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aline.nf
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#! /usr/bin/env nextflow
nextflow.enable.dsl=2
// Import
import static groovy.io.FileType.FILES
import java.nio.file.*
//*************************************************
// STEP 0 - parameters
//*************************************************
/*
* Default pipeline parameters. They can be overriden on the command line eg.
* given `params.foo` specify on the run command line `--foo some_value`.
*/
// Input/output params
params.reads = "/path/to/reads_{1,2}.fastq.gz"
params.genome = "/path/to/genome.fa"
params.outdir = "alignment_results"
params.reads_extension = ".fastq.gz" // Extension used to detect reads in folder
params.paired_reads_pattern = "{1,2}"
read_type_allowed = [ 'short_paired', 'short_single', 'pacbio', 'ont' ]
params.read_type = "short_paired"
params.relax = false // Avoid to automatically set option specific to ready type (e.g minimap, bwa-mem for long reads.).
// Read feature params
libtype_allowed = [ 'U', 'IU', 'MU', 'OU', 'ISF', 'ISR', 'MSF', 'MSR', 'OSF', 'OSR' ]
params.library_type = "auto"
params.skip_libray_usage = false // Avoid to use library type provided by library_type or auto
params.read_length = "" // Use by star to set the sjdbOverhang parameter
// annotation is used by different aligner (star, etc.). To avoid to duplicate processes according to the presence of the annotation file, a specific process is dedicated to create a fake file is none provided.
// If process receive a file wich is not the fake one it includes the file in the command. To append the options of aligner we will use the annotation_file variable
// While the processes will be called sending the "annotation" channel created by the prepare_annotation process.
params.annotation = ""
// Trimming params
params.trimming_fastp = false
// Aligner params
align_tools = [ 'bbmap', 'bowtie', 'bowtie2', 'bwaaln', 'bwamem', 'bwamem2', 'bwasw', 'graphmap2', 'hisat2', 'kallisto', 'last', 'minimap2', 'novoalign', 'nucmer', 'ngmlr', 'salmon', 'star', 'subread', 'sublong' ]
params.aligner = ''
params.bbmap_options = ''
params.bowtie_options = ''
params.bowtie2_options = ''
params.bwaaln_options = ''
params.bwamem_options = ''
params.bwamem2_options = ''
params.bwasw_options = ''
params.graphmap2_options = '' // owler option is possible
params.hisat2_options = ''
params.kallisto_options = ''
params.kallisto_index_options = '' // e.g. to use --distinguish, --make-unique, etc...
params.last_options = ''
params.last_index_options = ''
params.minimap2_options = ''
params.minimap2_index_options = '' // -k, -w, -H and -I
params.ngmlr_options = ''
params.novoalign_options = ''
params.novoalign_license = '' // license. You can ask for one month free trial license at http://www.novocraft.com/products/novoalign/
params.nucmer_options = ''
params.salmon_options = ''
params.salmon_index_options = ''
params.star_options = ''
params.star_index_options = ''
params.star_2pass = false
params.subread_options = '-t 0'// -t specifes the type of input sequencing data. Possible values include 0, denoting RNA-seq data, or 1, denoting genomic DNA-seq data.
params.sublong_options = '-X'// -X turn on the RNA-seq mode.
// Report params
params.fastqc = false
params.samtools_stats = false
params.multiqc_config = "$baseDir/config/multiqc_conf.yml"
// other
params.help = null
params.seqtk_sample_size = 10000 // number of reads to sample for seqtk - used to determnine the library type
bbmap_tool = "bbmap.sh"
star_tool = "STAR"
params.debug = false
//*************************************************
// STEP 1 - HELP
//*************************************************
log.info header()
if (params.help) { exit 0, helpMSG() }
//*************************************************
// STEP 1 - PARAMS CHECK
//*************************************************
def stop_pipeline = false
log.info """check aligner provided: ${params.aligner} ..."""
// Check aligner params. Can be a list (comma or space separated)
def aligner_list=[]
if( !params.aligner ){
exit 1, "Error: <aligner> parameter is empty, please provide a aligner(s) among this list ${align_tools}.\n"
} else {
str_list = params.aligner.tokenize(',')
str_list.each {
str_list2 = it.tokenize(' ')
str_list2.each {
if ( ! (it.toLowerCase() in align_tools) ){
exit 1, "Error: <${it}> aligner not acepted, please provide aligner(s) among this list ${align_tools}.\n"
}
else{
aligner_list.add(it.toLowerCase())
}
}
}
}
// check read type parameter
log.info """check read type parameter: ${params.read_type} ..."""
if( ! params.read_type ){
exit 1, "Error: <read_type> parameter is empty, please provide a read type among this list ${read_type_allowed}.\n"
} else {
if ( ! (params.read_type.toLowerCase() in read_type_allowed) ){
exit 1, "Error: <${params.read_type}> aligner not acepted, please provide a read type among this list ${read_type_allowed}.\n"
}
}
// check read library type parameter
log.info """check readlibrary type parameter: ${params.library_type} ..."""
if( ! params.library_type.contains("auto") ){
if ( ! (params.library_type.toUpperCase() in libtype_allowed) ){
exit 1, "Error: <${params.library_type}> library type is not accepted, please provide a library type among this list ${libtype_allowed}.\n"
}
}
// ---- check annotation file
def annotation_file
if (params.annotation){
if (params.annotation.startsWith('http:') || params.annotation.startsWith('https:') || params.annotation.startsWith('s3:') || params.annotation.startsWith('az:') || params.annotation.startsWith('gs:') || params.annotation.startsWith('ftp:') ) {
annotation_file = file(params.annotation)
annotation_file = annotation_file.getName()
} else {
File f = new File( "${params.annotation}" );
if (! f.exists() ){
log.error "Warning: Annotation file <${params.annotation}> does not exist.\n"
stop_pipeline = true
}
annotation_file = f.getName()
}
}
// ----------------------------------------------------------
// Add annotation file within the tool options if annotation provided
// Add specific options for aligner according to the read type
log.info """check alinger parameters ..."""
// --- bbmap tool ---
if ( "bbmap" in aligner_list && !params.relax ){
if (params.read_type == "pacbio" || params.read_type == "ont"){
bbmap_tool = "mapPacBio.sh"
log.warn "Long reads being used, using mapPacBio.sh to align with bbmap!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter to use bbmap.sh anyway."
// Function to check and set maxlen in params.bbmap_options when long_reads is set
// params is supposed to be a immutable. Using params.replace method might not be supported in the future
if ( !params.bbmap_options.contains("maxlen") ){
params.replace("bbmap_options", "${params.bbmap_options} maxlen=5000")
}
}
}
// --- bwa aln tool ---
if ( "bwaaln" in aligner_list ){
if ( params.read_type == "pacbio" || params.read_type == "ont"){
log.warn "Bwaaln aligner is not recommended to align long reads!\n"
}
}
// --- bwa mem tool ---
if ( "bwamem" in aligner_list && !params.relax ){
if (params.read_type == "pacbio"){
if ( !params.bwamem_options.contains(" pacbio") ){
params.replace("bwamem_options", "${params.bwamem_options} -x pacbio")
log.warn "Pacbio reads being used, setting -x pacbio to bwamem!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter and avoid this behavior."
}
}
if (params.read_type == "ont"){
if ( !params.bwamem_options.contains(" ont2d") ){
params.replace("bwamem_options", "${params.bwamem_options} -x ont2d")
log.warn "Ont reads being used, setting -x ont2d to bwamem!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter and avoid this behavior."
}
}
}
// --- bwa mem2 tool ---
if ("bwamem2" in aligner_list && !params.relax){
if (params.read_type == "pacbio"){
if ( !params.bwamem2_options.contains(" pacbio") ){
params.replace("bwamem2_options", "${params.bwamem2_options} -x pacbio")
log.warn "Pacbio reads being used, setting -x pacbio to bwamem2!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter and avoid this behavior."
}
}
if (params.read_type == "ont"){
if ( !params.bwamem2_options.contains(" ont2d") ){
params.replace("bwamem2_options", "${params.bwamem2_options} -x ont2d")
log.warn "Ont reads being used, setting -x ont2d to bwamem2!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter and avoid this behavior."
}
}
}
// --- bwa sw tool ---
if ( "bwasw" in aligner_list ){
if (params.read_type == "pacbio" || params.read_type == "ont"){
log.warn "Bwasw aligner is not recommended to align long reads!\n"
}
}
// --- graphmap2 tool ---
if ( "graphmap2" in aligner_list ){
if (! params.read_type == "pacbio" && ! params.read_type == "ont"){
log.warn "Graphmap2 aligner is not recommended to align short reads!\n"
}
if (annotation_file && !params.graphmap2_options.contains("--gtf ") ){
params.replace("graphmap2_options", "${params.graphmap2_options} --gtf ${annotation_file}")
}
}
// --- kallisto tool ---
if ( "kallisto" in aligner_list ){
if ( params.read_type == "ont" || params.read_type == "pacbio"){
log.warn "Kallisto aligner is not recommended to align long reads!\n"
}
}
// ---- minimap2 tool ---
// Force -a option to be sure to get sam output
if ("minimap2" in aligner_list && !params.relax){
if (params.read_type == "short_single" || params.read_type == "short_paired"){
if ( ! params.minimap2_options.contains("--sr ") ){
params.replace("minimap2_options", "--sr ${params.minimap2_options}")
}
}
if (params.read_type == "pacbio"){
if ( ! params.minimap2_options.contains(" ava-pb") and ! params.minimap2_options.contains(" splice:hq") and
! params.minimap2_options.contains(" map-hifi") and ! params.minimap2_options.contains(" map-pb") ){
log.warn("""Warn: <${params.minimap2_options}> minimap2 options missing or not accepted for pacbio data.
We set the default <map-pb> parameter. If you do not agree, please provide options among this list:
ava-pb, splice:hq, map-hifi, map-pb (see https://github.com/lh3/minimap2).
If you wish to use parameter not intended for pacbio data use the --relax parameter to skip this warning message.\n""")
params.replace("minimap2_options", "${params.minimap2_options} -x map-pb")
}
}
if (params.read_type == "ont"){
if ( ! params.minimap2_options.contains(" ava-ont") and ! params.minimap2_options.contains(" splice") and
! params.minimap2_options.contains(" lr:hq") and ! params.minimap2_options.contains(" map-ont") ){
log.warn("""Warn: <${params.minimap2_options}> minimap2 options missing or not accepted for ont data.
We set the default <map-ont> option. If you do not agree, please provide options among this list:
ava-ont, splice, lr:hq, map-ont (see https://github.com/lh3/minimap2).
If you wish to use parameter not intended for pacbio data use the --relax parameter to skip this warning message.\n""")
params.replace("minimap2_options", "${params.minimap2_options} -x map-ont")
}
}
}
// relax or not this option has to be used
if ( ! params.minimap2_options.contains("-a ") ){
params.replace("minimap2_options", "${params.minimap2_options} -a")
}
// ngmlr tool - check options
if ("ngmlr" in aligner_list ){
if (!params.relax) {
// for pacbio reads, set -g 20 and -x 0
if (params.read_type == "ont"){
if (! params.ngmlr_options.contains("-x ont")){
params.replace("ngmlr_options", "${params.ngmlr_options} -x ont")
log.warn "Ont reads being used, setting -x ont to ngmlr!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter and avoid this behavior."
}
}
}
if (! params.read_type == "pacbio" && ! params.read_type == "ont"){
log.warn "Ngmlr aligner is not recommended to align short reads!\n"
}
}
// novoalign tool - load license into the container
def novoalign_lic = ""
if ("novoalign" in aligner_list ){
if( params.novoalign_license ){
File f = new File( "${params.novoalign_license}" );
if (! f.exists() ){
log.warn ": NovoAlign aligner selected but license file <${params.novoalign_license}> does not exist.\n"
stop_pipeline = true
}
license_file = f.getName()
novoalign_lic = "-v ${params.novoalign_license}:/usr/local/bin/${license_file}"
} else {
log.warn ": NovoAlign aligner selected but no license provided. Please provide a license to run novoalign.\n"
stop_pipeline = true
}
if (!params.relax) {
// for pacbio reads, set -g 20 and -x 0
if (params.read_type == "pacbio" || params.read_type == "ont"){
if (! params.novoalign_options.contains("-g ")){
params.replace("novoalign_options", "${params.novoalign_options} -g 20")
log.warn "Long reads being used, setting -g 20 to Novoalign!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter and avoid this behavior."
}
if (! params.novoalign_options.contains("-x ")){
params.replace("novoalign_options", "${params.novoalign_options} -x 0")
log.warn "Long reads being used, setting -x 0 to Novoalign!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter and avoid this behavior."
}
}
}
}
// --- salmon tool ---
if ( "salmon" in aligner_list ){
if ( params.read_type == "ont" || params.read_type == "pacbio"){
log.warn "Salmon aligner is not recommended to align long reads!\n"
}
}
// --- star tool ---
if ( "star" in aligner_list ){
if (annotation_file && !params.star_options.contains("--sjdbGTFfile ") ){
params.replace("star_options", "${params.star_options} --sjdbGTFfile ${annotation_file}")
}
if (!params.relax){
if (params.read_type == "pacbio" || params.read_type == "ont"){
star_tool = "STARlong"
log.warn "Long reads being used, using STARlong to align with star!\nHowever, if you know what you are doing you can activate the AliNe --relax parameter to use star anyway."
}
}
}
// --- subread tool ---
if ( "subread" in aligner_list ){
if (annotation_file && !params.subread_options.contains("-a ") ){
params.replace("subread_options", "${params.subread_options} -a ${annotation_file}")
}
}
if ( "sublong" in aligner_list ){
if ( params.read_type == "short_single" || params.read_type == "short_paired"){
log.warn "Sublong aligner is not recommended to align short reads!\n"
}
}
if(stop_pipeline){
exit 1, "Please fix previous issues in order to run the pipeline.\n"
}
//*************************************************
// STEP 1 - LOG INFO
//*************************************************
log.info """
General Parameters
genome : ${params.genome}
reads : ${params.reads}
annotation : ${params.annotation}
aligner : ${params.aligner}
read_type : ${params.read_type}
paired_reads_pattern : ${params.paired_reads_pattern}
reads_extension : ${params.reads_extension}
library_type : ${params.library_type}
skip_libray_usage : ${params.skip_libray_usage}
outdir : ${params.outdir}
Report Parameters
fastqc : ${params.fastqc}
samtools_stats : ${params.samtools_stats}
multiqc_config : ${params.multiqc_config}
"""
log.info printAlignerOptions(aligner_list, annotation_file, params.star_index_options)
//*************************************************
// STEP 2 - Include needed modules
//*************************************************
include {read_length; set_tuple_withUserReadLength} from "$baseDir/modules/bash.nf"
include {bbmap_index; bbmap} from "$baseDir/modules/bbmap.nf"
include {bowtie_index; bowtie} from "$baseDir/modules/bowtie.nf"
include {bowtie2_index; bowtie2} from "$baseDir/modules/bowtie2.nf"
include {bwa_index; bwaaln; bwamem; bwasw} from "$baseDir/modules/bwa.nf"
include {bwamem2_index; bwamem2} from "$baseDir/modules/bwamem2.nf"
include {seqkit_convert} from "$baseDir/modules/seqkit.nf"
include {graphmap2_index; graphmap2} from "$baseDir/modules/graphmap2.nf"
include {fastp} from "$baseDir/modules/fastp.nf"
include {fastqc as fastqc_raw; fastqc as fastqc_fastp; fastqc as fastqc_ali_bbmap; fastqc as fastqc_ali_bowtie ; fastqc as fastqc_ali_bowtie2 ;
fastqc as fastqc_ali_bwaaln; fastqc as fastqc_ali_bwamem; fastqc as fastqc_ali_bwamem2; fastqc as fastqc_ali_bwasw; fastqc as fastqc_ali_graphmap2 ;
fastqc as fastqc_ali_hisat2; fastqc as fastqc_ali_kallisto; fastqc as fastqc_ali_last; fastqc as fastqc_ali_minimap2; fastqc as fastqc_ali_ngmlr;
fastqc as fastqc_ali_novoalign ; fastqc as fastqc_ali_nucmer; fastqc as fastqc_ali_salmon; fastqc as fastqc_ali_star; fastqc as fastqc_ali_subread ;
fastqc as fastqc_ali_sublong } from "$baseDir/modules/fastqc.nf"
include {hisat2_index; hisat2} from "$baseDir/modules/hisat2.nf"
include {kallisto_index; kallisto} from "$baseDir/modules/kallisto.nf"
include {last_index; last} from "$baseDir/modules/last.nf"
include {minimap2_index; minimap2} from "$baseDir/modules/minimap2.nf"
include {multiqc} from "$baseDir/modules/multiqc.nf"
include {ngmlr} from "$baseDir/modules/ngmlr.nf"
include {nucmer} from "$baseDir/modules/mummer4.nf"
include {novoalign_index; novoalign} from "$baseDir/modules/novoalign.nf"
include {salmon_index; salmon_guess_lib; set_tuple_withUserLib; salmon} from "$baseDir/modules/salmon.nf"
include {samtools_sam2bam_nucmer; samtools_sam2bam as samtools_sam2bam_bowtie; samtools_sam2bam as samtools_sam2bam_bowtie2;
samtools_sam2bam as samtools_sam2bam_bwaaln; samtools_sam2bam as samtools_sam2bam_bwamem; samtools_sam2bam as samtools_sam2bam_bwamem2;
samtools_sam2bam as samtools_sam2bam_bwasw; samtools_sam2bam as samtools_sam2bam_graphmap2; samtools_sam2bam as samtools_sam2bam_hisat2;
samtools_sam2bam as samtools_sam2bam_last; samtools_sam2bam as samtools_sam2bam_minimap2;
samtools_sam2bam as samtools_sam2bam_ngmlr; samtools_sam2bam as samtools_sam2bam_novoalign; samtools_sam2bam as samtools_sam2bam_salmon } from "$baseDir/modules/samtools.nf"
include {samtools_sort as samtools_sort_bbmap; samtools_sort as samtools_sort_bowtie; samtools_sort as samtools_sort_bowtie2; samtools_sort as samtools_sort_bwaaln;
samtools_sort as samtools_sort_bwamem; samtools_sort as samtools_sort_bwamem2; samtools_sort as samtools_sort_bwasw; samtools_sort as samtools_sort_graphmap2;
samtools_sort as samtools_sort_hisat2; samtools_sort as samtools_sort_last; samtools_sort as samtools_sort_minimap2; samtools_sort as samtools_sort_ngmlr;
samtools_sort as samtools_sort_novoalign; samtools_sort as samtools_sort_nucmer; samtools_sort as samtools_sort_salmon;
samtools_sort as samtools_sort_sublong; } from "$baseDir/modules/samtools.nf"
include {samtools_stats as samtools_stats_ali_bbmap; samtools_stats as samtools_stats_ali_bowtie; samtools_stats as samtools_stats_ali_bowtie2 ;
samtools_stats as samtools_stats_ali_bwaaln; samtools_stats as samtools_stats_ali_bwamem; samtools_stats as samtools_stats_ali_bwamem2;
samtools_stats as samtools_stats_ali_bwasw; samtools_stats as samtools_stats_ali_graphmap2; samtools_stats as samtools_stats_ali_hisat2;
samtools_stats as samtools_stats_ali_kallisto; samtools_stats as samtools_stats_ali_last; samtools_stats as samtools_stats_ali_minimap2; samtools_stats as samtools_stats_ali_ngmlr;
samtools_stats as samtools_stats_ali_novoalign ; samtools_stats as samtools_stats_ali_nucmer; samtools_stats as samtools_stats_ali_salmon; samtools_stats as samtools_stats_ali_star;
samtools_stats as samtools_stats_ali_subread; samtools_stats as samtools_stats_ali_sublong } from "$baseDir/modules/samtools.nf"
include {samtools_merge_bam} from "$baseDir/modules/samtools.nf"
include {seqtk_sample} from "$baseDir/modules/seqtk.nf"
include {subread_index; subread; sublong_index; sublong} from "$baseDir/modules/subread.nf"
include {prepare_star_index_options; star_index; star; star2pass} from "$baseDir/modules/star.nf"
//*************************************************
// STEP 3 - CHECK 2 for parameters
//*************************************************
// check profile
if (
workflow.profile.contains('singularity') ||
workflow.profile.contains('docker')
) { "executer selected" }
else { exit 1, "No executer selected: -profile docker/singularity"}
// --------- handle read input (file or folder / local or remote / paired or not) --------
def list_files = []
def pattern_reads
def fromFilePairs_input
def path_reads = params.reads
// check if paired reads
def per_pair = false // read the reads per pair
if (params.read_type == "short_paired") {
per_pair = true
}
// Case of remote data
def via_URL = false
def read_list=[]
if (path_reads.startsWith('https:') || path_reads.startsWith('s3:') || path_reads.startsWith('az:') || path_reads.startsWith('gs:') || path_reads.startsWith('ftp:') ) {
log.info "The input is a based on URLs! ${path_reads}\n"
via_URL = true
str_list = path_reads.tokenize(',')
str_list.each {
str_list2 = it.tokenize(' ')
str_list2.each {
read_list.add(file(it)) // use file insted of File for URL
}
}
fromFilePairs_input = read_list
}
// Case of local data
else {
File input_reads = new File(path_reads)
if(input_reads.exists()){
if ( input_reads.isDirectory()) {
// in case of folder provided, add a trailing slash if missing
path_reads = "${input_reads}" + "/"
}
}
if (params.read_type == "short_paired") {
pattern_reads = "${params.paired_reads_pattern}${params.reads_extension}"
fromFilePairs_input = "${path_reads}*${params.paired_reads_pattern}${params.reads_extension}"
} else{
pattern_reads = "${params.reads_extension}"
fromFilePairs_input = "${path_reads}*${params.reads_extension}"
}
if(input_reads.exists()){
if ( input_reads.isDirectory()) {
log.info "The input ${path_reads} is a folder!\n"
input_reads.eachFileRecurse(FILES){
if (it.name =~ ~/${pattern_reads}/){
list_files.add(it)
}
}
samples_number = list_files.size()
log.info "${samples_number} files in ${path_reads} with pattern ${pattern_reads}"
}
else {
log.info "The input ${path_reads} is a file!\n"
fromFilePairs_input = "${path_reads}"
if (params.read_type == "short_paired") {
log.error "Providing a file is not authorized for (local) paired data! Please provide a folder path or change <read_type> parameter to <short_single>.\n"
}
}
} else {
exit 1, "The input ${path_reads} does not exists!\n"
}
}
//*************************************************
// STEP 4 - Main Workflow
//*************************************************
workflow {
main:
// In case of URL paired data, we cannot use fromFilePairs because matching pattern impossible. We must recreate manually a structure similar
if (via_URL && per_pair){
my_samples = Channel.of(read_list)
reads = my_samples.flatten().map { it -> [it.name.split('_')[0], it] }
.groupTuple()
.ifEmpty { exit 1, "Cannot find reads matching ${path_reads}!\n" }
} else {
reads = Channel.fromFilePairs(fromFilePairs_input, size: per_pair ? 2 : 1, checkIfExists: true)
.ifEmpty { exit 1, "Cannot find reads matching ${path_reads}!\n" }
}
genome = Channel.fromPath(params.genome, checkIfExists: true)
.ifEmpty { exit 1, "Cannot find genome matching ${params.genome}!\n" }
if(annotation_file){
annotation = Channel.fromPath(params.annotation, checkIfExists: true)
.ifEmpty { exit 1, "Cannot find annotation matching ${annotation_file}!\n" }
} else {
annotation = Channel.of("$baseDir/config/aline_null.gtf") // use the fake file (not used by tools just for the processes to be called)
}
align(reads, genome, annotation, aligner_list)
}
//*************************************************
// STEP 4 - Workflow align
//*************************************************
workflow align {
take:
raw_reads
genome
annotation
aligner_list
main:
// Initialize channels
Channel.empty().set{logs}
Channel.empty().set{sorted_bam}
// ------------------------------------------------------------------------------------------------
// PREPROCESSING
// ------------------------------------------------------------------------------------------------
// ------------------- QC -----------------
if(params.fastqc){
fastqc_raw(raw_reads, "fastqc/raw", "raw")
logs.concat(fastqc_raw.out).set{logs} // save log
}
// ------------------- Standardize Score to Be Phred+33 ----------------
params.debug && log.info('Standardize Score to Be Phred+33')
seqkit_convert(raw_reads, "seqkit_score")
seqkit_convert.out.trimmed.set{raw_reads_standardized}
// ------------------------------------------------------------------------------------------------
// TRIMMING
// ------------------------------------------------------------------------------------------------
// ------------------- FASTP -----------------
if (params.trimming_fastp){
params.debug && log.info('fastp trimming')
fastp(raw_reads_standardized, "fastp")
fastp.out.trimmed.set{raw_reads_trim}
logs.concat(fastp.out.report).set{logs} // save log
if(params.fastqc){
fastqc_fastp(raw_reads_trim, "fastqc/trimming_fastp", "trimmed")
logs.concat(fastqc_fastp.out).set{logs} // save log
}
} else {
raw_reads_trim = raw_reads_standardized
}
// ------------------------------------------------------------------------------------------------
// LIBRARY TYPE GUESSING -
// here we subsample and do read length and library type guessing only if needed.
// ------------------------------------------------------------------------------------------------
// In which case I need the mean read length (kallisto, salmon, star)
// Need to list here all aligner that need the read length
params.debug && log.info('read_length guessing')
def guess_read_length = null
if ( (!params.read_length) &&
( ("kallisto" in aligner_list && params.read_type == "short_single" && ( !params.kallisto_options.contains("-l ") || !params.kallisto_options.contains("--fragment-length ") ) ) ||
( annotation.toString() != "aline_null.gtf" && !params.star_index_options.contains("--sjdbOverhang") ) ||
( "salmon" in aligner_list && params.read_type == "short_single" && !params.salmon.contains("--fldMean ") ) ) ){
guess_read_length=1
}
params.debug && log.info('subsample guessing')
// Should I subsample for library type guessing?
def subsample_lg = false
if (params.library_type.contains("auto")){
subsample_lg = true
}
// Subsample if needed (can be for library guessing or read length guessing)
if (subsample_lg || guess_read_length){
// ------------------- subsample -----------------
seqtk_sample(raw_reads_trim)
}
// ------------------- set libtype -----------------
if (params.library_type.contains("auto")){
// ------------------- guess libtype -------------------
salmon_index(genome.collect())
salmon_guess_lib(seqtk_sample.out.sampled, salmon_index.out.index, "salmon_libtype")
salmon_guess_lib.out.tuple_id_libtype.set{tuple_id_lib}
} else {
set_tuple_withUserLib(raw_reads_trim)
set_tuple_withUserLib.out.set{tuple_id_lib}
}
reads = raw_reads_trim.join(tuple_id_lib)
// ------------------- set read length -----------------
if (guess_read_length){
read_length(seqtk_sample.out.sampled, "mean_read_length")
read_length.out.tuple_id_readlength.set{tuple_id_readle}
}else{
set_tuple_withUserReadLength(raw_reads_trim)
set_tuple_withUserReadLength.out.set{tuple_id_readle}
}
reads = reads.join(tuple_id_readle)
// ------------------------------------------------------------------------------------------------
// ALIGNEMENT
// ------------------------------------------------------------------------------------------------
// ------------------- BBMAP -----------------
if ("bbmap" in aligner_list ){
// index
bbmap_index(genome.collect(), "alignment/bbmap/indicies")
// align
bbmap(reads, genome.collect(), bbmap_index.out.collect(), "alignment/bbmap")
logs.concat(bbmap.out.bbmap_summary).set{logs} // save log
// sort
samtools_sort_bbmap(bbmap.out.tuple_sample_bam, "alignment/bbmap")
samtools_sort_bbmap.out.tuple_sample_sortedbam.set{bbmap_ali} // set name
// save aligned reads
sorted_bam.concat(bbmap_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_bbmap(bbmap_ali, "fastqc/bbmap", "bbmap")
logs.concat(fastqc_ali_bbmap.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_bbmap(bbmap_ali, genome.collect(), "samtools_stats/bbmap", "bbmap")
logs.concat(samtools_stats_ali_bbmap.out).set{logs} // save log
}
}
// ------------------- BOWTIE -----------------
if ( "bowtie" in aligner_list ){ // &&
bowtie_index(genome.collect(), "alignment/bowtie/indicies") // index
bowtie(reads, genome.collect(), bowtie_index.out.collect(), "alignment/bowtie") // align
logs.concat(bowtie.out.bowtie_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_bowtie(bowtie.out.tuple_sample_sam)
// sort
samtools_sort_bowtie(samtools_sam2bam_bowtie.out.tuple_sample_bam, "alignment/bowtie")
samtools_sort_bowtie.out.tuple_sample_sortedbam.set{bowtie_ali} // set name
// save aligned reads
sorted_bam.concat(bowtie_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_bowtie(bowtie_ali, "fastqc/bowtie", "bowtie")
logs.concat(fastqc_ali_bowtie.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_bowtie(bowtie_ali, genome.collect(), "samtools_stats/bowtie", "bowtie")
logs.concat(samtools_stats_ali_bowtie.out).set{logs} // save log
}
}
// ------------------- BOWTIE2 -----------------
if ( "bowtie2" in aligner_list ){
// index
bowtie2_index(genome.collect(), "alignment/bowtie2/indicies")
// align
bowtie2(reads, genome.collect(), bowtie2_index.out.collect(), "alignment/bowtie2")
logs.concat(bowtie2.out.bowtie2_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_bowtie2(bowtie2.out.tuple_sample_sam)
// sort
samtools_sort_bowtie2(samtools_sam2bam_bowtie2.out.tuple_sample_bam, "alignment/bowtie2")
samtools_sort_bowtie2.out.tuple_sample_sortedbam.set{bowtie2_ali} // set name
// save aligned reads
sorted_bam.concat(bowtie2_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_bowtie2(bowtie2_ali, "fastqc/bowtie2", "bowtie2")
logs.concat(fastqc_ali_bowtie2.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_bowtie2(bowtie2_ali, genome.collect(), "samtools_stats/bowtie2", "bowtie2")
logs.concat(samtools_stats_ali_bowtie2.out).set{logs} // save log
}
}
// ------------------- BWA ALN/MEM/SW -----------------
if ("bwaaln" in aligner_list || "bwamem" in aligner_list || "bwasw" in aligner_list){
// index
bwa_index(genome.collect(), "alignment/bwa/indicies")
if ("bwaaln" in aligner_list){
// align
bwaaln(reads, genome.collect(), bwa_index.out.collect(), "alignment/bwa/bwaaln")
logs.concat(bwaaln.out.bwaaln_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_bwaaln(bwaaln.out.tuple_sample_sam)
// sort
samtools_sort_bwaaln(samtools_sam2bam_bwaaln.out.tuple_sample_bam, "alignment/bwa/bwaaln")
samtools_sort_bwaaln.out.tuple_sample_sortedbam.set{bwaaln_ali} // set name
// save aligned reads
sorted_bam.concat(bwaaln_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_bwaaln(bwaaln_ali, "fastqc/bwaaln", "bwaaln")
logs.concat(fastqc_ali_bwaaln.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_bwaaln(bwaaln_ali, genome.collect(), "samtools_stats/bwaaln", "bwaaln")
logs.concat(samtools_stats_ali_bwaaln.out).set{logs} // save log
}
}
if ("bwamem" in aligner_list){
// align
bwamem(reads, genome.collect(), bwa_index.out.collect(), "alignment/bwa/bwamem")
logs.concat(bwamem.out.bwamem_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_bwamem(bwamem.out.tuple_sample_sam)
// sort
samtools_sort_bwamem(samtools_sam2bam_bwamem.out.tuple_sample_bam, "alignment/bwa/bwamem")
samtools_sort_bwamem.out.tuple_sample_sortedbam.set{bwamem_ali} // set name
// save aligned reads
sorted_bam.concat(bwamem_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_bwamem(bwamem_ali, "fastqc/bwamem", "bwamem")
logs.concat(fastqc_ali_bwamem.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_bwamem(bwamem_ali, genome.collect(), "samtools_stats/bwamem", "bwamem")
logs.concat(samtools_stats_ali_bwamem.out).set{logs} // save log
}
}
if ("bwasw" in aligner_list){
// align
bwasw(reads, genome.collect(), bwa_index.out.collect(), "alignment/bwa/bwasw")
logs.concat(bwasw.out.bwasw_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_bwasw(bwasw.out.tuple_sample_sam)
// sort
samtools_sort_bwasw(samtools_sam2bam_bwasw.out.tuple_sample_bam, "alignment/bwa/bwasw")
samtools_sort_bwasw.out.tuple_sample_sortedbam.set{bwasw_ali} // set name
// save aligned reads
sorted_bam.concat(bwasw_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_bwasw(bwasw_ali, "fastqc/bwasw", "bwasw")
logs.concat(fastqc_ali_bwasw.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_bwasw(bwasw_ali, genome.collect(), "samtools_stats/bwasw", "bwasw")
logs.concat(samtools_stats_ali_bwasw.out).set{logs} // save log
}
}
}
// ------------------- BWA MEM2 -----------------
if ("bwamem2" in aligner_list){
// index
bwamem2_index(genome.collect(), "alignment/bwamem2/indicies")
// align
bwamem2(reads, genome.collect(), bwamem2_index.out.collect(), "alignment/bwamem2/")
logs.concat(bwamem2.out.bwamem2_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_bwamem2(bwamem2.out.tuple_sample_sam)
// sort
samtools_sort_bwamem2(samtools_sam2bam_bwamem2.out.tuple_sample_bam, "alignment/bwamem2/")
samtools_sort_bwamem2.out.tuple_sample_sortedbam.set{bwamem2_ali} // set name
// save aligned reads
sorted_bam.concat(bwamem2_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_bwamem2(bwamem2_ali, "fastqc/bwamem2", "bwamem2")
logs.concat(fastqc_ali_bwamem2.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_bwamem2(bwamem2_ali, genome.collect(), "samtools_stats/bwamem2", "bwamem2")
logs.concat(samtools_stats_ali_bwamem2.out).set{logs} // save log
}
}
// ------------------- GRAPHMAP2 -----------------
if ("graphmap2" in aligner_list ){
// index
graphmap2_index(genome.collect(), "alignment/graphmap2/indicies")
// align
graphmap2(reads, genome.collect(), graphmap2_index.out.collect(), annotation.collect(), "alignment/graphmap2")
logs.concat(graphmap2.out.graphmap2_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_graphmap2(graphmap2.out.tuple_sample_sam)
// sort
samtools_sort_graphmap2(samtools_sam2bam_graphmap2.out.tuple_sample_bam, "alignment/graphmap2")
samtools_sort_graphmap2.out.tuple_sample_sortedbam.set{graphmap2_ali} // set name
// save aligned reads
sorted_bam.concat(graphmap2_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_graphmap2(graphmap2_ali, "fastqc/graphmap2", "graphmap2")
logs.concat(fastqc_ali_graphmap2.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_graphmap2(graphmap2_ali, genome.collect(), "samtools_stats/graphmap2", "graphmap2")
logs.concat(samtools_stats_ali_graphmap2.out).set{logs} // save log
}
}
// ------------------- HISAT2 -----------------
if ("hisat2" in aligner_list){
// index
hisat2_index(genome.collect(), "alignment/hisat2/indicies")
// align
hisat2(reads, hisat2_index.out.collect(), "alignment/hisat2")
logs.concat(hisat2.out.hisat2_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_hisat2(hisat2.out.tuple_sample_sam)
// sort
samtools_sort_hisat2(samtools_sam2bam_hisat2.out.tuple_sample_bam, "alignment/hisat2")
samtools_sort_hisat2.out.tuple_sample_sortedbam.set{hisat2_ali} // set name
// save aligned reads
sorted_bam.concat(hisat2_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_hisat2(hisat2_ali, "fastqc/hisat2", "hisat2")
logs.concat(fastqc_ali_hisat2.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_hisat2(hisat2_ali, genome.collect(), "samtools_stats/hisat2", "hisat2")
logs.concat(samtools_stats_ali_hisat2.out).set{logs} // save log
}
}
// ------------------- KALLISTO -----------------
if ("kallisto" in aligner_list){
// index
kallisto_index(genome.collect(), "alignment/kallisto/indicies")
// align
kallisto(reads, kallisto_index.out.collect(), "alignment/kallisto")
logs.concat(kallisto.out.kallisto_summary).set{logs} // save log
kallisto.out.tuple_sample_bam.set{kallisto_ali} // set name
// save aligned reads
sorted_bam.concat(kallisto_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_kallisto(kallisto_ali, "fastqc/kallisto", "kallisto")
logs.concat(fastqc_ali_kallisto.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_kallisto(kallisto_ali, genome.collect(), "samtools_stats/kallisto", "kallisto")
logs.concat(samtools_stats_ali_kallisto.out).set{logs} // save log
}
}
// --------------------- LAST --------------------
if ("last" in aligner_list ){
// index
last_index(genome.collect(), "alignment/last/indicies")
// align
last(reads, genome.collect(), last_index.out.collect(), "alignment/last")
logs.concat(last.out.last_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_last(last.out.tuple_sample_sam)
// sort
samtools_sort_last(samtools_sam2bam_last.out.tuple_sample_bam, "alignment/last")
samtools_sort_last.out.tuple_sample_sortedbam.set{last_ali} // set name
// save aligned reads
sorted_bam.concat(last_ali).set{sorted_bam}
// stat on aligned reads
if (params.fastqc){
fastqc_ali_last(last_ali, "fastqc/last", "last")
logs.concat(fastqc_ali_last.out).set{logs} // save log
}
if (params.samtools_stats){
samtools_stats_ali_last(last_ali, genome.collect(), "samtools_stats/last", "last")
logs.concat(samtools_stats_ali_last.out).set{logs} // save log
}
}
// ------------------- minimap2 -----------------
if ("minimap2" in aligner_list ){
// index
minimap2_index(genome.collect(), "alignment/minimap2/indicies")
// align
minimap2(reads, genome.collect(), minimap2_index.out.collect(), "alignment/minimap2")
logs.concat(minimap2.out.minimap2_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_minimap2(minimap2.out.tuple_sample_sam)
// sort
samtools_sort_minimap2(samtools_sam2bam_minimap2.out.tuple_sample_bam, "alignment/minimap2")
samtools_sort_minimap2.out.tuple_sample_sortedbam.set{minimap2_ali} // set name
// save aligned reads
sorted_bam.concat(minimap2_ali).set{sorted_bam}
// stat on aligned reads
if(params.fastqc){
fastqc_ali_minimap2(minimap2_ali, "fastqc/minimap2", "minimap2")
logs.concat(fastqc_ali_minimap2.out).set{logs} // save log
}
if(params.samtools_stats){
samtools_stats_ali_minimap2(minimap2_ali, genome.collect(), "samtools_stats/minimap2", "minimap2")
logs.concat(samtools_stats_ali_minimap2.out).set{logs} // save log
}
}
// --------------------- NGMLR --------------------
if ("ngmlr" in aligner_list ){
// align
ngmlr(reads, genome.collect(), "alignment/ngmlr")
logs.concat(ngmlr.out.ngmlr_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_ngmlr(ngmlr.out.tuple_sample_sam)
// sort
samtools_sort_ngmlr(samtools_sam2bam_ngmlr.out.tuple_sample_bam, "alignment/ngmlr")
samtools_sort_ngmlr.out.tuple_sample_sortedbam.set{ngmlr_ali} // set name
// save aligned reads
sorted_bam.concat(ngmlr_ali).set{sorted_bam}
// stat on aligned reads
if (params.fastqc){
fastqc_ali_ngmlr(ngmlr_ali, "fastqc/ngmlr", "ngmlr")
logs.concat(fastqc_ali_ngmlr.out).set{logs} // save log
}
if (params.samtools_stats){
samtools_stats_ali_ngmlr(ngmlr_ali, genome.collect(), "samtools_stats/ngmlr", "ngmlr")
logs.concat(samtools_stats_ali_ngmlr.out).set{logs} // save log
}
}
// ------------------- novoalign -----------------
if ("novoalign" in aligner_list ){
// index
novoalign_index(genome.collect(), "alignment/minimap2/indicies")
// align
novoalign(reads, genome.collect(), novoalign_index.out.collect(), novoalign_lic, "alignment/novoalign")
// convert sam to bam
samtools_sam2bam_novoalign(novoalign.out.tuple_sample_sam)
// sort
samtools_sort_novoalign(samtools_sam2bam_novoalign.out.tuple_sample_bam, "alignment/novoalign")
samtools_sort_novoalign.out.tuple_sample_sortedbam.set{novoalign_ali} // set name
// save aligned reads
sorted_bam.concat(novoalign_ali).set{sorted_bam}
// stat on aligned reads
if (params.fastqc){
fastqc_ali_novoalign(novoalign_ali, "fastqc/novoalign", "novoalign")
logs.concat(fastqc_ali_novoalign.out).set{logs} // save log
}
if (params.samtools_stats){
samtools_stats_ali_novoalign(novoalign_ali, genome.collect(), "samtools_stats/novoalign", "novoalign")
logs.concat(samtools_stats_ali_novoalign.out).set{logs} // save log
}
}
// ------------------- nucmer (mummer4) -----------------
if ("nucmer" in aligner_list ){
// align
nucmer(reads, genome.collect(), "alignment/nucmer")
// No summary available. To get one we could run show-coords see https://mummer4.github.io/tutorial/tutorial.html
// convert sam to bam
samtools_sam2bam_nucmer(nucmer.out.tuple_sample_sam, genome.collect())
// sort
samtools_sort_nucmer(samtools_sam2bam_nucmer.out.tuple_sample_bam, "alignment/nucmer")
samtools_sort_nucmer.out.tuple_sample_sortedbam.set{nucmer_ali} // set name
// save aligned reads
sorted_bam.concat(nucmer_ali).set{sorted_bam}
// stat on aligned reads
if (params.fastqc){
fastqc_ali_nucmer(nucmer_ali, "fastqc/nucmer", "nucmer")
logs.concat(fastqc_ali_nucmer.out).set{logs} // save log
}
if (params.samtools_stats){
samtools_stats_ali_nucmer(nucmer_ali, genome.collect(), "samtools_stats/nucmer", "nucmer")
logs.concat(samtools_stats_ali_nucmer.out).set{logs} // save log
}
}
if ("salmon" in aligner_list ){
// index
if (! params.library_type.contains("auto")){ // run salmon index only if library type is not provided otherwise it has been already ran
salmon_index(genome.collect(), "alignment/salmon/indicies")
}
// align
salmon(reads, salmon_index.out.collect(), "alignment/salmon")
logs.concat(salmon.out.salmon_summary).set{logs} // save log
// convert sam to bam
samtools_sam2bam_salmon(salmon.out.tuple_sample_sam)
// sort
samtools_sort_salmon(samtools_sam2bam_salmon.out.tuple_sample_bam, "alignment/salmon")
samtools_sort_salmon.out.tuple_sample_sortedbam.set{salmon_ali} // set name