Snakemake pipeline for detection limit test from laser-microdissected samples

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A Snakemake workflow for assessing detection limit from laser-microdissected samples.

Usage

  1. Requirements

    1. git-lfs

    2. miniconda / mamba

    3. snakemake

  2. Clone the repository Clone the repository, and set it as the working directory. 

git clone --recursive https://github.com/3d-omics/detection_limit_test.git
cd detection_limit_test
  1. Run the pipeline with the test data (takes 5 minutes) 
snakemake \ --use-conda \ --conda-frontend mamba \ -j 8

  1. Edit the following files:

    1. config/samples.tsv : the control file with the sequencing libraries and their location.

    2. config/features.tsv : the references against which to map the libraries: human, chicken / pig, MAG catalogue.

    3. config/params.tsv : parameters for every program. The defaults are reasonable.

  2. Run the pipeline on a cluster with slurm and go for a walk: 

./run_slurm

Brief description

  1. Trim reads and remove adaptors with fastp

  2. Map to human, chicken / pig, mag catalogue:

    1. Map to the reference with bowtie2

    2. Extract the reads that have one of both ends unmapped with samtools

    3. Map those unmapped reads to the next reference

  3. Generate MAG statistics with

    1. coverm

    2. singlem

    3. nonpareil

  4. Generate lots of reports in the reports/ folder

Rulegraph

rulegraph

Code Snippets

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shell:
    """
    bowtie2-build \
        --threads {threads} \
        {params.extra} \
        {input.reference} \
        {output.mock} \
    2> {log} 1>&2
    """
SnakeMake Bowtie 2 From line 21 of rules/bowtie2_hosts.smk 
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shell:
    """
    if [[ {params.is_paired} = "True" ]] ; then
        (bowtie2 \
            -x {input.mock} \
            -1 {input.forward_} \
            -2 {input.reverse_} \
            --threads {threads} \
            --rg-id '{params.rg_id}' \
            --rg '{params.rg_extra}' \
            {params.extra} \
        | samtools sort \
            --threads {threads} \
            -m {params.samtools_mem} \
        | samtools rmdup - - \
        | samtools view \
            --reference {input.reference} \
            --output {output.cram} \
            --output-fmt cram,level=9,nthreads={threads} \
            --write-index \
        ) 2> {log} 1>&2
    else
        (bowtie2 \
            -x {input.mock} \
            -U {input.forward_} \
            --threads {threads} \
            --rg-id '{params.rg_id}' \
            --rg '{params.rg_extra}' \
            {params.extra} \
        | samtools sort \
            --threads {threads} \
            -m {params.samtools_mem} \
        | samtools rmdup -s - - \
        | samtools view \
            --reference {input.reference} \
            --output {output.cram} \
            --output-fmt cram,level=9,nthreads={threads} \
            --write-index \
        ) 2> {log} 1>&2
    fi
    """
SnakeMake SAMtools From line 59 of rules/bowtie2_hosts.smk 
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shell:
    """
    if [[ {params.is_paired} = "True" ]] ; then
        (samtools view \
            --reference {input.reference} \
            --threads {threads} \
            -u \
            -o /dev/stdout \
            -f 12 \
            {input.cram} \
        | samtools fastq \
            -1 {output.forward_} \
            -2 {output.reverse_} \
            -0 /dev/null \
            -c 9 \
            --threads {threads} \
        ) 2> {log} 1>&2
    else
        (samtools view \
            --reference {input.reference} \
            --threads {threads} \
            -u \
            -o /dev/stdout \
            -f 4 \
            {input.cram} \
        | samtools fastq \
            -0 {output.forward_} \
            -c 9 \
            --threads {threads} \
        ) 2> {log} 1>&2
    fi
    """
SnakeMake SAMtools From line 123 of rules/bowtie2_hosts.smk 
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shell:
    """
    bowtie2-build \
        --threads {threads} \
        {params.extra} \
        {input.reference} \
        {output.mock} \
    2> {log} 1>&2
    """
SnakeMake Bowtie 2 From line 21 of rules/bowtie2_mags.smk 
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shell:
    """
    if [[ {params.is_paired} == "True" ]] ; then
        (bowtie2 \
            -x {input.mock} \
            -1 {input.forward_} \
            -2 {input.reverse_} \
            --threads {threads} \
            --rg-id '{params.rg_id}' \
            --rg '{params.rg_extra}' \
            {params.extra} \
        | samtools sort \
            -l 9 \
            -M \
            -m {params.samtools_mem} \
            -o {output.cram} \
            --reference {input.reference} \
            --threads {threads} \
            --write-index \
        ) 2> {log} 1>&2
    else
        (bowtie2 \
            -x {input.mock} \
            -U {input.forward_} \
            --threads {threads} \
            --rg-id '{params.rg_id}' \
            --rg '{params.rg_extra}' \
            {params.extra} \
        | samtools sort \
            -l 9 \
            -M \
            -m {params.samtools_mem} \
            -o {output.cram} \
            --reference {input.reference} \
            --threads {threads} \
            --write-index \
        ) 2> {log} 1>&2
    fi
    """
SnakeMake SAMtools From line 59 of rules/bowtie2_mags.smk 
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shell:
    """
    if [[ {params.is_paired} = True ]] ; then
        fastp \
            --in1 {input.forward_} \
            --in2 {input.reverse_} \
            --out1 >(bgzip -l 1 -@ {threads} > {output.forward_}) \
            --out2 >(bgzip -l 1 -@ {threads} > {output.reverse_}) \
            --unpaired1 >(bgzip -l 1 -@ {threads} > {output.unpaired1}) \
            --unpaired2 >(bgzip -l 1 -@ {threads} > {output.unpaired2}) \
            --html {output.html} \
            --json {output.json} \
            --compression 1 \
            --verbose \
            --adapter_sequence {params.adapter_forward} \
            --adapter_sequence_r2 {params.adapter_reverse} \
            --length_required {params.length_required} \
            --thread {threads} \
            {params.extra} \
        2> {log} 1>&2
    else
        fastp \
            --in1 {input.forward_} \
            --out1 >(bgzip -l 1 -@ {threads} > {output.forward_}) \
            --html {output.html} \
            --json {output.json} \
            --compression 1 \
            --verbose \
            --adapter_sequence {params.adapter_forward} \
            --length_required {params.length_required} \
            --thread {threads} \
            {params.extra} \
        2> {log} 1>&2
    fi
    """
SnakeMake fastp From line 26 of rules/fastp.smk 
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shell:
    "fastqc {input} 2> {log} 1>&2"
SnakeMake FastQC From line 12 of rules/fastqc.smk 
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shell:
    """
    if [[ {params.is_paired} = "True" ]] ; then
        kraken2 \
            --db {input.database} \
            --threads {threads} \
            --paired \
            --gzip-compressed \
            --output >(pigz > {output.out_gz}) \
            --report {output.report} \
            {input.forward_} \
            {input.reverse_} \
        > {log} 2>&1
    else
        kraken2 \
            --db {input.database} \
            --threads {threads} \
            --gzip-compressed \
            --output >(pigz > {output.out_gz}) \
            --report {output.report} \
            {input.forward_} \
        > {log} 2>&1
    fi
    """
SnakeMake kraken2 From line 20 of rules/kraken2.smk 
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shell:
    """
    ln --symbolic $(readlink --canonicalize {input.forward_}) {output.forward_} 2> {log}
    ln --symbolic $(readlink --canonicalize {input.reverse_}) {output.reverse_} 2> {log}
    """
SnakeMake From line 13 of rules/reads.smk 
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shell:
    """
    (gzip \
        --decompres \
        --stdout {input.fa_gz} \
    | bgzip \
        --compress-level 9 \
        --threads {threads} \
        --stdout \
        /dev/stdin \
    > {output.fa_gz} \
    ) 2> {log}
    """
SnakeMake From line 12 of rules/reference.smk 
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shell:
    """
    (gzip \
        --decompres \
        --stdout {input.fa_gz} \
    | bgzip \
        --compress-level 9 \
        --threads {threads} \
        --stdout \
        /dev/stdin \
    > {output.fa_gz} \
    ) 2> {log}
    """
SnakeMake From line 43 of rules/reference.smk 
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shell:
    """
    multiqc \
        --title {params.library} \
        --force \
        --filename {params.library} \
        --outdir {params.out_dir} \
        --dirs \
        --dirs-depth 1 \
        {input} \
    2> {log} 1>&2
    """
SnakeMake MultiQC From line 18 of rules/report_library.smk 
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shell:
    """
    multiqc \
        --filename reads \
        --title reads \
        --force \
        --outdir {params.dir} \
        {input} \
    2> {log} 1>&2
    """
SnakeMake MultiQC From line 13 of rules/report_step.smk 
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shell:
    """
    multiqc \
        --title fastp \
        --force \
        --filename fastp \
        --outdir {params.dir} \
        {input} \
    2> {log} 1>&2
    """
SnakeMake MultiQC fastp From line 37 of rules/report_step.smk 
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shell:
    """
    multiqc \
        --title {params.title} \
        --force \
        --filename {params.title} \
        --outdir {params.dir} \
        --module kraken \
        {input} \
    2> {log} 1>&2
    """
SnakeMake MultiQC Kraken From line 61 of rules/report_step.smk 
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shell:
    """
    multiqc \
        --title {params.title} \
        --force \
        --filename {params.title} \
        --outdir {params.dir} \
        --dirs \
        --dirs-depth 1 \
        {input.reports} \
    2> {log} 1>&2
    """
SnakeMake MultiQC From line 97 of rules/report_step.smk 
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shell:
    """
    multiqc \
        --title {params.title} \
        --force \
        --filename {params.title} \
        --outdir {params.dir} \
        --dirs \
        --dirs-depth 1 \
        {input.reports} \
    2> {log} 1>&2
    """
SnakeMake MultiQC From line 135 of rules/report_step.smk 
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shell:
    "samtools flagstats {input.cram} > {output.txt} 2> {log}"
SnakeMake SAMtools From line 12 of rules/samtools.smk 
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shell:
    "samtools idxstats {input.cram} > {output.tsv} 2> {log}"
SnakeMake SAMtools From line 27 of rules/samtools.smk 
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shell:
    """
    samtools stats \
        --reference {input.reference} \
        {input.cram} \
    > {output.tsv} \
    2> {log}
    """
SnakeMake SAMtools From line 43 of rules/samtools.smk 
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shell:
    """
    samtools stats \
        --reference {input.reference} \
        {input.cram} \
    > {output.tsv} \
    2> {log}
    """
SnakeMake SAMtools From line 64 of rules/samtools.smk 
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shell:
    """
    gzip -dc {input.forward_} > {output.forward_fq} 2> {log}

    nonpareil \
        -s {output.forward_fq} \
        -T kmer \
        -b {params.prefix} \
        -f fastq \
        -t {threads} \
    2>> {log} 1>&2 || true
    """
SnakeMake nonpareil From line 28 of rules/stats.smk 
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shell:
    """
    Rscript --no-init-file workflow/scripts/aggregate_nonpareil.R \
        --input-folder {params.input_dir} \
        --output-file {output} \
    2> {log} 1>&2
    """
SnakeMake nonpareil From line 64 of rules/stats.smk 
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shell:
    """
    if [[ {params.is_paired} = "True" ]] ; then
        singlem pipe \
            --forward {input.forward_} \
            --reverse {input.reverse_} \
            --otu-table {output.otu_table} \
            --archive-otu-table {output.archive_otu_table} \
            --taxonomic-profile {output.condense} \
            --metapackage {input.data} \
            --threads {threads} \
            --assignment-threads {threads} \
        2> {log} 1>&2 || true
    else
        singlem pipe \
            --forward {input.forward_} \
            --otu-table {output.otu_table} \
            --archive-otu-table {output.archive_otu_table} \
            --taxonomic-profile {output.condense} \
            --metapackage {input.data} \
            --threads {threads} \
            --assignment-threads {threads} \
        2> {log} 1>&2 || true
    fi
    """
SnakeMake singlem From line 99 of rules/stats.smk 
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shell:
    """
    singlem condense \
        --input-archive-otu-tables {input.archive_otu_tables} \
        --taxonomic-profile {output.condense} \
        --metapackage {input.data} \
    2> {log} 1>&2
    """
SnakeMake singlem From line 151 of rules/stats.smk 
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shell:
    """
    samtools view \
        -F 4 \
        --threads {threads} \
        --reference {input.reference} \
        --output {output.bam} \
        --fast \
        {input.cram} \
    2> {log}
    """
SnakeMake SAMtools From line 190 of rules/stats.smk 
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shell:
    """
    coverm genome \
        --bam-files {input.bam} \
        --methods {params.method} \
        --separator {params.separator} \
        --min-covered-fraction {params.min_covered_fraction} \
    > {output} 2> {log} || true \
    """
SnakeMake CoverM From line 219 of rules/stats.smk 
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shell:
    """
    Rscript --no-init-file workflow/scripts/aggregate_coverm.R \
        --input-folder {params.input_dir} \
        --output-file {output} \
    2> {log} 1>&2
    """
SnakeMake From line 245 of rules/stats.smk 
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shell:
    """
    coverm contig \
        --bam-files {input.bam} \
        --methods {params.method} \
        --proper-pairs-only \
    > {output} 2> {log} || true
    """
SnakeMake CoverM From line 276 of rules/stats.smk 
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shell:
    """
    Rscript --no-init-file workflow/scripts/aggregate_coverm.R \
        --input-folder {params.input_dir} \
        --output-file {output} \
    2> {log} 1>&2
    """
SnakeMake From line 301 of rules/stats.smk
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/3d-omics/bioinfo_detection_limit_test
Name: bioinfo_detection_limit_test
Version: 1
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Copyright: Public Domain
License: MIT License
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