01_VirReport/${sampleid}/blastx/NT", mode: 'link', overwrite: true
tag "$sampleid"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(cap3_fasta), file(top5Hits)
output:
file "${cap3_fasta.baseName}_blastx_vs_NT.bls"
file "${cap3_fasta.baseName}_blastx_vs_NT_topHits.txt"
file "${cap3_fasta.baseName}_blastx_vs_NT_topHits_virus_viroids_final.txt"
file "summary_${cap3_fasta.baseName}_blastx_vs_NT_topHits_virus_viroids_final.txt"
script:
"""
#To extract the taxonomy, copy the taxonomy databases associated with your blast NT database
if [[ ! -f ${params.blast_db_dir}/taxdb.btd || ! -f ${params.blast_db_dir}/taxdb.bti ]]; then
perl ${projectDir}/bin/update_blastdb.pl taxdb
tar -xzf taxdb.tar.gz
else
cp ${params.blast_db_dir}/taxdb.btd .
cp ${params.blast_db_dir}/taxdb.bti .
fi
#extract contigs with blastn results
cut -f1 ${top5Hits} | sort | uniq > denovo_contig_name_ids_with_blastn_hits.txt
#extract all contigs names from de novo assembly
grep ">" ${cap3_fasta.baseName}.fasta | sed 's/>//' | sort | uniq > denovo_contig_name_ids.txt
#extract contigs with no blastn results
grep -v -F -f denovo_contig_name_ids_with_blastn_hits.txt denovo_contig_name_ids.txt | sort > denovo_contig_name_ids_unassigned.txt || [[ \$? == 1 ]]
perl ${projectDir}/bin/faSomeRecords.pl -f ${cap3_fasta.baseName}.fasta -l denovo_contig_name_ids_unassigned.txt -o ${cap3_fasta.baseName}_no_blastn_hits.fasta
extract_seqs_rename.py ${cap3_fasta.baseName}_no_blastn_hits.fasta ${params.blastx_len} \
| sed "s/CONTIG/${sampleid}_${params.minlen}-${params.maxlen}_/" \
> ${cap3_fasta.baseName}_no_blastn_hits_${params.blastx_len}nt.fasta
blastx -query ${cap3_fasta.baseName}_no_blastn_hits_${params.blastx_len}nt.fasta \
-db ${blastp_db_name} \
-out ${cap3_fasta.baseName}_blastx_vs_NT.bls \
-evalue ${params.blastx_evalue} \
-num_threads ${task.cpus} \
-outfmt '6 qseqid sseqid pident nident length mismatch gapopen gaps qstart qend qlen qframe sstart send slen evalue bitscore qcovhsp sallseqid sscinames' \
-max_target_seqs 1
#grep ">" ${cap3_fasta} | sed 's/>//' > ${cap3_fasta.baseName}.ids
cut -f1 ${cap3_fasta.baseName}_blastx_vs_NT.bls | sed 's/ //' | sort | uniq > ${cap3_fasta.baseName}.ids
#fetch top blastn hits
touch ${cap3_fasta.baseName}_blastx_vs_NT_topHits.txt
for i in `cat ${cap3_fasta.baseName}.ids`; do
grep \$i ${cap3_fasta.baseName}_blastx_vs_NT.bls | head -n1 >> ${cap3_fasta.baseName}_blastx_vs_NT_topHits.txt;
done
grep -i "Virus" ${cap3_fasta.baseName}_blastx_vs_NT_topHits.txt > ${cap3_fasta.baseName}_blastx_vs_NT_topHits_virus_viroids.txt || [[ \$? == 1 ]]
grep -i "Viroid" ${cap3_fasta.baseName}_blastx_vs_NT_topHits.txt >> ${cap3_fasta.baseName}_blastx_vs_NT_topHits_virus_viroids.txt || [[ \$? == 1 ]]
sed 's/ /_/g' ${cap3_fasta.baseName}_blastx_vs_NT_topHits_virus_viroids.txt | awk -v OFS='\\t' '{ print \$2,\$1,\$3,\$4,\$5,\$6,\$7,\$8,\$9,\$10,\$11,\$12,\$13,\$14,\$15,\$16,\$17,\$18,\$19,\$20}' > ${cap3_fasta.baseName}_blastx_vs_NT_topHits_virus_viroids_final.txt
java -jar ${projectDir}/bin/BlastTools.jar -t blastp ${cap3_fasta.baseName}_blastx_vs_NT_topHits_virus_viroids_final.txt
rm taxdb.btd
rm taxdb.bti
"""
}
process VIRUS_DETECT {
tag "$sampleid"
label "setting_6"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), file(fastqfile), file(fastq_filt_by_size)
output:
tuple val(sampleid),
path(fastq_filt_by_size),
path("${sampleid}_${size_range}.combined"),
emit: virusdetect
script:
"""
virus_detect.pl --thread_num ${task.cpus} \
--reference ${params.virusdetect_db_path} \
${fastq_filt_by_size} \