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main.nf
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#!/usr/bin/env nextflow
nextflow.enable.dsl = 2
def helpMessage () {
log.info """
ONTViSc - ONT-based Viral Screening for Biosecurity
Marie-Emilie Gauthier 23/05/2023
Magda Antczak
Craig Windell
Roberto Barrero
Usage:
Run the command
nextflow run eresearchqut/ontvisc {optional arguments}...
Optional arguments:
-resume Resume a failed run
--outdir Path to save the output file
'results'
--samplesheet '[path/to/file]' Path to the csv file that contains the list of
samples to be analysed by this pipeline.
Default: 'index.csv'
Contents of samplesheet csv:
sampleid,sample_files
SAMPLE01,/user/folder/sample.fastq.gz
SAMPLE02,/user/folder/*.fastq.gz
sample_files can refer to a folder with a number of
files that will be merged in the pipeline
#### Pre-processing and QC options ####
--qc_only Only perform preliminary QC step using Nanoplot
--analysis_mode clustering, denovo_assembly, read_classification, map2ref
Default: ''
--adapter_trimming Run porechop step
[False]
--porechop_options Porechop_ABI options
[null]
--qual_filt Run quality filtering step
[False]
--chopper_options Chopper options
[null]
--host_filtering Run host filtering step using Minimap2
Default: false
--host_fasta Fasta file of nucleotide sequences to filter
[null]
--canu Use Canu for de novo assembly step
Default: false
--canu_options Canu options
Default: ''
--flye Use Flye for de novo assembly step
--flye_ont_mode Select from nano-raw, nano-corr, nano-hq
Default: 'nano-raw'
--flye_options Flye options
Default: ''
--rattle_clustering_options Rattle clustering options
Default: ''
--rattle_polishing_options Rattle polishing options
Default: ''
--final_primer_check Performs a final primer check after performing de novo assembly step
Default: false
""".stripIndent()
}
// Show help message
if (params.help) {
helpMessage()
exit 0
}
if (params.blastn_db != null) {
blastn_db_name = file(params.blastn_db).name
blastn_db_dir = file(params.blastn_db).parent
}
if (params.reference != null) {
reference_name = file(params.reference).name
reference_dir = file(params.reference).parent
}
if (params.host_fasta != null) {
host_fasta_dir = file(params.host_fasta).parent
}
if (params.kaiju_nodes != null & params.kaiju_dbname != null & params.kaiju_names != null) {
kaiju_dbs_dir = file(params.kaiju_nodes).parent
}
if (params.krkdb != null) {
krkdb_dir = file(params.krkdb).parent
}
if (params.porechop_custom_primers == true) {
porechop_custom_primers_dir = file(params.porechop_custom_primers_path).parent
}
switch (workflow.containerEngine) {
case "singularity":
bindbuild = "";
if (params.blastn_db != null) {
bindbuild = (bindbuild + "-B ${blastn_db_dir} ")
}
if (params.reference != null) {
bindbuild = (bindbuild + "-B ${reference_dir} ")
}
if (params.kaiju_nodes != null & params.kaiju_dbname != null & params.kaiju_names != null) {
bindbuild = (bindbuild + "-B ${kaiju_dbs_dir} ")
}
if (params.krkdb != null) {
bindbuild = (bindbuild + "-B ${krkdb_dir} ")
}
if (params.host_fasta != null) {
bindbuild = (bindbuild + "-B ${host_fasta_dir} ")
}
if (params.porechop_custom_primers) {
bindbuild = (bindbuild + "-B ${porechop_custom_primers_dir} ")
}
bindOptions = bindbuild;
break;
default:
bindOptions = "";
}
/*
process MERGE {
//publishDir "${params.outdir}/${sampleid}/merge", pattern: '*.fastq.gz', mode: 'link'
tag "${sampleid}"
label 'setting_1'
input:
tuple val(sampleid), path(lanes)
output:
tuple val(sampleid), path("${sampleid}.fastq.gz"), emit: merged
script:
"""
cat ${lanes} > ${sampleid}.fastq.gz
"""
}
*/
process QCREPORT {
publishDir "${params.outdir}/qc_report", mode: 'copy', overwrite: true
containerOptions "${bindOptions}"
input:
path multiqc_files
output:
path("run_qc_report_*txt")
path("run_qc_report_*html")
script:
"""
seq_run_qc_report.py --host_filtering ${params.host_filtering} --adapter_trimming ${params.adapter_trimming} --quality_trimming ${params.qual_filt}
"""
}
process CUTADAPT {
publishDir "${params.outdir}/${sampleid}/assembly", pattern: '*_filtered.fa', mode: 'link'
publishDir "${params.outdir}/${sampleid}/assembly", pattern: '*_cutadapt.log', mode: 'link'
tag "${sampleid}"
label 'medium'
input:
tuple val(sampleid), path(sample)
output:
file("${sampleid}_cutadapt.log")
file("${sample.baseName}_filtered.fa")
tuple val(sampleid), path("${sample.baseName}_filtered.fa"), emit: trimmed
script:
"""
cutadapt -j ${task.cpus} -g "AAGCAGTGGTATCAACGCAGAGTACGCGGG;min_overlap=14" -a "CCCGCGTACTCTGCGTTGATACCACTGCTT;min_overlap=14" -o ${sample.baseName}_filtered.fa ${sample} > ${sampleid}_cutadapt.log
sed -i 's/len=[0-9]* reads/reads/' ${sample.baseName}_filtered.fa
sed -i 's/ trim=.*\$//' ${sample.baseName}_filtered.fa
"""
}
process CHOPPER {
publishDir "${params.outdir}/${sampleid}/preprocessing/chopper", pattern: '*_chopper.log', mode: 'link'
tag "${sampleid}"
label 'setting_3'
input:
tuple val(sampleid), path(sample)
output:
path("${sampleid}_chopper.log")
tuple val(sampleid), path("${sampleid}_filtered.fastq.gz"), emit: chopper_filtered_fq
script:
def chopper_options = (params.chopper_options) ? " ${params.chopper_options}" : ''
"""
gunzip -c ${sample} | chopper ${chopper_options} 2> ${sampleid}_chopper.log | gzip > ${sampleid}_filtered.fastq.gz
"""
}
/*
#recommended settings for CANU using metagnomics data
https://github.com/marbl/canu/issues/2079
genomeSize=20m
maxInputCoverage=10000 corOutCoverage=10000
corMhapSensitivity=high
corMinCoverage=0
redMemory=32 oeaMemory=32 batMemory=64
useGrid=false
minReadLength=200
minOverlapLength=50
maxThreads=4
minInputCoverage=0
stopOnLowCoverage=0
maxInputCoverage=10000 corOutCoverage=10000 corMhapSensitivity=high corMinCoverage=0 redMemory=32 oeaMemory=32 batMemory=64 useGrid=false minReadLength=200 minOverlapLength=50 maxThreads=4 minInputCoverage=0 stopOnLowCoverage=0
*/
process CANU {
publishDir "${params.outdir}/${sampleid}/assembly/canu", mode: 'copy', pattern: '{*.fasta,*.log}'
tag "${sampleid}"
label 'setting_8'
input:
tuple val(sampleid), path(fastq)
output:
path("${sampleid}_canu_assembly.fasta")
path("${sampleid}_canu.log")
tuple val(sampleid), path("${sampleid}_canu.fastq.gz"), path("${sampleid}_canu_assembly.fasta"), emit: assembly
tuple val(sampleid), path("${sampleid}_canu_assembly.fasta"), emit: assembly2
script:
def canu_options = (params.canu_options) ? " ${params.canu_options}" : ''
"""
canu -p ${sampleid} -d ${sampleid} \
genomeSize=${params.canu_genome_size} \
-nanopore ${fastq} ${canu_options} 2> ${sampleid}_canu.log
if [[ ! -s ${sampleid}/${sampleid}.contigs.fasta ]]
then
touch ${sampleid}_canu_assembly.fasta
else
cat ${sampleid}/${sampleid}.contigs.fasta ${sampleid}/${sampleid}.unassembled.fasta > ${sampleid}_canu_assembly.fasta
fi
cp ${fastq} ${sampleid}_canu.fastq.gz
"""
}
process FLYE {
publishDir "${params.outdir}/${sampleid}/assembly/flye", mode: 'copy', pattern: '{*.fasta,*.log}'
tag "${sampleid}"
label 'setting_9'
input:
tuple val(sampleid), path(fastq)
output:
path("${sampleid}_flye_assembly.fasta")
path("${sampleid}_flye.log")
tuple val(sampleid), path("${sampleid}_flye.fastq.gz"), path("${sampleid}_flye_assembly.fasta"), emit: assembly
tuple val(sampleid), path("${sampleid}_flye_assembly.fasta"), emit: assembly2
script:
def flye_options = (params.flye_options) ? " ${params.flye_options}" : ''
"""
flye ${flye_options} --out-dir outdir --threads ${task.cpus} --${params.flye_mode} ${fastq}
if [[ ! -s outdir/assembly.fasta ]]
then
touch ${sampleid}_flye_assembly.fasta
else
cp outdir/assembly.fasta ${sampleid}_flye_assembly.fasta
cp outdir/flye.log ${sampleid}_flye.log
fi
cp ${fastq} ${sampleid}_flye.fastq.gz
"""
}
/*
errorStrategy 'ignore'
*/
process BLASTN2REF {
publishDir "${params.outdir}/${sampleid}", mode: 'copy', pattern: '*/*/*txt'
tag "${sampleid}"
label 'setting_1'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(assembly)
output:
path "*/*/${sampleid}_blastn_reference_vs_*.txt"
script:
"""
if [[ ${assembly} == *_assembly*.fa* ]] ;
then
if [[ ${assembly} == *canu_assembly*.fa* ]] ;
then
mkdir -p assembly/blast_to_ref
blastn -query ${assembly} -subject ${reference_dir}/${reference_name} -evalue 1e-3 -out ${sampleid}_blastn_reference_vs_canu_assembly_tmp.txt \
-outfmt '6 qseqid sacc length pident mismatch gapopen qstart qend qlen sstart send slen evalue bitscore qcovhsp qcovs' -max_target_seqs 5
echo "qseqid\tsacc\tlength\tpident\tmismatch\tgapopen\tqstart\tqend\tqlen\tsstart\tsend\tslen\tevalue\tbitscore\tqcovhsp\tqcovs" > header
cat header ${sampleid}_blastn_reference_vs_canu_assembly_tmp.txt > assembly/blast_to_ref/${sampleid}_blastn_reference_vs_canu_assembly.txt
elif [[ ${assembly} == *flye_assembly*.fasta ]] ;
then
mkdir -p assembly/blast_to_ref
blastn -query ${assembly} -subject ${reference_dir}/${reference_name} -evalue 1e-3 -out ${sampleid}_blastn_reference_vs_flye_assembly_tmp.txt \
-outfmt '6 qseqid sacc length pident mismatch gapopen qstart qend qlen sstart send slen evalue bitscore qcovhsp qcovs' -max_target_seqs 5
echo "qseqid\tsacc\tlength\tpident\tmismatch\tgapopen\tqstart\tqend\tqlen\tsstart\tsend\tslen\tevalue\tbitscore\tqcovhsp\tqcovs" > header
cat header ${sampleid}_blastn_reference_vs_flye_assembly_tmp.txt > assembly/blast_to_ref/${sampleid}_blastn_reference_vs_flye_assembly.txt
fi
elif [[ ${assembly} == *clustering.fasta ]] ;
then
mkdir -p clustering/blast_to_ref
blastn -query ${assembly} -subject ${reference_dir}/${reference_name} -evalue 1e-3 -out ${sampleid}_blastn_reference_vs_clustering_tmp.txt \
-outfmt '6 qseqid sacc length pident mismatch gapopen qstart qend qlen sstart send slen evalue bitscore qcovhsp qcovs' -max_target_seqs 5
echo "qseqid\tsacc\tlength\tpident\tmismatch\tgapopen\tqstart\tqend\tqlen\tsstart\tsend\tslen\tevalue\tbitscore\tqcovhsp\tqcovs" > header
cat header ${sampleid}_blastn_reference_vs_clustering_tmp.txt > clustering/blast_to_ref/${sampleid}_blastn_reference_vs_clustering_assembly.txt
fi
"""
}
process MINIMAP2_REF {
tag "${sampleid}"
label 'setting_2'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(sample)
output:
tuple val(sampleid), file("${sampleid}_aln.sam"), emit: aligned_sample
script:
"""
minimap2 -ax map-ont --MD --sam-hit-only ${reference_dir}/${reference_name} ${sample} > ${sampleid}_aln.sam
"""
}
process SAMTOOLS {
publishDir "${params.outdir}/${sampleid}/mapping", mode: 'copy'
tag "${sampleid}"
label 'setting_2'
input:
tuple val(sampleid), path(sample)
output:
path "${sampleid}_aln.sorted.bam"
path "${sampleid}_aln.sorted.bam.bai"
path "${sampleid}_coverage.txt"
path "${sampleid}_histogram"
tuple val(sampleid), path("${sampleid}_aln.sorted.bam"), path("${sampleid}_aln.sorted.bam.bai"), emit: sorted_sample
script:
"""
samtools view -Sb -F 4 ${sample} | samtools sort -o ${sampleid}_aln.sorted.bam
samtools index ${sampleid}_aln.sorted.bam
samtools coverage ${sampleid}_aln.sorted.bam > ${sampleid}_histogram.txt > ${sampleid}_coverage.txt
samtools coverage -A -w 50 ${sampleid}_aln.sorted.bam > ${sampleid}_histogram
"""
}
/*
process BAMCOVERAGE {
publishDir "${params.outdir}/${sampleid}/mapping", mode: 'link'
tag "${sampleid}"
label 'setting_11'
input:
tuple val(sampleid), path(bam), path(bai)
output:
path "${sampleid}.bw"
script:
"""
bamCoverage -b ${bam} -o ${sampleid}.bw
"""
}
process BAMCOVERAGE {
publishDir "${params.outdir}/${sampleid}/mapping", mode: 'link'
tag "${sampleid}"
label 'setting_11'
input:
tuple val(sampleid), path(bam), path(bai)
output:
path "${sampleid}.bamcov.txt"
script:
"""
bamcov -H ${bam} > ${sampleid}.bamcov.txt
"""
}
*/
process PORECHOP_ABI {
tag "${sampleid}"
publishDir "$params.outdir/${sampleid}/preprocessing/porechop", mode: 'link', pattern: '*_porechop.log'
label "setting_9"
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(sample)
output:
file("${sampleid}_porechop_trimmed.fastq.gz")
file("${sampleid}_porechop.log")
tuple val(sampleid), file("${sampleid}_porechop_trimmed.fastq.gz"), emit: porechopabi_trimmed_fq
script:
def porechop_options = (params.porechop_options) ? " ${params.porechop_options}" : ''
"""
if [[ ${params.porechop_custom_primers} == true ]]; then
porechop_abi -i ${sample} -t ${task.cpus} -o ${sampleid}_porechop_trimmed.fastq.gz --custom_adapters ${params.porechop_custom_primers_path} ${porechop_options} > ${sampleid}_porechop.log
else
porechop_abi -i ${sample} -t ${task.cpus} -o ${sampleid}_porechop_trimmed.fastq.gz ${porechop_options} > ${sampleid}_porechop.log
fi
"""
}
//trims fastq read names after the first whitespace
process REFORMAT {
tag "${sampleid}"
label "setting_3"
publishDir "$params.outdir/${sampleid}/preprocessing", mode: 'copy'
input:
tuple val(sampleid), path(fastq)
output:
tuple val(sampleid), path("${sampleid}_preprocessed.fastq.gz"), emit: reformatted_fq
script:
"""
reformat.sh in=${fastq} out=${sampleid}_preprocessed.fastq.gz trd qin=33
"""
}
process CAP3 {
tag "${sampleid}"
label "setting_3"
time "3h"
publishDir "$params.outdir/$sampleid/clustering/cap3", mode: 'copy', pattern: '*_clustering.fasta'
publishDir "$params.outdir/$sampleid/clustering/rattle", mode: 'copy', pattern: '*_rattle.fasta'
input:
tuple val(sampleid), path(fasta)
output:
file("${sampleid}_rattle.fasta")
tuple val(sampleid), path("${sampleid}_clustering.fasta"), emit: scaffolds
script:
"""
cap3 ${fasta}
cat ${fasta}.cap.singlets ${fasta}.cap.contigs > ${sampleid}_clustering.fasta
cp ${fasta} ${sampleid}_rattle.fasta
"""
}
process EXTRACT_VIRAL_BLAST_HITS {
tag "${sampleid}"
label "setting_2"
publishDir "$params.outdir/$sampleid", mode: 'copy', pattern: '*/*/*txt', overwrite: true
publishDir "$params.outdir/$sampleid", mode: 'copy', pattern: '*/*/*html', overwrite: true
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(blast_results)
output:
file "*/*/${sampleid}*_blastn_top_hits.txt"
file "*/*/${sampleid}*_blastn_top_viral_hits*.txt"
file "*/*/${sampleid}*_blastn_top_viral_spp_hits.txt"
file "*/*/${sampleid}*_queryid_list_with_viral_match.txt"
file "*/*/${sampleid}*_viral_spp_abundance.txt"
file "*/*/*report*html"
tuple val(sampleid), path("*/*/${sampleid}*_blastn_top_viral_spp_hits.txt"), path("*/*/${sampleid}*_queryid_list_with_viral_match.txt"), path("*/*/${sampleid}*_viral_spp_abundance.txt"), emit: blast_results
script:
"""
if [[ ${blast_results} == *clustering_blastn.bls ]] ;
then
mkdir -p clustering/blastn
cat ${blast_results} > clustering/blastn/${sampleid}_blastn.txt
cd clustering/blastn
select_top_blast_hit.py --sample_name ${sampleid} --blastn_results ${sampleid}_blastn.txt --analysis_method clustering --mode ${params.blast_mode}
elif [[ ${blast_results} == *assembly*_blastn.bls ]] ;
then
mkdir -p assembly/blastn
cat ${blast_results} > assembly/blastn/${sampleid}_blastn.txt
cd assembly/blastn
select_top_blast_hit.py --sample_name ${sampleid} --blastn_results ${sampleid}_blastn.txt --analysis_method assembly --mode ${params.blast_mode}
fi
"""
}
process EXTRACT_VIRAL_BLAST_SPLIT_HITS {
tag "${sampleid}"
label "setting_2"
publishDir "$params.outdir/$sampleid/read_classification", mode: 'copy', pattern: 'homology_search/*txt', overwrite: true
publishDir "$params.outdir/$sampleid/read_classification", mode: 'copy', pattern: 'homology_search/*html', overwrite: true
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(blast_results)
output:
file "*/${sampleid}*_blastn_top_hits.txt"
file "*/${sampleid}*_blastn_top_viral_hits.txt"
file "*/${sampleid}*_blastn_top_viral_spp_hits.txt"
file "*/${sampleid}*_queryid_list_with_viral_match.txt"
file "*/${sampleid}*_viral_spp_abundance.txt"
file "*/*report*html"
tuple val(sampleid), path("*/${sampleid}*_viral_spp_abundance.txt"), emit: blast_results
tuple val(sampleid), path("*/${sampleid}*_viral_spp_abundance_filtered.txt"), emit: blast_results_filt
script:
"""
mkdir homology_search
cat ${blast_results} > homology_search/${sampleid}_blastn.txt
cd homology_search
select_top_blast_hit.py --sample_name ${sampleid} --blastn_results ${sampleid}_blastn.txt --analysis_method read_classification --mode ${params.blast_mode}
"""
}
//tuple val(sampleid), path("*/${sampleid}*_blastn_top_viral_hits.txt"), emit: blast_results
process CONCATENATE_FASTA {
tag "${sampleid}"
label "setting_2"
publishDir "${params.outdir}/${sampleid}", mode: 'link'
input:
tuple val(sampleid), path("${sampleid}_canu_assembly.fasta")
tuple val(sampleid), path("${sampleid}_cap3.fasta")
tuple val(sampleid), path("${sampleid}.fasta")
output:
file "${sampleid}_merged.fasta"
tuple val(sampleid), path("*_merged.fasta"), emit: assembly
script:
"""
seqtk seq -l0 ${sampleid}_canu_assembly.fasta > ${sampleid}_canu_assembly_1l.fasta
seqtk seq -l0 ${sampleid}_cap3.fasta > ${sampleid}_cap3_1l.fasta
seqtk seq -l0 ${sampleid}.fasta > ${sampleid}_1l.fasta
cat ${sampleid}_canu_assembly_1l.fasta ${sampleid}_cap3_1l.fasta ${sampleid}.fasta > ${sampleid}_merged.fasta
"""
}
/*
KAIJU notes
The default run mode is Greedy with three allowed mismatches.
The number of allowed mismatches can be changed using option -e.
In Greedy mode, matches are filtered by a minimum length and score and their E-value (similar to blastp)
The cutoffs for minimum required match length and match score can be changed using the options -m (default: 11) and -s (default: 65)
Minimum E-value can be adjusted with the option -E (default 0.01).
For fastest classification, , use MEM mode with option '-a mem' and multiple parallel threads (-z)
Greedy run mode yields a higher sensitivity compared with MEM mode.
For lowest memory usage use the proGenomes reference database. The number of parallel threads has only little impact on memory usage.
Further, the choice of the minimum required match length (-m) in MEM mode or match score (-s) in Greedy mode governs the trade-off between
sensitivity and precision of the classification. Please refer to the paper for a discussion on this topic.
Option -x enables filtering of query sequences containing low-complexity regions by using the SEG algorithm from the blast+ package.
It is enabled by default and can be disabled by the -X option. SEG filtering is always recommended in order to avoid
false positive taxon assignments that are caused by spurious matches due to simple repeat patterns or other sequencing noise.
The accuracy of the classification depends both on the choice of the reference database and the chosen options when running Kaiju.
These choices also affect the speed and memory usage of Kaiju.
For highest sensitivity, it is recommended to use the nr database (+eukaryotes) as a reference database because it is the most comprehensive
set of protein sequences. Alternatively, use proGenomes over Refseq for increased sensitivity.
NOTE, viroid will not be detected using this approach
Mandatory arguments:
-t FILENAME Name of nodes.dmp file
-f FILENAME Name of database (.fmi) file
-i FILENAME Name of input file containing reads in FASTA or FASTQ format
Optional arguments:
-j FILENAME Name of second input file for paired-end reads
-o FILENAME Name of output file. If not specified, output will be printed to STDOUT
-z INT Number of parallel threads for classification (default: 1)
-a STRING Run mode, either "mem" or "greedy" (default: greedy)
-e INT Number of mismatches allowed in Greedy mode (default: 3) #set to 1 in kodoja
-m INT Minimum match length (default: 11) #set to 15 in kodoja
-s INT Minimum match score in Greedy mode (default: 65) #set to 85 in Kodoja
-E FLOAT Minimum E-value in Greedy mode
-x Enable SEG low complexity filter (enabled by default)
-X Disable SEG low complexity filter
-p Input sequences are protein sequences
-v Enable verbose output
*/
process KAIJU {
publishDir "${params.outdir}/${sampleid}/read_classification/kaiju", mode: 'copy'
label 'setting_4'
containerOptions "${bindOptions}"
tag "${sampleid}"
input:
tuple val(sampleid), path(fastq)
output:
file "${sampleid}_kaiju_name.tsv"
file "${sampleid}_kaiju_summary*.tsv"
file "${sampleid}_kaiju.krona"
tuple val(sampleid), path("${sampleid}_kaiju_summary_viral.tsv"), emit: kaiju_results
tuple val(sampleid), path("*kaiju.krona"), emit: krona_results
script:
"""
c1grep() { grep "\$@" || test \$? = 1; }
kaiju \
-z ${params.kaiju_threads} \
-t ${params.kaiju_nodes} \
-f ${params.kaiju_dbname} \
-o ${sampleid}_kaiju.tsv \
-i ${fastq} \
-v
kaiju-addTaxonNames -t ${params.kaiju_nodes} -n ${params.kaiju_names} -i ${sampleid}_kaiju.tsv -o ${sampleid}_kaiju_name.tsv
kaiju2table -e -t ${params.kaiju_nodes} -r species -n ${params.kaiju_names} -o ${sampleid}_kaiju_summary.tsv ${sampleid}_kaiju.tsv
kaiju2krona -t ${params.kaiju_nodes} -n ${params.kaiju_names} -i ${sampleid}_kaiju.tsv -o ${sampleid}_kaiju.krona
c1grep "taxon_id\\|virus\\|viroid\\|viricota\\|viridae\\|viriform\\|virales\\|virinae\\|viricetes\\|virae\\|viral" ${sampleid}_kaiju_summary.tsv > ${sampleid}_kaiju_summary_viral.tsv
awk -F'\\t' '\$2>=0.05' ${sampleid}_kaiju_summary_viral.tsv > ${sampleid}_kaiju_summary_viral_filtered.tsv
"""
}
/*
process KAIJU_HTML {
tag "${sampleid}"
label "local"
publishDir "$params.outdir/$sampleid/read_classification/kaiju", mode: 'link'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(kaiju_report)
output:
file("*_kaiju_report.html")
script:
"""
kraken_html_report.py --sample ${sampleid}
"""
}
*/
process READ_CLASSIFICATION_HTML {
publishDir "${params.outdir}/${sampleid}/read_classification/Summary", mode: 'copy', overwrite: true
label 'local'
containerOptions "${bindOptions}"
tag "${sampleid}"
input:
tuple val(sampleid), path(results)
output:
file "*_read_classification_report.html"
script:
"""
summary_read_classification.py --sample ${sampleid} --mode ${params.blast_mode}
"""
}
process KRONA {
publishDir "${params.outdir}/${sampleid}/read_classification/kaiju", mode: 'link'
label 'setting_3'
containerOptions "${bindOptions}"
tag "${sampleid}"
input:
tuple val(sampleid), path(krona_input)
output:
file "${sampleid}_krona.html"
script:
"""
ktImportText -o ${sampleid}_krona.html ${krona_input}
"""
}
/*
KRAKEN2
https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown
If you get the Loading database information...classify: Error reading in hash table error allocate more memory to the job. Keep incrementing the memory request until you see a status message in the job log like
Usage: kraken2 [options] <filename(s)>
Options:
--db NAME Name for Kraken 2 DB
(default: none)
--threads NUM Number of threads (default: 1)
--quick Quick operation (use first hit or hits)
--unclassified-out FILENAME
Print unclassified sequences to filename
--classified-out FILENAME
Print classified sequences to filename
--output FILENAME Print output to filename (default: stdout); "-" will
suppress normal output
--confidence FLOAT Confidence score threshold (default: 0.0); must be
in [0, 1].
--minimum-base-quality NUM
Minimum base quality used in classification (def: 0,
only effective with FASTQ input).
--report FILENAME Print a report with aggregrate counts/clade to file
--use-mpa-style With --report, format report output like Kraken 1's
kraken-mpa-report
--report-zero-counts With --report, report counts for ALL taxa, even if
counts are zero
--report-minimizer-data With --report, report minimizer and distinct minimizer
count information in addition to normal Kraken report
--memory-mapping Avoids loading database into RAM
--paired The filenames provided have paired-end reads
--use-names Print scientific names instead of just taxids
--gzip-compressed Input files are compressed with gzip
--bzip2-compressed Input files are compressed with bzip2
--minimum-hit-groups NUM
Minimum number of hit groups (overlapping k-mers
sharing the same minimizer) needed to make a call
(default: 3)
--help Print this message
--version Print version information
32 min using 4 cpus and 164 Gb of mem, test with 2 cpus instead
*/
process KRAKEN2 {
tag "${sampleid}"
label 'setting_5'
publishDir "$params.outdir/$sampleid/read_classification/kraken", mode: 'copy'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(fastq)
output:
file("${sampleid}.kraken2")
file("${sampleid}_kraken_report.txt")
file("${sampleid}_unclassified.fastq")
tuple val(sampleid), path("${sampleid}_kraken_report.txt"), emit: results
script:
"""
kraken2 --db ${params.krkdb} \
--use-names \
--threads ${task.cpus} \
--report ${sampleid}_kraken_report.txt \
--report-minimizer-data \
--minimum-hit-groups 3 \
--unclassified-out ${sampleid}_unclassified.fastq \
${fastq} > ${sampleid}.kraken2
"""
}
process BRACKEN {
tag "${sampleid}"
label 'setting_2'
publishDir "$params.outdir/$sampleid/read_classification/kraken", mode: 'copy'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(kraken_report)
output:
file("${sampleid}_bracken_report*.txt")
tuple val(sampleid), path("${sampleid}_bracken_report_viral.txt"), emit: bracken_results
script:
"""
c1grep() { grep "\$@" || test \$? = 1; }
est_abundance.py -i ${kraken_report} \
-k ${params.krkdb}/database50mers.kmer_distrib \
-t 1 \
-l S -o ${sampleid}_bracken_report.txt
c1grep "taxonomy_id\\|virus\\|viroid" ${sampleid}_bracken_report.txt > ${sampleid}_bracken_report_viral.txt
awk -F'\\t' '\$7>=0.0001' ${sampleid}_bracken_report_viral.txt > ${sampleid}_bracken_report_viral_filtered.txt
"""
}
process BRACKEN_HTML {
tag "${sampleid}"
label "local"
publishDir "$params.outdir/$sampleid/read_classification/kraken", mode: 'link'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(braken_report)
output:
file("*_bracken_report.html")
script:
"""
kraken_html_report.py --sample ${sampleid}
"""
}
process RATTLE {
publishDir "${params.outdir}/${sampleid}/clustering/rattle", mode: 'copy', pattern: 'transcriptome.fq'
tag "${sampleid}"
label 'setting_7'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(fastq)
output:
file("transcriptome.fq")
tuple val(sampleid), path("transcriptome.fq"), emit: clusters
tuple val(sampleid), path(fastq), path("transcriptome.fq"), emit: clusters2
def rattle_polishing_options = (params.rattle_polishing_options) ? " ${params.rattle_polishing_options}" : ''
def rattle_clustering_options = (params.rattle_clustering_options) ? " ${params.rattle_clustering_options}" : ''
script:
"""
rattle cluster -i ${fastq} -t ${task.cpus} ${rattle_clustering_options} -o .
rattle cluster_summary -i ${fastq} -c clusters.out > ${sampleid}_cluster_summary.txt
mkdir clusters
rattle extract_clusters -i ${fastq} -c clusters.out -l ${sampleid} -o clusters --fastq
rattle correct -i ${fastq} -c clusters.out -t ${task.cpus} -l ${sampleid}
rattle polish -i consensi.fq -t ${task.cpus} --summary ${rattle_polishing_options}
"""
}
process MEDAKA {
publishDir "${params.outdir}/${sampleid}/mapping", mode: 'copy'
tag "${sampleid}"
label 'setting_3'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(bam), path(bai)
output:
tuple val(sampleid), path("${sampleid}_medaka.annotated.unfiltered.vcf"), emit: unfilt_vcf
script:
def medaka_consensus_options = (params.medaka_consensus_options) ? " ${params.medaka_consensus_options}" : ''
"""
medaka consensus ${bam} ${sampleid}_medaka_consensus_probs.hdf \
${medaka_consensus_options} --threads ${task.cpus}
medaka variant ${reference_dir}/${reference_name} ${sampleid}_medaka_consensus_probs.hdf ${sampleid}_medaka.vcf
medaka tools annotate --dpsp ${sampleid}_medaka.vcf ${reference_dir}/${reference_name} ${bam} \
${sampleid}_medaka.annotated.unfiltered.vcf
"""
}
process FILTER_VCF {
publishDir "${params.outdir}/${sampleid}/mapping", mode: 'copy'
tag "${sampleid}"
label 'setting_3'
containerOptions "${bindOptions}"
input:
tuple val(sampleid), path(vcf)
output:
path("${sampleid}_medaka.consensus.fasta")
path("${sampleid}_medaka.annotated.vcf.gz")
script:
"""
bcftools reheader ${vcf} -s <(echo '${sampleid}') \
| bcftools filter \
-e 'INFO/DP < ${params.bcftools_min_coverage}' \
-s LOW_DEPTH \
-Oz -o ${sampleid}_medaka.annotated.vcf.gz
# create consensus
bcftools index ${sampleid}_medaka.annotated.vcf.gz
bcftools consensus -f ${reference_dir}/${reference_name} ${sampleid}_medaka.annotated.vcf.gz \
-i 'FILTER="PASS"' \
-o ${sampleid}_medaka.consensus.fasta
"""
}
process FASTCAT {
publishDir "${params.outdir}/${sampleid}/qc/fastcat", mode: 'copy'
tag "${sampleid}"
label "setting_2"
input:
tuple val(sampleid), path(fastq)
output:
path("${sampleid}_stats.tsv")
path("histograms/*")
tuple val(sampleid), path("${sampleid}.fastq.gz"), emit: merged
script:
"""
fastcat \
-s ${sampleid} \
-f ${sampleid}_stats.tsv \
--histograms histograms \
${fastq} \
| bgzip > ${sampleid}.fastq.gz
"""
}
include { MINIMAP2_ALIGN_RNA } from './modules.nf'
include { EXTRACT_READS as EXTRACT_READS_STEP1 } from './modules.nf'
include { EXTRACT_READS as EXTRACT_READS_STEP2 } from './modules.nf'
include { EXTRACT_READS as EXTRACT_READS_STEP3 } from './modules.nf'
include { MINIMAP2_ALIGN_DNA as MAP_BACK_TO_CONTIGS } from './modules.nf'
include { MINIMAP2_ALIGN_DNA as MAP_BACK_TO_CLUSTERS } from './modules.nf'
include { FASTQ2FASTA } from './modules.nf'
include { FASTQ2FASTA as FASTQ2FASTA_STEP1} from './modules.nf'
include { FASTQ2FASTA as FASTQ2FASTA_STEP2} from './modules.nf'
include { NANOPLOT as QC_PRE_DATA_PROCESSING } from './modules.nf'
include { NANOPLOT as QC_POST_DATA_PROCESSING } from './modules.nf'
include { BLASTN as READ_CLASSIFICATION_BLASTN } from './modules.nf'
include { BLASTN as ASSEMBLY_BLASTN } from './modules.nf'
include { BLASTN as CLUSTERING_BLASTN } from './modules.nf'
workflow {
if (params.samplesheet) {
Channel
.fromPath(params.samplesheet, checkIfExists: true)
.splitCsv(header:true)
.map{ row-> tuple((row.sampleid), file(row.sample_files)) }
.set{ ch_sample }
} else { exit 1, "Input samplesheet file not specified!" }
if ( params.analysis_mode == 'clustering' | params.analysis_mode == 'denovo_assembly' | (params.analysis_mode == 'read_classification' & params.megablast)) {
if (!params.blast_vs_ref) {
if ( params.blastn_db == null) {
error("Please provide the path to a blast database using the parameter --blastn_db.")
}
}
else if (params.blast_vs_ref ) {
if ( params.reference == null) {
error("Please provide the path to a reference fasta file with the parameter --reference.")
}
}
}
else if ( params.analysis_mode == 'map2ref' ) {
if ( params.reference == null) {
error("Please provide the path to a reference fasta file with the parameter --reference.")
}
}
if (params.merge) {
FASTCAT ( ch_sample )
QC_PRE_DATA_PROCESSING ( FASTCAT.out.merged )
fq = FASTCAT.out.merged
}
else {
fq = ch_sample
QC_PRE_DATA_PROCESSING ( fq )
}
// Data pre-processing
// Remove adapters uisng either PORECHOP_ABI or CUTADAPT
if (!params.qc_only) {
if (params.adapter_trimming) {
PORECHOP_ABI ( fq )
trimmed_fq = PORECHOP_ABI.out.porechopabi_trimmed_fq
}
else {
trimmed_fq = fq
}
// Quality filtering of reads using chopper
if (params.qual_filt) {
CHOPPER ( trimmed_fq)
filtered_fq = CHOPPER.out.chopper_filtered_fq
}
else { filtered_fq = trimmed_fq
}
REFORMAT( filtered_fq )
if ( params.qual_filt & params.adapter_trimming | !params.qual_filt & params.adapter_trimming | params.qual_filt & !params.adapter_trimming) {
QC_POST_DATA_PROCESSING ( filtered_fq )
}
if (params.host_filtering) {
if ( params.host_fasta == null) {
error("Please provide the path to a fasta file of host sequences that need to be filtered with the parameter --host_fasta.")
}