nf_bisulfite_scPBAT_DirtyHarry

#!/usr/bin/env nextflow
nextflow.enable.dsl=2

// Last modified 24 02 2021
// This pipeline will first run paired-end alignments, with --unmapped specified, and then
// run single-end alignments afterwards

params.outdir = "."
params.genome = ""

params.pbat = '9' // default is 9, but this can be changed

params.verbose = false
params.single_end = false  // default mode is auto-detect. NOTE: params are handed over automatically

params.fastqc_args = ''
params.fastq_screen_args = ''
params.trim_galore_args = ''
params.bismark_args = ''
params.deduplicate_bismark_args = ''
params.bismark_methylation_extractor_args = ''
params.bismark2bedGraph_args = ''
params.bismark2summary_args = ''
params.bismark2report_args= ''
params.multiqc_args = ''


params.help = false
// Show help message and exit
if (params.help){
    helpMessage()
    exit 0
}

params.list_genomes = false;
if (params.list_genomes){
    println ("[WORKLFOW] List genomes selected")
}

if (params.verbose){
    println ("[WORKFLOW] FASTQC ARGS: "           + params.fastqc_args)
    println ("[WORKFLOW] FASTQ SCREEN ARGS ARE: " + params.fastq_screen_args)
    println ("[WORKFLOW] TRIM GALORE ARGS: "      + params.trim_galore_args)
    println ("[WORKFLOW] BISMARK ARGS ARE: "      + params.bismark_args)
    println ("[WORKFLOW] BISMARK DEDUPLICATION ARGS ARE: "         + params.deduplicate_bismark_args)
    println ("[WORKFLOW] BISMARK METHYLATION EXTRACTOR ARGS ARE: " + params.bismark_methylation_extractor_args)
}

include { makeFilesChannel; getFileBaseNames } from './nf_modules/files.mod.nf'
include { getGenome }                             from './nf_modules/genomes.mod.nf'
include { listGenomes }                           from './nf_modules/genomes.mod.nf'

if (params.list_genomes){
    listGenomes()  // this lists all available genomes, and exits
}

genome = getGenome(params.genome)

include { FASTQC }                               from './nf_modules/fastqc.mod.nf'                         
include { FASTQC as FASTQC2 }                    from './nf_modules/fastqc.mod.nf' 

include { FASTQ_SCREEN }                         from './nf_modules/fastq_screen.mod.nf' params(bisulfite: '--bisulfite')

// Adding pbat flag for PBAT processing. For Trim Galore we pass on the number of bp that need to be clipped at the 5' end
include { TRIM_GALORE }                          from './nf_modules/trim_galore.mod.nf' params(pbat: params.pbat)
// Adding single cell flag for non-directional processing
include { BISMARK }                              from './nf_modules/bismark.mod.nf' params(genome: genome, singlecell: true, unmapped: true)
include { BISMARK as BISMARK_UN1 }               from './nf_modules/bismark.mod.nf' params(genome: genome, singlecell: true, read_identity: "1")
include { BISMARK as BISMARK_UN2 }               from './nf_modules/bismark.mod.nf' params(genome: genome, singlecell: true, read_identity: "2")

include { BISMARK_DEDUPLICATION }                from './nf_modules/bismark_deduplication.mod.nf'
include { BISMARK_DEDUPLICATION as BISMARK_DEDUP1}    from './nf_modules/bismark_deduplication.mod.nf'
include { BISMARK_DEDUPLICATION as BISMARK_DEDUP2}    from './nf_modules/bismark_deduplication.mod.nf'

// Adding pbat flag for PBAT processing (same as sc flag)
include { BISMARK_METHYLATION_EXTRACTOR }        from './nf_modules/bismark_methylation_extractor.mod.nf' params(pbat: true)
include { BISMARK_METHYLATION_EXTRACTOR as METHXTRACT_UN1 }   from './nf_modules/bismark_methylation_extractor.mod.nf'
include { BISMARK_METHYLATION_EXTRACTOR as METHXTRACT_UN2 }   from './nf_modules/bismark_methylation_extractor.mod.nf'

include { BISMARK2BEDGRAPH }                     from './nf_modules/bismark2bedGraph.mod.nf' params(dirty_harry: true)

include { BISMARK2REPORT }                       from './nf_modules/bismark2report.mod.nf'
include { BISMARK2SUMMARY }                      from './nf_modules/bismark2summary.mod.nf'

include { MULTIQC }                              from './nf_modules/multiqc.mod.nf' 

file_ch = makeFilesChannel(args)

workflow {

    main:
        FASTQC                          (file_ch, params.outdir, params.fastqc_args, params.verbose)
        TRIM_GALORE                     (file_ch, params.outdir, params.trim_galore_args, params.verbose)
        FASTQ_SCREEN                    (TRIM_GALORE.out.reads, params.outdir, params.fastq_screen_args, params.verbose)
        FASTQC2                         (TRIM_GALORE.out.reads, params.outdir, params.fastqc_args, params.verbose)
        BISMARK                         (TRIM_GALORE.out.reads, params.outdir, params.bismark_args, params.verbose)
        BISMARK_DEDUPLICATION           (BISMARK.out.bam, params.outdir, params.deduplicate_bismark_args, params.verbose)

        BISMARK_UN1                     (BISMARK.out.unmapped1, params.outdir, params.bismark_args, params.verbose)
        BISMARK_DEDUP1                  (BISMARK_UN1.out.bam, params.outdir, params.deduplicate_bismark_args, params.verbose)
        
        BISMARK_UN2                     (BISMARK.out.unmapped2, params.outdir, params.bismark_args, params.verbose) 
        BISMARK_DEDUP2                  (BISMARK_UN2.out.bam, params.outdir, params.deduplicate_bismark_args, params.verbose)
               
        BISMARK_METHYLATION_EXTRACTOR   (BISMARK_DEDUPLICATION.out.bam, params.outdir, params.bismark_methylation_extractor_args, params.verbose)
        METHXTRACT_UN1                  (BISMARK_DEDUP1.out.bam, params.outdir, params.bismark_methylation_extractor_args, params.verbose)
        METHXTRACT_UN2                  (BISMARK_DEDUP2.out.bam, params.outdir, params.bismark_methylation_extractor_args, params.verbose)
        
        // BISMARK_METHYLATION_EXTRACTOR.out.context_files_CG.view()
        // METHXTRACT_UN1.out.context_files_CG.view()
        // METHXTRACT_UN2.out.context_files_CG.view()

        // To merge all relevant CpG calls, we will need a join statement instead of mix
        // left.join(right).view(); Joining occurs with the first element as the key
        ch_bedGraph_unmapped = METHXTRACT_UN1.out.context_files_CG
                         .join(METHXTRACT_UN2.out.context_files_CG, by: [0])
                         // .view()

        ch_bismark2bedGraph =  BISMARK_METHYLATION_EXTRACTOR.out.context_files_CG
                         .join(ch_bedGraph_unmapped, by: [0])
                         .map { it ->  [ it[0], [it[1], it[2], it[3]].flatten() ] }
                         //.view()
       
        BISMARK2BEDGRAPH                (ch_bismark2bedGraph, params.outdir, params.bismark2bedGraph_args, params.verbose)
      
        // merging channels for Bismark reports
        bismark_report_ch = BISMARK.out.bam.mix(
            BISMARK.out.report,
            BISMARK_UN1.out.bam.ifEmpty([]),
            BISMARK_UN1.out.report.ifEmpty([]),
            BISMARK_UN2.out.bam.ifEmpty([]),
            BISMARK_UN2.out.report.ifEmpty([]),
            BISMARK_DEDUPLICATION.out.report.ifEmpty([]),
            BISMARK_DEDUP1.out.report.ifEmpty([]),
            BISMARK_DEDUP2.out.report.ifEmpty([]),
            BISMARK_METHYLATION_EXTRACTOR.out.report.ifEmpty([]),
            BISMARK_METHYLATION_EXTRACTOR.out.mbias.ifEmpty([]),
            METHXTRACT_UN1.out.report.ifEmpty([]),
            METHXTRACT_UN1.out.mbias.ifEmpty([]),
            METHXTRACT_UN2.out.report.ifEmpty([]),
            METHXTRACT_UN2.out.mbias.ifEmpty([]),
        ).collect()

        BISMARK2REPORT                   (bismark_report_ch, params.outdir, params.bismark2report_args, params.verbose)

        // merging channels for Bismark summary report
        // PAIRED-END only as it fails with a mixture of single-end and paired-end files
        bismark_summary_report_ch = BISMARK.out.bam.mix(
            BISMARK.out.report,
            BISMARK_DEDUPLICATION.out.report.ifEmpty([]),
            BISMARK_METHYLATION_EXTRACTOR.out.report.ifEmpty([]),
            BISMARK_METHYLATION_EXTRACTOR.out.mbias.ifEmpty([]),
        ).collect()
        // bismark_report_ch.subscribe {  println "Got: $it"  }

        BISMARK2SUMMARY                  (bismark_summary_report_ch, params.outdir, params.bismark2summary_args, params.verbose)
        
        // merging channels for MultiQC
        multiqc_ch = FASTQC.out.report.mix(
            TRIM_GALORE.out.report,
            FASTQ_SCREEN.out.report.ifEmpty([]),
            FASTQC2.out.report.ifEmpty([]),
            BISMARK.out.report.ifEmpty([]),
            BISMARK_UN1.out.report.ifEmpty([]),
            BISMARK_UN2.out.report.ifEmpty([]),
            BISMARK_DEDUPLICATION.out.report.ifEmpty([]),
            BISMARK_DEDUP1.out.report.ifEmpty([]),
            BISMARK_DEDUP2.out.report.ifEmpty([]),
            BISMARK_METHYLATION_EXTRACTOR.out.report.ifEmpty([]),
            BISMARK_METHYLATION_EXTRACTOR.out.mbias.ifEmpty([]),
            METHXTRACT_UN1.out.report.ifEmpty([]),
            METHXTRACT_UN1.out.mbias.ifEmpty([]),
            METHXTRACT_UN2.out.report.ifEmpty([]),
            METHXTRACT_UN2.out.mbias.ifEmpty([]),
        ).collect()

        MULTIQC                          (multiqc_ch, params.outdir, params.multiqc_args, params.verbose)
}

// Since workflows with very long command lines tend to fail to get rendered at all, I was experimenting with a
// minimal execution summary report so we at least know what the working directory was...
workflow.onComplete {

    def msg = """\
        Pipeline execution summary
        ---------------------------
        Jobname     : ${workflow.runName}
        Completed at: ${workflow.complete}
        Duration    : ${workflow.duration}
        Success     : ${workflow.success}
        workDir     : ${workflow.workDir}
        exit status : ${workflow.exitStatus}
        """
    .stripIndent()

    sendMail(to: "${workflow.userName}@babraham.ac.uk", subject: 'Minimal pipeline execution report', body: msg)
}

def helpMessage() {
 
    log.info"""
    >>


    SYNOPSIS:
    PBAT - Dirty Harry: an abomination for paired-end data...

    This workflow runs an entire Bisulfite-seq processing pipeline for PBAT (post-bisulfite adaptor tagging) samples
    on FastQ files, first for paired-end end file, and then in single-end mode for unmapped files. This includes QC,
    quality-/adapter trimming, contamination QC (post-trimming), alignments to a genome using Bismark (see below
    deduplication, methylation extraction, coverage file generation, and finally generates aa aggregate MultiQC report.
    The workflow is suitable for paired-end PBAT-type bisulfite sequencing experiments, by default assuming 9N oligos
    for the pulldown reaction. The term ‘Dirty Harry’ was coined in ~2014 because it doesn’t seem to be the cleanest of methods…:

    (1) PE alignment (--pbat) to start, while grabbing the unmapped R1 and R2 reads out the end using the option ‘--unmapped’. 
    Properly aligned PE reads should be methylation extracted using --no_overlap.
    (2) unmapped R1 is then mapped SE (--pbat)
    (3) unmapped R2 is then mapped SE (default mode, i.e. directional)
    
    Single-end aligned R1 and R2 can then be methylation extracted normally as they should in theory map to different places
    in the genome anyway so don’t require attention to overlapping reads. Note that paired-end and single-end processing is
    PE/SE specific until the methylation extraction stage. [CpG]* methylation calls of PE as well as both SE aligments are then
    fed to a single bismark2bedGraph process to generate a single, merged coverage file (ends in "_DH.cov.gz").

    Here is a graphical representation of the workflow:

    --- FastQC
    --- Trim Galore
        |
        --- FastQ Screen
        --- FastQC
        --- Bismark (PE)
            |
            --- Bismark deduplication (PE)
                |
                --- Bismark methylation extraction (PE) 
            ---unmapped R1
               |
               ---Bismark deduplication (SE R1)
                  |
                  --- Bismark methylation extraction (SE R1) 
            ---unmapped R2
               |
               --- Bismark deduplication (SE R2)
                 |
                 --- Bismark methylation extraction (SE R2) 
        ---bismark2bedGraph (PE, SE R1, SE R2)
    --- bismark2report*
    --- bismark2summary*
    --- MultiQC*
        
    * These steps run only once ALL other jobs have completed.
     
    By default all these steps are submitted as jobs to the Babraham stone compute cluster.

    By default, the involved tools are run in the following way:
    ------------------------------------------------------------
    FastQC:                           defaults ('-q')
    FastQ Screen:                     '--bisulfite'
    Trim Galore:                      adapter auto-detection; '--paired --clip_r1 9 --clip_r2 9'
    Bismark:                          PE: '--unmapped --pbat', SE R1: '--pbat', SE R2: defaults
    Bismark deduplication:            defaults
    Bismark methylation extraction:   '--bedGraph --buffer 10G --parallel 4'
    bismark2bedGraph                  'dirty_harry'

    To add additional parameters to any of the programs, consider supplying tool-specific arguments (see --toolname_args="..." below).


            ==============================================================================================================


    USAGE:
    
    nf_bisulfite_PBAT_DirtyHarry [options] --genome <genomeID> <input files>
    
    Mandatory arguments:
    ====================

      <input files>                   List of input files, e.g. '*fastq.gz' or '*fq.gz'. Files are automatically processed as
                                      single-end (SE) or paired end (PE) files (if file pairs share the same base-name, and differ only
                                      by a read number, e.g. 'base_name_R1.fastq.gz' and 'base_name_R2.fastq.gz' (or R3, R4). For
                                      PE files, only Read 1 is run through FastQ Screen (as typically R1 and R2 produce nearly identical
                                      contamination profiles). To run PE files in single-end mode, please see '--single_end' below.

      --genome [str]                  Genome build ID to be used for the alignment, e.g. GRCh38 (latest human genome) or GRCm38
                                      (latest mouse genome build). To list all available genomes, see '--list_genomes' below.


    Tool-specific options:
    ======================

    For all following options, please note that the format: ="your options" needs to be strictly adhered to in order to work correctly.

      --fastqc_args="[str]"           This option can take any number of options that are compatible with FastQC to modify its default
                                      behaviour. For more detailed information on available options please refer to the FastQC documentation,
                                      or run 'fastqc --help' on the command line. As an example, to run FastQC without grouping of bases when
                                      reads are >50bp and use a specific file with non-default adapter sequences, use: 
                                      ' --fastqc_args="--nogroup --adapters ./non_default_adapter_file.txt" '. [Default: None]

      --fastq_screen_args="[str]"     This option can take any number of options that are compatible with FastQ Screen to modify its
                                      default behaviour. For more detailed information on available options please refer to the FastQ Screen
                                      documentation, or run 'fastq_screen --help' on the command line. For instance, to process a bisulfite
                                      converted library with fairly relaxed parameters, you could use: 
                                      ' --fastq_screen_args="--bisulfite --score_min L,0,-0.6" '. [Default: None]

      --trim_galore_args="[str]"      This option can take any number of options that are compatible with Trim Galore to modify its
                                      default trimming behaviour. For more detailed information on available options please refer
                                      to the Trim Galore User Guide, or run 'trim_galore --help' on the command line. As an example, to trim
                                      off the first 10bp from the 5' of R1 and 5bp of R2, use: 
                                      ' --trim_galore_args="--clip_r1 10 --clip_r2 5" '. [Default: None]

      --bismark_args="[str]"          This option can take any number of options that are compatible with Bismark to modify its
                                      default mapping behaviour. For more detailed information on available options please refer
                                      to the Bismark User Guide, or run 'bismark --help' on the comamnd line. As an example, to run somewhat
                                      relaxed alignments, use: ' --bismark_args="--score_min L,0,-0.4" '. [Default: None]

      --bismark_deduplication_args="[str]"           This option can take any number of options that are compatible with deduplicate_bismark
                                      to modify its default deduplication behaviour. For more detailed information on available options please
                                      refer to the Bismark User Guide, or run 'deduplicate_bismark --help'. As an example, to take UMI sequences
                                      into account, use: ' --bismark_deduplication_args="--barcode" '. [Default: None]

      --bismark_methylation_extractor_args="[str]"   This option can take any number of options that are compatible with bismark_methylation_extractor
                                      to modify its default exctraction behaviour. For more detailed information on available options please refer
                                      to the Bismark User Guide, or run 'bismark_methylation_extractor --help' on the command line. As an example,
                                      to run the extraction and coverage file generation for a genome containing thousands of scaffolds
                                      or contigs, use: ' --bismark_methylation_extractor_args="--gazillion" '. [Default: None]

      --bismark2bedGraph_args="[str]" This option can take any number of options that are compatible with bismark2bedGraph to modify its
                                      default behaviour. For more detailed information on available options please refer to the Bismark
                                      User Guide, or run 'bismark2bedGraph --help' on the command line. As an example, to run the coverage file
                                      generation for a genome containing thousands of scaffolds or contigs, use: 
                                      ' --bismark2bedGraph_args="--gazillion" '. [Default: None]                           


    Other options:
    ==============

      --outdir [str]                  Path to the output directory. [Default: current working directory]

      --pbat [int]                    Number of nucleotides used for the PBAT pulldown reaction. This number of bases will be removed
                                      from the 5'-end of both R1 and R2 prior to mapping. Default: 9.

      --list_genomes                  List all genome builds that are currently available to choose from. To see this list
                                      of available genomes with more detailed information about paths and indexes, run
                                      the command as '--list_genomes --verbose'
    
      --single_end                    Force files of a read pair to be treated as single-end files. [Default: auto-detect]
      
      --verbose                       More verbose status messages. [Default: OFF]
      --help                          Displays this help message and exits.

    Workflow options:
    =================

    Please note the single '-' hyphen for the following options!

      -resume                         If a pipeline workflow has been interrupted or stopped (e.g. by accidentally closing a laptop),
                                      this option will attempt to resume the workflow at the point it got interrupted by using
                                      Nextflow's caching mechanism. This may save a lot of time.

      -bg                             Sends the entire workflow into the background, thus disconnecting it from the terminal session.
                                      This option launches a daemon process (which will keep running on the headnode) that watches over
                                      your workflow, and submits new jobs to the SLURM queue as required. Use this option for big pipeline
                                      jobs, or whenever you do not want to watch the status progress yourself. Upon completion, the
                                      pipeline will send you an email with the job details. This option is HIGHLY RECOMMENDED!

      -process.executor=local         Temporarily changes where the workflow is executed to the 'local' machine. See also Nextflow config
                                      file for more details. [Default: slurm] 
    

    <<
    """.stripIndent()

}