Snakefile_fastq_singlelane

include:
    'configs/config_HuR.mouse.py'

workdir: OUT_DIR

from itertools import chain, combinations
from os.path import join
import glob
import re

SAMPLES = glob.glob('{}**/*.fastq.gz'.format(RAWDATA_DIR), recursive=True)
SAMPLES = [sample.replace('{}/'.format(RAWDATA_DIR),'').replace('.fastq.gz','') for sample in SAMPLES]
SAMPLES = [('_').join(sample.split('_')[:-2])  for sample in SAMPLES]

PLOT_PREFIXES = combinations(SAMPLES, 2)
PLOT_PREFIXES_1 = [x[0] for x in PLOT_PREFIXES]
PLOT_PREFIXES_2 = [x[1] for x in PLOT_PREFIXES]

rule all:
    input:
        STAR_INDEX,
        expand('qc/{sample}_R1_001_fastqc.html', sample=SAMPLES),
        expand('qc/{sample}_R2_001_fastqc.html', sample=SAMPLES),
        expand('mapped/counts_strict/{sample}.counts.tsv', sample=SAMPLES),
        expand('mapped/plots/tpm_scatter/{sample1}_VS_{sample2}.png', sample1=PLOT_PREFIXES_1, sample2=PLOT_PREFIXES_2),
        #expand('mapped/integenic_reads/{sample}.intergenic.bam', sample=SAMPLES),
        #expand('mapped/fpkm/{sample}.FPKM.xls', sample=SAMPLES),
        'mapped/tpm/HTSeq/masterTPM.tsv',
        'mapped/tpm/featureCounts/masterTPM.tsv',
        'mapped/DE_analysis/HTSeq/'+GENOME_BUILD+'.HTSeq.DESeq2.all.tsv',
        'mapped/DE_analysis/featureCounts/'+GENOME_BUILD+'.featureCounts.DESeq2.sig.tsv',
        'multiqc_report/multiqc_report.html',


rule create_index:
    input:
        GENOME_FASTA,
        GTF
    output: STAR_INDEX
    threads: 16
    shell:
        r'''mkdir -p {output} && STAR --runThreadN 16\
            --runMode genomeGenerate \
            --genomeDir {output} \
            --genomeFastaFiles {input[0]}\
            --sjdbGTFfile {input[1]}'''


rule perform_qc:
    input:
        R1=RAWDATA_DIR+'/{sample}_R1_001.fastq.gz',
        R2=RAWDATA_DIR+'/{sample}_R2_001.fastq.gz'
    params:
        out_dir = 'qc'
    output:
       'qc/{sample}_R1_001_fastqc.html',
       'qc/{sample}_R1_001_fastqc.zip',
       'qc/{sample}_R2_001_fastqc.html',
       'qc/{sample}_R2_001_fastqc.zip',
    shell:
        r'''
            fastqc -o {params.out_dir} -f fastq {input.R1} {input.R2}
        '''

rule perfom_trimming:
    input:
        R1=RAWDATA_DIR+'/{sample}_R1_001.fastq.gz',
        R2=RAWDATA_DIR+'/{sample}_R2_001.fastq.gz'
    params:
        out_dir='preprocessed/{sample}',
        phred_cutoff=5
    output:
        'preprocessed/{sample}/{sample}_R1_001_val_1.fq.gz',
        'preprocessed/{sample}/{sample}_R2_001_val_2.fq.gz',
    shell:
        r'''
            trim_galore --paired -o {params.out_dir} -q {params.phred_cutoff} {input.R1} {input.R2}
        '''

"""
rule map_star:
    input:
        R1='preprocessed/{sample}/{sample}_R1_001_val_1.fq.gz',
        R2='preprocessed/{sample}/{sample}_R2_001_val_2.fq.gz',
        index=STAR_INDEX
    output: 'mapped/bams/{sample}.bam'
    params:
        prefix = 'mapped/bams/{sample}',
        unmapped = 'unmapped/fastq/{sample}',
        starlogs = 'mapped/starlogs'
    threads: 16
    shell:
        r'''
        STAR --runThreadN {threads}\
             --genomeDir {input.index}\
             --outFileNamePrefix {params.prefix} --readFilesIn {input.R1} {input.R2}\
             --outSAMtype BAM SortedByCoordinate\
             --outFilterMatchNmin 50\
             --outFilterMismatchNmax 100\
             --readFilesCommand zcat\
             --outReadsUnmapped {params.unmapped} && mv {params.prefix}Aligned.sortedByCoord.out.bam {output} && mkdir -p {params.starlogs} && mv {params.prefix}Log.final.out {params.prefix}Log.out {params.prefix}Log.progress.out {params.starlogs}
        '''
"""

rule map_starlong:
    input:
        R1='preprocessed/{sample}/{sample}_R1_001_val_1.fq.gz',
        R2='preprocessed/{sample}/{sample}_R2_001_val_2.fq.gz',
        index=STAR_INDEX
    output: 'mapped/bams/{sample}.bam'
    params:
        prefix = 'mapped/bams/{sample}',
        unmapped = 'unmapped/fastq/{sample}',
        starlogs = 'mapped/starlogs'
    threads: 16
    shell:
        r'''
        STARlong --runThreadN {threads}\
             --genomeDir {input.index}\
             --outFileNamePrefix {params.prefix} --readFilesIn {input.R1} {input.R2}\
             --outSAMtype BAM SortedByCoordinate\
             --readFilesCommand zcat\
             --outReadsUnmapped {params.unmapped}\
            --outFilterMultimapScoreRange 20\
            --outFilterScoreMinOverLread 0\
            --outFilterMatchNminOverLread 0.66\
            --outFilterMismatchNmax 100\
            --winAnchorMultimapNmax 200\
            --seedPerReadNmax 100000 --seedPerWindowNmax 100\
            && mv {params.prefix}Aligned.sortedByCoord.out.bam {output} &&\
            mkdir -p {params.starlogs} && mv {params.prefix}Log.final.out\
            {params.prefix}Log.out {params.prefix}Log.progress.out {params.starlogs}
        '''
rule sort_by_name:
    input: 'mapped/bams/{sample}.bam'
    output: 'mapped/bams/{sample}.sortedByName.bam'
    shell:
        r'''
            samtools sort -on {input} -T /tmp/ -o {output}
        '''
rule count:
    input: 'mapped/bams/{sample}.sortedByName.bam'
    params:
        annotation=GTF,
        phred_cutoff=5
    output: 'mapped/counts_strict/{sample}.counts.tsv'
    shell:
        r'''
        source activate clipseq2 && htseq-count --order=name --format=bam --mode=intersection-strict --stranded=no --minaqual={params.phred_cutoff} --type=exon --idattr=gene_id {input} {params.annotation} > {output}
        '''
rule format_counts:
    input: 'mapped/counts_strict/{sample}.counts.tsv'
    output: 'mapped/counts_strict/{sample}.counts.noversion.tsv'
    shell:
        r'''
        cat {input} | sed -E 's/\.[0-9]+//' > {output}

        '''

rule featurecounts:
    input: expand('mapped/bams/{sample}.sortedByName.bam', sample=set(SAMPLES))
    params:
        annotation=GTF
    output: 'mapped/featureCounts/fcounts.tsv'
    threads: 16
    shell:
        r'''featureCounts -a {params.annotation} -o {output} -t exon -g gene_id -Q 4 -T {threads} {input}'''

rule format_fcounts:
    input: 'mapped/featureCounts/fcounts.tsv'
    output: 'mapped/featureCounts/fcounts.noversion.tsv'
    shell:
        r'''
        cat {input} | sed -E 's/\.[0-9]+//' > {output}

        '''
    output: 'mapped/featureCounts/fcounts.noversion.tsv'

rule run_deseq:
    input: expand('mapped/counts_strict/{sample}.counts.noversion.tsv', sample=SAMPLES)
    output:
        'mapped/DE_analysis/HTSeq/'+GENOME_BUILD+'.HTSeq.DESeq2.all.tsv',
        'mapped/DE_analysis/HTSeq/'+GENOME_BUILD+'.HTSeq.DESeq2.sig.tsv'

    params:
        basedir = 'mapped/counts_strict',
        inprefix = 'counts.noversion',
        gene_annotations  = GENE_NAMES,
        outprefix = 'mapped/DE_analysis/HTSeq/'+GENOME_BUILD+'.HTSeq'
    shell:
        r'''
        Rscript {SRC_DIR}/do_DE_analysis.R --basedir={params.basedir} \
            --gene_annotations={params.gene_annotations} \
            --design_file={DESIGN_FILE} \
            --outprefix={params.outprefix} \
            --inprefix={params.inprefix}

        '''

rule run_deseq_featureCounts:
    input: 'mapped/featureCounts/fcounts.noversion.tsv'
    output:
        'mapped/DE_analysis/featureCounts/'+GENOME_BUILD+'.featureCounts.DESeq2.all.tsv',
        'mapped/DE_analysis/featureCounts/'+GENOME_BUILD+'.featureCounts.DESeq2.sig.tsv'
    params:
        gene_annotations  = GENE_NAMES,
        outprefix = 'mapped/DE_analysis/featureCounts/'+GENOME_BUILD+'.featureCounts'
    shell:
        r'''
        Rscript {SRC_DIR}/do_DE_analysis_featureCounts.R --counts={input} \
            --gene_annotations={params.gene_annotations} \
            --design_file={DESIGN_FILE} \
            --outprefix={params.outprefix}

        '''

rule run_picardmetrics:
    input: 'mapped/bams/{sample}.bam'
    output: 'mapped/bam_metrics/{sample}.metrics'
    shell:
        r'''
        picard CollectInsertSizeMetrics I={input} H={output}.insertsize.pdf O={output}

        '''

rule create_insertsize_tsv:
    input: 'mapped/bam_metrics/{sample}.metrics'
    output: 'mapped/bam_metrics/{sample}.insertsizes.tsv'
    shell:
        r'''
        python {SRC_DIR}/collect_picard_metrics.py {input} {output}

        '''

rule counts_to_tpm:
    input:
        count = expand('mapped/counts_strict/{sample}.counts.noversion.tsv', sample=SAMPLES),
        insert_size = expand('mapped/bam_metrics/{sample}.insertsizes.tsv', sample=SAMPLES),
    output: 'mapped/tpm/HTSeq/masterTPM.tsv'
    params:
        gene_lengths=GENE_LENGTHS,
        name=expand('{sample}', sample=set(SAMPLES)),
        outprefix='mapped/tpm/HTSeq',
        gene_map=GENE_NAMES
    run:
        counts_input = (',').join(input.count)
        sizes_input = (',').join(input.insert_size)
        names = (',').join(params.name)
        shell('Rscript {SRC_DIR}/counts_to_tpm.R --counts={counts_input} --insert_sizes={sizes_input} --gene_lengths={params.gene_lengths} --inprefix={names} --gene_map={params.gene_map} --outprefix={params.outprefix}')

rule featurecounts_to_tpm:
    input:
        insert_size = expand('mapped/bam_metrics/{sample}.insertsizes.tsv', sample=set(SAMPLES)),
        fcounts = 'mapped/featureCounts/fcounts.noversion.tsv'
    output: 'mapped/tpm/featureCounts/masterTPM.tsv'
    params:
        gene_lengths=GENE_LENGTHS,
        name=expand('{sample}', sample=set(SAMPLES)),
        outprefix='mapped/tpm/featureCounts',
        gene_map=GENE_NAMES
    run:
        sizes_input = (',').join(input.insert_size)
        names = (',').join(params.name)
        shell('Rscript {SRC_DIR}/featurecounts_to_tpm.R --counts={input.fcounts} --insert_sizes={sizes_input} --gene_lengths={params.gene_lengths} --inprefix={names} --gene_map={params.gene_map} --outprefix={params.outprefix}')

rule plot_tpm:
    input: 'mapped/tpm/HTSeq/masterTPM.tsv'
    output: 'mapped/plots/tpm_scatter/{sample1}_VS_{sample2}.png'
    params:
        prefix = 'mapped/plots/tpm_scatter/'
    shell:
        r'''
        python {SRC_DIR}/plot_tpm_scatter.py --master {input} --outprefix  {params.prefix}
        '''

rule perform_qualimap_qc:
    input:  'mapped/bams/{sample}.bam',
    output: 'mapped/post_mapping_qualimap/{sample}/qualimapReport.html',
    params:
        outdir='mapped/post_mapping_qualimap/{sample}',
        gtf=GTF
    shell:
        r'''
        qualimap rnaseq -bam {input} -gtf {params.gtf} --outdir {params.outdir} --java-mem-size=8G

        '''

rule get_duplication_estimate:
    input:  'mapped/bams/{sample}.bam'
    output: 'mapped/post_mapping_deduplication/{sample}/output.DupRate_plot.r'
    params:
        outprefix='mapped/post_mapping_deduplication/{sample}/output'
    shell:
        r'''
        source activate clipseq2 && read_duplication.py -i {input} -o {params.outprefix}
        '''

rule run_multiqc:
    input:
        expand('qc/{sample_name}_R1_001_fastqc.html', sample_name=SAMPLES),
        expand('qc/{sample_name}_R2_001_fastqc.html', sample_name=SAMPLES),
        expand('mapped/post_mapping_deduplication/{sample}/output.DupRate_plot.r', sample=SAMPLES),
        expand('mapped/bam_metrics/{sample}.metrics', sample=SAMPLES),
        expand('mapped/post_mapping_qualimap/{sample}/qualimapReport.html', sample=SAMPLES),
        expand('mapped/bams/{sample}.bam', sample=SAMPLES),
        'mapped/DE_analysis/HTSeq/'+GENOME_BUILD+'.HTSeq.DESeq2.sig.tsv',
        'mapped/DE_analysis/featureCounts/'+GENOME_BUILD+'.featureCounts.DESeq2.sig.tsv',
    output:
        'multiqc_report/multiqc_report.html'
    shell:
        'export LC_ALL=en_US.UTF-8 && multiqc -f --outdir multiqc_report .'


rule calc_fpkm:
    input: 'mapped/bams/{sample}.bam'
    output: 'mapped/fpkm/{sample}.FPKM.xls'
    params:
        prefix='mapped/fpkm/{sample}'
    shell:
        r'''
        source activate clipseq2 && FPKM_count.py -i {input} -o {params.prefix} -r {GENE_BED}
        '''