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Example Snakefile

#Data downloaded from: https://osf.io/vzfc6/

# Default rule to run entire workflow, only works once inputs/outputs correctly filled in all rules
rule all:
    input: "variants.vcf"

# Download raw sequence data
rule download_data:
    output: "SRR2584857_1.fastq.gz"
    shell:
        "wget https://osf.io/4rdza/download -O {output}"

# Download reference genome
rule download_genome:
    output: "ecoli-rel606.fa.gz"
    shell:
        "wget https://osf.io/8sm92/download -O {output}"

# The .gz file must be uncompressed before it can be accessed
rule uncompress_genome:
    input: "ecoli-rel606.fa.gz"
    output: "ecoli-rel606.fa"
    shell:
        "gunzip -c {input} > {output}"

# Create an index for the reference genome for bwa
rule index_genome_bwa:
    input: "ecoli-rel606.fa"
    output:
        expand("ecoli-rel606.fa.{ext}", ext=['sa', 'amb', 'ann', 'pac', 'bwt'])
    shell:
        "bwa index {input}"

# Map the raw reads to the reference genome
rule map_reads:
    input:
        genome = "ecoli-rel606.fa",
        reads = "SRR2584857_1.fastq.gz",
        idxfile = expand("ecoli-rel606.fa.{ext}", ext=['sa', 'amb', 'ann', 'pac', 'bwt'])
    output: "SRR2584857.sam"
    shell:
        "bwa mem -t 4 {input.genome} {input.reads} > {output}"

# Create an index for the reference genome fasta file for samtools
# Note: this indexing step is different and unrelated to the one above for bwa
rule index_genome_samtools:
    input: "ecoli-rel606.fa"
    output: "ecoli-rel606.fa.fai"
    shell:
        "samtools faidx {input}"

# Convert .sam to .bam file
rule samtools_import:
    input:
        index="ecoli-rel606.fa.fai",
        samfile="SRR2584857.sam"
    output:"SRR2584857.bam"
    shell:
        """
        samtools view -bt {input.index} -o {output} {input.samfile}
        """
        # original command with samtools v1.9
        ## samtools import {input.index} {input.samfile} {output}
        # but it gave segmentation fault error with samtools v1.10, so here we use samtools view instead

# Sort the bam alignment file
rule samtools_sort:
    input: "SRR2584857.bam"
    output: "SRR2584857.sorted.bam"
    shell:
        "samtools sort {input} -o {output}"

# Create an index for the sorted bam file
# again, this is different from the above indexing steps
rule samtools_index_sorted:
    input: "SRR2584857.sorted.bam"
    output: "SRR2584857.sorted.bam.bai"
    shell: "samtools index {input}"

# Generate pileup file with samtools, then call variants with bcftools
# From samtools doc: 'Pileup format consists of TAB-separated lines, with each line representing the pileup of reads at a single genomic position.'
rule samtools_mpileup:
    input:
        index="ecoli-rel606.fa",
        sorted="SRR2584857.sorted.bam",
        sorted_bai="SRR2584857.sorted.bam.bai"
    output:
        "variants.raw.bcf"
    shell:
        """
        samtools mpileup -u -t DP -f {input.index} {input.sorted} | \
        bcftools call -mv -Ob -o - > {output}
        """

# Convert bcf to vcf file so we can look at the resulting mappings
rule make_vcf:
    input: "variants.raw.bcf"
    output: "variants.vcf"
    shell: "bcftools view {input} > {output}"

# at end, run this command in the terminal to view the mapping:
## samtools tview -p ecoli:4202391 SRR2584857.sorted.bam ecoli-rel606.fa

Last update: October 10, 2020