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index 0000000000000000000000000000000000000000..cf3cafc1813c56806454a1f75979b52f2d8ff4f6
--- /dev/null
+++ b/src/orelob.nf
@@ -0,0 +1,330 @@
+#!/usr/bin/env nextflow
+
+nextflow.enable.dsl=2
+//syntax extension DSL2
+
+/*
+========================================================================================================================
+ Orelob
+========================================================================================================================
+
+bolero pipeline :
+ * Pipeline dedicated to transcription terminaison analysis of Hepatitis B Virus from nanopore seq
+ * Preprocessing, filtration, alignment, quantification.
+
+ ****************************************************************
+ Help Message Definition
+ ****************************************************************
+*/
+
+def helpMessage() {
+ log.info"""
+ Usage:
+ The typical command for running the pipeline is as follows:
+
+ nextflow ./src/orelob.nf -c ./src/nextflow.config -profile singularity
+
+ Nextflow parameters:
+ -profile [str] Configuration profile to use.
+ Available: docker, singularity, podman, psmn, ccin2p3
+
+ Mandatory arguments:
+ --input [path] Path to the folder containing fast5 files.
+ If skip basecalling option enabled, path to fastq files folder.
+ --adapt [file] Sequence of 3'RACE adapter.
+ --gsp [file] Sequence of gene-specific primer used in 3'RACE amplification step.
+
+ References:
+ --genome [file] Path to the fasta file containing the genome.
+ --gtf [file] Path to the gtf file containing the genome annotation.
+
+ Nanopore basecalling:
+ --skipBC [boolean] Skip basecalling step. If true, give fastq folder as input. Default: true.
+ --flowcell [str] Nanopore flowcell. Default = FLO-MIN106.
+ --kit [str] Nanopore kit. Default = SQK-PBK004.
+ --gpu_mode [boolean] Guppy basecaller configuration. Default: false.
+ "gpu" mode is dedicated to NVIDIA Cuda compatible system according to Guppy specifications.
+
+ Nanopore barcoding:
+ --kit_barcoding Nanopore barcoding kit.
+ --config_file Nanopore configuration file.
+
+ GPU basecalling parameters:
+ --min_qscore [float] Minimum quality score threshold, default = 7.0.
+ --gpu_runners_per_device [int] Number of runner per device, default = 32 (refer to guppy manual).
+ --num_callers [int] Number of callers, default = 16 (refer to guppy manual).
+ --chunks_per_runner [int] Number of chunks per runner, default = 512 (refer to guppy manual).
+ --chunk_size [int] Chunck size, default = 1900 (refer to guppy manual).
+
+ Help:
+ --help | --h Display this help message.
+
+ """.stripIndent()
+}
+
+// Show help message
+
+params.help = ""
+params.h = ""
+
+if (params.help || params.h) {
+ helpMessage()
+ exit 0
+}
+
+/*
+ ****************************************************************
+ Default Parameters
+ ****************************************************************
+*/
+
+/* Params in */
+
+params.skipBC = true
+params.gpu_mode = false
+params.adapt = ""
+params.gsp = ""
+params.genome = "/home/xavier/Data/Genome/202201_Full-length_HBV_GTFv3/20230516_HBV_FL_preCore_reference.fasta"
+params.gtf = "/home/xavier/Data/Genome/202201_Full-length_HBV_GTFv3/20230516_GTF_preCore_FL_HBV_XGR.gtf"
+
+params.flowcell = "FLO-MIN106"
+params.kit = "SQK-PBK004"
+params.min_qscore = 7.0
+params.gpu_runners_per_device = 32
+params.num_callers = 16
+params.chunks_per_runner = 512
+params.chunk_size = 1900
+params.config_file = ""
+params.kit_barcoding = ""
+
+
+/* Params out */
+
+params.basecalling_out = "01_basecalling/"
+params.barcoding_out = "02_barcoding/"
+params.fastq_out = "03_fastq/"
+params.seqkit_grep_out = "03_fastq/"
+params.porechop_out = "03_fastq/"
+params.cutadapt_out = "04_cutadapt/"
+params.minimap2_genome_out = "05_minimap2/"
+params.filtered_bam_out = "05_minimap2/"
+params.start_position_counts_out = "06_start_positions/"
+params.nanosplicer_out = "07_nanosplicer/"
+params.rna_count_out = "08_RNA_count/"
+params.rna_qc_out = "09_quality_control/"
+
+/*
+ ****************************************************************
+ Logs
+ ****************************************************************
+*/
+
+log.info "fast5/q folder : ${params.input}"
+log.info "3'RACE adapter sequence : ${params.adapt}"
+log.info "Gene specific primer : ${params.gsp}"
+if(!params.skipBC) log.info "Guppy basecalling calculation using GPU mode : ${params.gpu_mode}."
+log.info "Genome file : ${params.genome}"
+log.info "Genome annotation file : ${params.gtf}"
+
+/*
+ ****************************************************************
+ Channel definitions
+ ****************************************************************
+*/
+
+Channel
+ .of( params.input )
+ .ifEmpty { error "No fast5/q folder defined." }
+ .set { input }
+
+Channel
+ .fromPath( params.genome )
+ .ifEmpty { error "No genome defined, a fasta file containing the full length preC RNA from HBV genome." }
+ .set { genome }
+
+Channel
+ .fromPath( params.gtf )
+ .ifEmpty { error "No annotation defined, a gtf file describing transcripts and splice variants." }
+ .set { gtf }
+
+Channel
+ .fromPath(params.input+'*/', type: 'dir')
+ .map(it -> [it.baseName, it])
+ .set{barcodes}
+
+
+/*
+ ****************************************************************
+ Imports
+ ****************************************************************
+*/
+
+if(!params.skipBC) {
+ /* Hardware configuration, if Nvidia CUDA compatible graphic card is installed, use guppy-gpu, else guppy-cpu (much slower)*/
+ if(params.gpu_mode) {
+ include { basecall_fast5_gpu } from "./nf_modules/ont-guppy/main.nf"
+ include { barcoding_gpu } from "./nf_modules/ont-guppy/main.nf"
+ }
+ else {
+ include { basecall_fast5_cpu } from "./nf_modules/ont-guppy/main.nf"
+ include { barcoding_cpu } from "./nf_modules/ont-guppy/main.nf"
+ }
+}
+
+include { barcoding_cpu } from "./nf_modules/ont-guppy/main.nf"
+include { control_basecalling } from "./nf_modules/pycoqc/main.nf"
+include { control_bam } from "./nf_modules/pycoqc/main.nf"
+include { concatenate } from "./nf_modules/seqkit/main.nf"
+include { cut_5pRACE } from "./nf_modules/cutadapt/main.nf"
+include { hbv_genome } from "./nf_modules/minimap2/main.nf"
+include { seqkit_grep } from "./nf_modules/seqkit/main.nf"
+include { sort_bam } from './nf_modules/samtools/main.nf' addParams(sort_bam_out: params.minimap2_genome_out)
+include { index_bam } from './nf_modules/samtools/main.nf' addParams(index_bam_out: params.minimap2_genome_out)
+include { sort_index_bam } from './nf_modules/samtools/main.nf' addParams(indexed_bam_out: params.minimap2_genome_out)
+include { filter_as } from './nf_modules/samtools/main.nf'
+include { start_position_counts } from "./nf_modules/samtools/main.nf"
+include { start_position_individuals } from "./nf_modules/start_positions/main.nf"
+include { jwr_checker } from "./nf_modules/nanosplicer/main.nf"
+include { junctions_nanosplicer } from "./nf_modules/junction_nanosplicer/main.nf"
+include { rna_count } from "./nf_modules/rna_count/main.nf"
+
+include { porechop } from "./nf_modules/porechop/main.nf"
+include { trimmming_pychopper } from "./nf_modules/pychopper/main.nf"
+
+/*
+ ****************************************************************
+ Workflow
+ ****************************************************************
+*/
+
+workflow {
+
+ if(params.skipBC) { // we take fastq files as input and skip basecalling
+ concatenate(barcodes)
+ }
+
+ else { // we take fast5 files as input and proceed to basecalling with guppy
+ if(params.gpu_mode) {
+ basecall_fast5_gpu(input)
+ if(params.kit_barcoding != ""){
+ barcoding_gpu(basecall_fast5_gpu.out.pass)
+ barcoding_gpu.out.barcodes
+ .flatten()
+ .map{it -> [it.name, it]}
+ .set{tuples_barcode}
+ concatenate(tuples_barcode)
+ }
+ else{
+ basecall_fast5_gpu.out.pass
+ .map{it -> ["Sample", it]}
+ .set{tuple_sample}
+ concatenate(tuple_sample)
+ }
+
+ }
+ else {
+ basecall_fast5_cpu(input)
+ if(params.kit_barcoding != ""){
+ barcoding_cpu(basecall_fast5_cpu.out.pass)
+ barcoding_cpu.out.barcodes
+ .flatten()
+ .map{it -> [it.name, it]}
+ .set{tuples_barcode}
+ concatenate(tuples_barcode)
+ }
+ else{
+ basecall_fast5_cpu.out.pass
+ .map{it -> ["Sample", it]}
+ .set{tuple_sample}
+ concatenate(tuple_sample)
+ }
+ }
+ }
+
+
+
+ //####################### PREPROCESSING #######################
+
+
+ //Filtration (seqkit_grep looks for the 5'RACE and the gsp patterns in the reads to keep only mature ARNs)
+ seqkit_grep(concatenate.out.merged_fastq, params.adapt, params.gsp)
+
+ //Trimming with porechop
+ porechop(seqkit_grep.out.filtered_fastq)
+
+ //Trimming with pychopper
+ //trimmming_pychopper(seqkit_grep.out.filtered_fastq)
+
+ //Cut of the 5'RACE sequence
+ cut_5pRACE(porechop.out.porechoped_fastq, params.adapt)
+ //cut_5pRACE(trimmming_pychopper.out.pychoped_fastq, params.adapt)
+ //cut_5pRACE(seqkit_grep.out.filtered_fastq, params.adapt)
+
+ //########################## MAPPING ##########################
+
+ hbv_genome(cut_5pRACE.out.fastq_cutadapt, genome.collect())
+
+ //Filter
+ filter_as(hbv_genome.out.bam)
+
+ //Index
+ sort_index_bam(filter_as.out.filtered_bam)
+
+ //Quality control
+ if(params.skipBC == false) {
+ if(params.gpu_mode) {
+ control_bam(basecall_fast5_gpu.out.sequencing_summary.collect(), sort_index_bam.out.indexed_bam)
+ }
+ else {
+ control_bam(basecall_fast5_cpu.out.sequencing_summary.collect(), sort_index_bam.out.indexed_bam)
+ }
+ }
+ //###################### START POSITIONS #######################
+
+ //Identification of start positions
+ start_position_counts(sort_index_bam.out.indexed_bam)
+
+ //Identification of RNA
+ start_position_individuals(start_position_counts.out.count)
+
+ //#################### VARIANTS D'EPISSAGE ####################
+
+ //Identification of splicing junction sites
+ jwr_checker(sort_index_bam.out.indexed_bam)
+
+ start_position_individuals.out.classification_of_reads
+ .combine(jwr_checker.out.nanosplicer_jwr, by: 0)
+ .set{files_for_nanosplicer}
+
+ //Identification of variants
+ junctions_nanosplicer(files_for_nanosplicer)
+
+ //#################### VARIANTS D'EPISSAGE ####################
+
+ junctions_nanosplicer.out.identified_SPvariants
+ .combine(start_position_individuals.out.classification_of_reads, by: 0)
+ .set{files_for_rna_count}
+
+ //Variants count
+ rna_count(files_for_rna_count)
+
+}
+
+// End message:
+
+workflow.onComplete {
+
+ println ( workflow.success ? """
+ DUPFinder tools execution summary
+ ---------------------------
+ Completed at : ${workflow.complete}
+ Duration : ${workflow.duration}
+ Success : ${workflow.success}
+ workDir : ${workflow.workDir}
+ exit status : ${workflow.exitStatus}
+ """ : """
+ Failed: ${workflow.errorReport}
+ exit status : ${workflow.exitStatus}
+ """
+ )
+}