Example Snakefile for the new Tuxedo RNA-Seq pipeline (local)

Snakefile

# The Snakefile that loads raw data and genome reference locally
GENOME_FA = "griffithlab_brain_vs_uhr/GRCh38_Ens87_chr22_ERCC/chr22_ERCC92.fa"
GENOME_GTF = "griffithlab_brain_vs_uhr/GRCh38_Ens87_chr22_ERCC/genes_chr22_ERCC92.gtf"
HISAT2_INDEX_PREFIX = "hisat2_index/chr22_ERCC92"

SAMPLES, *_ = glob_wildcards('griffithlab_brain_vs_uhr/HBR_UHR_ERCC_ds_10pc/{sample}.read1.fastq.gz')

from pathlib import Path


rule extract_genome_splice_sites:
    input: GENOME_GTF
    output: "hisat2_index/chr22_ERCC92.ss"
    shell: "hisat2_extract_splice_sites.py {input} > {output}"


rule extract_genome_exons:
    input: GENOME_GTF
    output: "hisat2_index/chr22_ERCC92.exon"
    shell: "hisat2_extract_exons.py {input} > {output}"


rule build_hisat_index:
    input:
        genome_fa=GENOME_FA,
        splice_sites="hisat2_index/chr22_ERCC92.ss",
        exons="hisat2_index/chr22_ERCC92.exon",
    output: expand(f"{HISAT2_INDEX_PREFIX}.{{ix}}.ht2", ix=range(1, 9))
    log: "hisat2_index/build.log"
    threads: 8
    shell:
        "hisat2-build -p {threads} {input.genome_fa} "
        "--ss {input.splice_sites} --exon {input.exons} {HISAT2_INDEX_PREFIX} "
        "2>{log}"


rule align_hisat:
    input:
        hisat2_index=expand(f"{HISAT2_INDEX_PREFIX}.{{ix}}.ht2", ix=range(1, 9)),
        fastq1="griffithlab_brain_vs_uhr/HBR_UHR_ERCC_ds_10pc/{sample}.read1.fastq.gz",
        fastq2="griffithlab_brain_vs_uhr/HBR_UHR_ERCC_ds_10pc/{sample}.read2.fastq.gz",
    output: "align_hisat2/{sample}.bam"
    log: "align_hisat2/{sample}.log"
    threads: 4
    shell:
        "hisat2 -p {threads} --dta -x {HISAT2_INDEX_PREFIX} "
        "-1 {input.fastq1} -2 {input.fastq2} 2>{log} | "
        "samtools sort -@ {threads} -o {output}"


rule align_all_samples:
    input: expand("align_hisat2/{sample}.bam", sample=SAMPLES)


rule stringtie_assemble:
    input:
        genome_gtf=GENOME_GTF,
        bam="align_hisat2/{sample}.bam"
    output: "stringtie/assembled/{sample}.gtf"
    threads: 4
    shell:
        "stringtie -p {threads} -G {input.genome_gtf} "
        "-o {output} -l {wildcards.sample} {input.bam}"


rule stringtie_merge_list:
    input: expand("stringtie/assembled/{sample}.gtf", sample=SAMPLES)
    output: "stringtie/merged_list.txt"
    run:
        with open(output[0], 'w') as f:
            for gtf in input:
                print(Path(gtf).resolve(), file=f)


rule stringtie_merge:
    input:
        genome_gtf=GENOME_GTF,
        merged_list="stringtie/merged_list.txt",
        sample_gtfs=expand("stringtie/assembled/{sample}.gtf", sample=SAMPLES)
    output: "stringtie/merged.gtf"
    threads: 4
    shell:
        "stringtie --merge -p {threads} -G {input.genome_gtf} "
        "-o {output} {input.merged_list}"


rule stringtie_quant:
    input:
        merged_gtf="stringtie/merged.gtf",
        sample_bam="align_hisat2/{sample}.bam"
    output:
        gtf="stringtie/quant/{sample}/{sample}.gtf",
        gene_abund_tab="stringtie/quant/{sample}/gene_abund.tab",
        ctabs=expand(
            "stringtie/quant/{{sample}}/{name}.ctab",
            name=['i2t', 'e2t', 'i_data', 'e_data', 't_data']
        )
    threads: 4
    shell:
        "stringtie -e -B -p {threads} -G {input.merged_gtf} "
        "-A {output.gene_abund_tab} -o {output.gtf} {input.sample_bam}"


rule quant_all_samples:
    input: expand("stringtie/quant/{sample}/{sample}.gtf", sample=SAMPLES)


rule clean:
    shell:
        "rm -rf align_hisat2 hisat2_index stringtie"