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RNA Seq pipeline
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| --- | |
| use: | |
| - Slurp | |
| - Data::Dumper | |
| global: | |
| - indir: data/processed/rename | |
| - outdir: data/processed | |
| - PROJECT: /scratch/jdr400/projects/RNASeq | |
| - PROCESSED: data/processed | |
| - ROOT: data/processed | |
| - file_rule: (Sample.*)$ | |
| - by_sample_outdir: 1 | |
| - find_by_dir: 1 | |
| - trimmomatic_dir: "data/processed/{$sample}/trimmomatic" | |
| - analysis_dir: "data/processed/analysis" | |
| - data_dir: "/scratch/Reference_Genomes/Public/Invertebrate/Drosophila_melanogaster/ENSEMBL-release-81-BDGP6" | |
| - ANNOTATION: "{$self->data_dir}/Drosophila_melanogaster.BDGP6.81.gtf" | |
| - REFERENCE: "{$self->data_dir}/Drosophila_melanogaster.BDGP6.dna.toplevel.fa" | |
| - htseq_dir: "data/processed/analysis/Counts" | |
| - htseq_regexp: Sample_(\w+)_(\w+) | |
| rules: | |
| - raw_fastqc: | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 gcc/4.9.1 jdk/1.8.0_31 zlib/1.2.8 openssl/1.0.1i anyenv/1 | |
| #HPC mem=24GB | |
| #HPC walltime=24:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC queue=adserial | |
| #HPC commands_per_node=3 | |
| #HPC jobname=raw_fastqc | |
| process: | | |
| #NOTE job_tags={$sample} | |
| mkdir -p {$self->{outdir}}/{$sample}_R1_FASTQC && \ | |
| cat {$self->indir}/*_R1_*gz > {$self->outdir}/{$sample}_read1.fastq.gz && \ | |
| /scratch/jdr400/software/FastQC/fastqc {$self->outdir}/{$sample}_read1.fastq.gz --extract -o {$self->{outdir}}/{$sample}_R1_FASTQC/ -t 4 | |
| #NOTE job_tags={$sample} | |
| mkdir -p {$self->{outdir}}/{$sample}_R2_FASTQC && \ | |
| cat {$self->indir}/*_R2_*gz > {$self->outdir}/{$sample}_read2.fastq.gz && \ | |
| /scratch/jdr400/software/FastQC/fastqc {$self->outdir}/{$sample}_read2.fastq.gz --extract -o {$self->{outdir}}/{$sample}_R2_FASTQC/ -t 4 | |
| - trimmomatic: | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 gcc/4.9.1 jdk/1.8.0_31 zlib/1.2.8 openssl/1.0.1i anyenv/1 trimmomatic | |
| #HPC mem=48GB | |
| #HPC walltime=24:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC queue=adserial | |
| #HPC commands_per_node=1 | |
| #HPC jobname=trimmomatic | |
| # | |
| process: | | |
| #NOTE job_tags={$sample} | |
| java -jar /share/apps/NYUAD/trimmomatic/0.32/trimmomatic-0.32.jar PE -threads 12 -phred33 -trimlog {$self->outdir}/{$sample}_trimmomatic.log \ | |
| {$self->indir}/{$sample}_read1.fastq.gz {$self->indir}/{$sample}_read2.fastq.gz \ | |
| {$self->outdir}/{$sample}_read1_trimmomatic_1PE {$self->outdir}/{$sample}_read1_trimmomatic_1SE \ | |
| {$self->outdir}/{$sample}_read2_trimmomatic_2PE {$self->outdir}/{$sample}_read2_trimmomatic_2SE \ | |
| ILLUMINACLIP:/scratch/nd48/Tools/bin/trimmomatic_adapter.fa:2:30:10 TRAILING:3 LEADING:3 SLIDINGWINDOW:4:15 MINLEN:36 | |
| - trimmomatic_fastqc: | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 gcc/4.9.1 jdk/1.8.0_31 zlib/1.2.8 openssl/1.0.1i anyenv/1 trimmomatic | |
| #HPC mem=48GB | |
| #HPC walltime=48:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC queue=adserial | |
| #HPC commands_per_node=3 | |
| #HPC jobname=trimmomatic_fastqc | |
| # | |
| process: | | |
| #NOTE job_tags={$sample} | |
| mkdir -p {$self->outdir}/{$sample}_FASTQC_read1_TRIMMED && \ | |
| /scratch/jdr400/software/FastQC/fastqc {$self->indir}/{$sample}_read1_trimmomatic_1PE --extract -o {$self->outdir}/{$sample}_FASTQC_read1_TRIMMED/ -t 4 | |
| #NOTE job_tags={$sample} | |
| mkdir -p {$self->outdir}/{$sample}_FASTQC_read2_TRIMMED && \ | |
| /scratch/jdr400/software/FastQC/fastqc {$self->indir}/{$sample}_read2_trimmomatic_2PE --extract -o {$self->outdir}/{$sample}_FASTQC_read2_TRIMMED/ -t 4 | |
| - trimmomatic_gzip: | |
| local: | |
| - indir: "data/processed/{$sample}" | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 gcc/4.9.1 jdk/1.8.0_31 zlib/1.2.8 openssl/1.0.1i anyenv/1 trimmomatic | |
| #HPC mem=24GB | |
| #HPC walltime=5:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC queue=adserial | |
| #HPC commands_per_node=4 | |
| #HPC jobname=trimmomatic_gzip | |
| # | |
| process: | | |
| #NOTE job_tags={$sample} | |
| gzip {$self->indir}/trimmomatic/{$sample}_read1_trimmomatic_1PE | |
| #NOTE job_tags={$sample} | |
| gzip {$self->indir}/trimmomatic/{$sample}_read2_trimmomatic_2PE | |
| - analysis: | |
| override_process: 1 | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 apps tuxedo/2.0 | |
| #HPC mem=24GB | |
| #HPC walltime=00:10:00 | |
| #HPC queue=adserial | |
| #HPC cpus_per_task=12 | |
| #HPC jobname=analysis_dirs | |
| # | |
| process: | | |
| mkdir -p {$self->{analysis_dir}}/FPKMs && \ | |
| mkdir -p {$self->{analysis_dir}}/BAMs && \ | |
| mkdir -p {$self->{analysis_dir}}/Transcripts && \ | |
| mkdir -p {$self->{analysis_dir}}/CUFFDIFF && \ | |
| mkdir -p {$self->{analysis_dir}}/Counts && \ | |
| mkdir -p {$self->{analysis_dir}}/Counts/DESeq3 | |
| - bowtie: | |
| override_process: 1 | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 apps tuxedo/2.0 | |
| #HPC mem=24GB | |
| #HPC walltime=01:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC queue=adserial | |
| #HPC jobname=bowtie2 | |
| # | |
| process: | | |
| bowtie2-inspect {$self->REFERENCE} > {$self->outdir}/Drosophila_melanogaster.BDGP6.dna.toplevel.fa.fa && \ | |
| cp -rf {$self->data_dir}/Dros* {$self->outdir} | |
| - tophat2: | |
| override_process: 0 | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 apps tuxedo/2.0 | |
| #HPC mem=24GB | |
| #HPC walltime=48:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC queue=adserial | |
| #HPC procs=1 | |
| #HPC jobname=tophat2 | |
| #HPC commands_per_node=2 | |
| # | |
| - trimmomatic_dir: "data/processed/{$sample}/trimmomatic" | |
| - bowtie_dir: "data/processed/{$sample}/bowtie" | |
| - REFERENCE: "data/processed/bowtie/Drosophila_melanogaster.BDGP6.dna.toplevel.fa" | |
| - ANNOTATION: "data/processed/bowtie/Drosophila_melanogaster.BDGP6.81.gtf" | |
| process: | | |
| #NOTE job_tags={$sample} | |
| tophat2 --no-novel-juncs -G {$self->ANNOTATION} -p 12 -o {$self->outdir}/ \ | |
| {$self->{REFERENCE}} {$self->trimmomatic_dir}/{$sample}_read1_trimmomatic_1PE.gz \ | |
| {$self->trimmomatic_dir}/{$sample}_read2_trimmomatic_2PE.gz | |
| - cufflinks: | |
| override_process: 0 | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 apps tuxedo/2.0 | |
| #HPC mem=24GB | |
| #HPC walltime=48:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC procs=1 | |
| #HPC commands_per_node=2 | |
| #HPC jobname=cufflinks | |
| # | |
| process: | | |
| #NOTE job_tags={$sample} | |
| cufflinks -p 12 -o {$self->outdir} -G {$self->ANNOTATION} {$self->indir}/accepted_hits.bam && \ | |
| cp {$self->indir}/accepted_hits.bam {$self->analysis_dir}/BAMs/{$sample}_accepted_hits.bam && \ | |
| cp {$self->outdir}/gene* {$self->analysis_dir}/FPKMs/{$sample}_gene_fpkms.txt && \ | |
| cp {$self->outdir}/transcrip* {$self->analysis_dir}/Transcripts/{$sample}_transcripts.gtf | |
| - samtools_sort: | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 gcc/4.9.1 zlib/1.2.8 ncurses/5.9 samtools/1.2 openssl/1.0.1i anyenv/1 | |
| #HPC mem=24GB | |
| #HPC walltime=24:00:00 | |
| #HPC queue=adserial | |
| #HPC cpus_per_task=12 | |
| #HPC commands_per_node=2 | |
| #HPC procs=1 | |
| #HPC jobname=samtools_sort | |
| # | |
| process: | | |
| #NOTE job_tags={$sample} | |
| samtools sort -n -@ 12 -O bam -T {$self->analysis_dir}/BAMs/sorted.{$sample}.bam -o {$self->analysis_dir}/BAMs/sorted_bam_{$sample}.bam {$self->analysis_dir}/BAMs/{$sample}_accepted_hits.bam | |
| - htseq_count: | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 gcc/4.9.1 zlib/1.2.8 ncurses/5.9 samtools/1.2 openssl/1.0.1i anyenv/1 | |
| #HPC mem=24GB | |
| #HPC walltime=24:00:00 | |
| #HPC queue=adserial | |
| #HPC cpus_per_task=12 | |
| #HPC commands_per_node=2 | |
| #HPC jobname=htseq_count | |
| # | |
| process: | | |
| #NOTE job_tags={$sample} | |
| name=`echo {$sample}| sed s/Sample_//` && \ | |
| htseq-count -f bam -s no -t exon -i gene_id {$self->analysis_dir}/BAMs/sorted_bam_{$sample}.bam {$self->{ANNOTATION}} > {$self->analysis_dir}/Counts/htseq_$name.txt | |
| - deseq2: | |
| override_process: 1 | |
| local: | |
| - before_meta: | | |
| HPC THINGS | |
| # | |
| #HPC module=NYUAD/2.0 gcc/4.9.1 zlib/1.2.8 openssl/1.0.1i anyenv/1 | |
| #HPC mem=24GB | |
| #HPC walltime=24:00:00 | |
| #HPC cpus_per_task=12 | |
| #HPC queue=adserial | |
| #HPC jobname=deseq2 | |
| # | |
| process: | | |
| { | |
| my $data = {self => \$self}; | |
| my $project_dir = {$self->PROJECT}; | |
| open(my $fh, ">$self->{PROJECT}/script/make_deseq2.R"); | |
| Text::Template::fill_in_file("$self->{PROJECT}/conf/make_deseq2.tpl", HASH => $data, OUTPUT => $fh); | |
| $OUT .= "Rscript $self->{PROJECT}/script/make_deseq2.R" | |
| } |
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