Differential Gene Expression Analysis Workflow Using STAR and Deseq2

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rna-seq-star-deseq2
rna-seq-star-deseq2
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This workflow performs a differential gene expression analysis with STAR and Deseq2.

Usage

The usage of this workflow is described in the Snakemake Workflow Catalog .

If you use this workflow in a paper, don't forget to give credits to the authors by citing the URL of this (original) repository and its DOI (see above).

Code Snippets

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wrapper:
    "0.77.0/bio/star/align"
SnakeMake From line 16 of rules/align.smk 
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script:
    "../scripts/count-matrix.py"
SnakeMake From line 16 of rules/diffexp.smk 
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script:
    "../scripts/gtf2bed.py"
SnakeMake From line 14 of rules/qc.smk 
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shell:
    "junction_annotation.py {params.extra} -i {input.bam} -r {input.bed} -o {params.prefix} "
    "> {log[0]} 2>&1"
SnakeMake From line 32 of rules/qc.smk 
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shell:
    "junction_saturation.py {params.extra} -i {input.bam} -r {input.bed} -o {params.prefix} "
    "> {log} 2>&1"
SnakeMake From line 51 of rules/qc.smk 
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shell:
    "bam_stat.py -i {input} > {output} 2> {log}"
SnakeMake From line 66 of rules/qc.smk 
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shell:
    "infer_experiment.py -r {input.bed} -i {input.bam} > {output} 2> {log}"
SnakeMake From line 81 of rules/qc.smk 
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shell:
    "inner_distance.py -r {input.bed} -i {input.bam} -o {params.prefix} > {log} 2>&1"
SnakeMake From line 98 of rules/qc.smk 
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shell:
    "read_distribution.py -r {input.bed} -i {input.bam} > {output} 2> {log}"
SnakeMake From line 113 of rules/qc.smk 
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shell:
    "read_duplication.py -i {input} -o {params.prefix} > {log} 2>&1"
SnakeMake From line 129 of rules/qc.smk 
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shell:
    "read_GC.py -i {input} -o {params.prefix} > {log} 2>&1"
SnakeMake From line 145 of rules/qc.smk 
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wrapper:
    "0.75.0/bio/multiqc"
SnakeMake MultiQC From line 195 of rules/qc.smk 
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wrapper:
    "0.77.0/bio/reference/ensembl-sequence"
SnakeMake From line 12 of rules/ref.smk 
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wrapper:
    "0.77.0/bio/reference/ensembl-annotation"
SnakeMake From line 28 of rules/ref.smk 
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wrapper:
    "0.77.0/bio/samtools/faidx"
SnakeMake From line 40 of rules/ref.smk 
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wrapper:
    "0.77.0/bio/bwa/index"
SnakeMake From line 54 of rules/ref.smk 
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wrapper:
    "0.77.0/bio/star/index"
SnakeMake From line 70 of rules/ref.smk 
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wrapper:
    "0.77.0/bio/sra-tools/fasterq-dump"
SnakeMake From line 7 of rules/trim.smk 
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shell:
    "cat {input} > {output} 2> {log}"
SnakeMake From line 21 of rules/trim.smk 
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wrapper:
    "0.77.0/bio/cutadapt/pe"
SnakeMake From line 38 of rules/trim.smk 
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wrapper:
    "0.77.0/bio/cutadapt/se"
SnakeMake From line 54 of rules/trim.smk 
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import sys

# logging
sys.stderr = open(snakemake.log[0], "w")

import pandas as pd


def get_column(strandedness):
    if pd.isnull(strandedness) or strandedness == "none":
        return 1  # non stranded protocol
    elif strandedness == "yes":
        return 2  # 3rd column
    elif strandedness == "reverse":
        return 3  # 4th column, usually for Illumina truseq
    else:
        raise ValueError(
            (
                "'strandedness' column should be empty or have the "
                "value 'none', 'yes' or 'reverse', instead has the "
                "value {}"
            ).format(repr(strandedness))
        )


counts = [
    pd.read_table(
        f, index_col=0, usecols=[0, get_column(strandedness)], header=None, skiprows=4
    )
    for f, strandedness in zip(snakemake.input, snakemake.params.strand)
]

for t, sample in zip(counts, snakemake.params.samples):
    t.columns = [sample]

matrix = pd.concat(counts, axis=1)
matrix.index.name = "gene"
# collapse technical replicates
matrix = matrix.groupby(matrix.columns, axis=1).sum()
matrix.to_csv(snakemake.output[0], sep="\t")
Python Pandas From line 1 of scripts/count-matrix.py 
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import gffutils

db = gffutils.create_db(
    snakemake.input[0],
    dbfn=snakemake.output.db,
    force=True,
    keep_order=True,
    merge_strategy="merge",
    sort_attribute_values=True,
    disable_infer_genes=True,
    disable_infer_transcripts=True,
)

with open(snakemake.output.bed, "w") as outfileobj:
    for tx in db.features_of_type("transcript", order_by="start"):
        bed = [s.strip() for s in db.bed12(tx).split("\t")]
        bed[3] = tx.id
        outfileobj.write("{}\n".format("\t".join(bed)))
Python From line 1 of scripts/gtf2bed.py 
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__author__ = "Julian de Ruiter"
__copyright__ = "Copyright 2017, Julian de Ruiter"
__email__ = "[email protected]"
__license__ = "MIT"


from os import path

from snakemake.shell import shell


input_dirs = set(path.dirname(fp) for fp in snakemake.input)
output_dir = path.dirname(snakemake.output[0])
output_name = path.basename(snakemake.output[0])
log = snakemake.log_fmt_shell(stdout=True, stderr=True)

shell(
    "multiqc"
    " {snakemake.params}"
    " --force"
    " -o {output_dir}"
    " -n {output_name}"
    " {input_dirs}"
    " {log}"
)
Python Snakemake MultiQC From line 3 of multiqc/wrapper.py 
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__author__ = "Patrik Smeds"
__copyright__ = "Copyright 2016, Patrik Smeds"
__email__ = "[email protected]"
__license__ = "MIT"

from os import path

from snakemake.shell import shell

log = snakemake.log_fmt_shell(stdout=False, stderr=True)

# Check inputs/arguments.
if len(snakemake.input) == 0:
    raise ValueError("A reference genome has to be provided!")
elif len(snakemake.input) > 1:
    raise ValueError("Only one reference genome can be inputed!")

# Prefix that should be used for the database
prefix = snakemake.params.get("prefix", "")

if len(prefix) > 0:
    prefix = "-p " + prefix

# Contrunction algorithm that will be used to build the database, default is bwtsw
construction_algorithm = snakemake.params.get("algorithm", "")

if len(construction_algorithm) != 0:
    construction_algorithm = "-a " + construction_algorithm

shell(
    "bwa index" " {prefix}" " {construction_algorithm}" " {snakemake.input[0]}" " {log}"
)
Python Snakemake BWA From line 1 of index/wrapper.py 
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__author__ = "Julian de Ruiter"
__copyright__ = "Copyright 2017, Julian de Ruiter"
__email__ = "[email protected]"
__license__ = "MIT"


from snakemake.shell import shell


n = len(snakemake.input)
assert n == 2, "Input must contain 2 (paired-end) elements."

extra = snakemake.params.get("extra", "")
adapters = snakemake.params.get("adapters", "")
log = snakemake.log_fmt_shell(stdout=False, stderr=True)

assert (
    extra != "" or adapters != ""
), "No options provided to cutadapt. Please use 'params: adapters=' or 'params: extra='."

shell(
    "cutadapt"
    " {adapters}"
    " {extra}"
    " -o {snakemake.output.fastq1}"
    " -p {snakemake.output.fastq2}"
    " -j {snakemake.threads}"
    " {snakemake.input}"
    " > {snakemake.output.qc} {log}"
)
Python Snakemake Cutadapt From line 3 of pe/wrapper.py 
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__author__ = "Julian de Ruiter"
__copyright__ = "Copyright 2017, Julian de Ruiter"
__email__ = "[email protected]"
__license__ = "MIT"


from snakemake.shell import shell


n = len(snakemake.input)
assert n == 1, "Input must contain 1 (single-end) element."

extra = snakemake.params.get("extra", "")
adapters = snakemake.params.get("adapters", "")
log = snakemake.log_fmt_shell(stdout=False, stderr=True)

assert (
    extra != "" or adapters != ""
), "No options provided to cutadapt. Please use 'params: adapters=' or 'params: extra='."

shell(
    "cutadapt"
    " {snakemake.params.adapters}"
    " {snakemake.params.extra}"
    " -j {snakemake.threads}"
    " -o {snakemake.output.fastq}"
    " {snakemake.input[0]}"
    " > {snakemake.output.qc} {log}"
)
Python Snakemake Cutadapt From line 3 of se/wrapper.py 
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__author__ = "Johannes Köster"
__copyright__ = "Copyright 2019, Johannes Köster"
__email__ = "[email protected]"
__license__ = "MIT"

import subprocess
import sys
from snakemake.shell import shell

species = snakemake.params.species.lower()
release = int(snakemake.params.release)
fmt = snakemake.params.fmt
build = snakemake.params.build
flavor = snakemake.params.get("flavor", "")

branch = ""
if release >= 81 and build == "GRCh37":
    # use the special grch37 branch for new releases
    branch = "grch37/"

if flavor:
    flavor += "."

log = snakemake.log_fmt_shell(stdout=False, stderr=True)

suffix = ""
if fmt == "gtf":
    suffix = "gtf.gz"
elif fmt == "gff3":
    suffix = "gff3.gz"

url = "ftp://ftp.ensembl.org/pub/{branch}release-{release}/{fmt}/{species}/{species_cap}.{build}.{release}.{flavor}{suffix}".format(
    release=release,
    build=build,
    species=species,
    fmt=fmt,
    species_cap=species.capitalize(),
    suffix=suffix,
    flavor=flavor,
    branch=branch,
)

try:
    shell("(curl -L {url} | gzip -d > {snakemake.output[0]}) {log}")
except subprocess.CalledProcessError as e:
    if snakemake.log:
        sys.stderr = open(snakemake.log[0], "a")
    print(
        "Unable to download annotation data from Ensembl. "
        "Did you check that this combination of species, build, and release is actually provided?",
        file=sys.stderr,
    )
    exit(1)
Python Snakemake From line 1 of ensembl-annotation/wrapper.py 
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__author__ = "Johannes Köster"
__copyright__ = "Copyright 2019, Johannes Köster"
__email__ = "[email protected]"
__license__ = "MIT"

import subprocess as sp
import sys
from itertools import product
from snakemake.shell import shell

species = snakemake.params.species.lower()
release = int(snakemake.params.release)
build = snakemake.params.build

branch = ""
if release >= 81 and build == "GRCh37":
    # use the special grch37 branch for new releases
    branch = "grch37/"

log = snakemake.log_fmt_shell(stdout=False, stderr=True)

spec = ("{build}" if int(release) > 75 else "{build}.{release}").format(
    build=build, release=release
)

suffixes = ""
datatype = snakemake.params.get("datatype", "")
chromosome = snakemake.params.get("chromosome", "")
if datatype == "dna":
    if chromosome:
        suffixes = ["dna.chromosome.{}.fa.gz".format(chromosome)]
    else:
        suffixes = ["dna.primary_assembly.fa.gz", "dna.toplevel.fa.gz"]
elif datatype == "cdna":
    suffixes = ["cdna.all.fa.gz"]
elif datatype == "cds":
    suffixes = ["cds.all.fa.gz"]
elif datatype == "ncrna":
    suffixes = ["ncrna.fa.gz"]
elif datatype == "pep":
    suffixes = ["pep.all.fa.gz"]
else:
    raise ValueError("invalid datatype, must be one of dna, cdna, cds, ncrna, pep")

if chromosome:
    if not datatype == "dna":
        raise ValueError(
            "invalid datatype, to select a single chromosome the datatype must be dna"
        )

success = False
for suffix in suffixes:
    url = "ftp://ftp.ensembl.org/pub/{branch}release-{release}/fasta/{species}/{datatype}/{species_cap}.{spec}.{suffix}".format(
        release=release,
        species=species,
        datatype=datatype,
        spec=spec.format(build=build, release=release),
        suffix=suffix,
        species_cap=species.capitalize(),
        branch=branch,
    )

    try:
        shell("curl -sSf {url} > /dev/null 2> /dev/null")
    except sp.CalledProcessError:
        continue

    shell("(curl -L {url} | gzip -d > {snakemake.output[0]}) {log}")
    success = True
    break

if not success:
    print(
        "Unable to download requested sequence data from Ensembl. "
        "Did you check that this combination of species, build, and release is actually provided?",
        file=sys.stderr,
    )
    exit(1)
Python Snakemake From line 1 of ensembl-sequence/wrapper.py 
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__author__ = "Michael Chambers"
__copyright__ = "Copyright 2019, Michael Chambers"
__email__ = "[email protected]"
__license__ = "MIT"


from snakemake.shell import shell

log = snakemake.log_fmt_shell(stdout=False, stderr=True)

shell(
    "samtools faidx {snakemake.params} {snakemake.input[0]} > {snakemake.output[0]} {log}"
)
Python Snakemake SAMtools From line 1 of faidx/wrapper.py 
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__author__ = "Johannes Köster, Derek Croote"
__copyright__ = "Copyright 2020, Johannes Köster"
__email__ = "[email protected]"
__license__ = "MIT"

import os
import tempfile
from snakemake.shell import shell


log = snakemake.log_fmt_shell(stdout=True, stderr=True)
extra = snakemake.params.get("extra", "")


outdir = os.path.dirname(snakemake.output[0])
if outdir:
    outdir = f"--outdir {outdir}"


compress = ""
for output in snakemake.output:
    out_name, out_ext = os.path.splitext(output)
    if out_ext == ".gz":
        compress += f"pigz -p {snakemake.threads} {out_name}; "
    elif out_ext == ".bz2":
        compress += f"pbzip2 -p{snakemake.threads} {out_name}; "


with tempfile.TemporaryDirectory() as tmp:
    shell(
        "(fasterq-dump --temp {tmp} --threads {snakemake.threads} "
        "{extra} {outdir} {snakemake.wildcards.accession}; "
        "{compress}"
        ") {log}"
    )
Python Snakemake From line 1 of fasterq-dump/wrapper.py 
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__author__ = "Johannes Köster"
__copyright__ = "Copyright 2016, Johannes Köster"
__email__ = "[email protected]"
__license__ = "MIT"


import os
from snakemake.shell import shell

extra = snakemake.params.get("extra", "")
log = snakemake.log_fmt_shell(stdout=True, stderr=True)

fq1 = snakemake.input.get("fq1")
assert fq1 is not None, "input-> fq1 is a required input parameter"
fq1 = (
    [snakemake.input.fq1]
    if isinstance(snakemake.input.fq1, str)
    else snakemake.input.fq1
)
fq2 = snakemake.input.get("fq2")
if fq2:
    fq2 = (
        [snakemake.input.fq2]
        if isinstance(snakemake.input.fq2, str)
        else snakemake.input.fq2
    )
    assert len(fq1) == len(
        fq2
    ), "input-> equal number of files required for fq1 and fq2"
input_str_fq1 = ",".join(fq1)
input_str_fq2 = ",".join(fq2) if fq2 is not None else ""
input_str = " ".join([input_str_fq1, input_str_fq2])

if fq1[0].endswith(".gz"):
    readcmd = "--readFilesCommand zcat"
else:
    readcmd = ""

outprefix = os.path.dirname(snakemake.output[0]) + "/"

shell(
    "STAR "
    "{extra} "
    "--runThreadN {snakemake.threads} "
    "--genomeDir {snakemake.params.index} "
    "--readFilesIn {input_str} "
    "{readcmd} "
    "--outFileNamePrefix {outprefix} "
    "--outStd Log "
    "{log}"
)
Python Snakemake STAR From line 1 of align/wrapper.py 
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__author__ = "Thibault Dayris"
__copyright__ = "Copyright 2019, Dayris Thibault"
__email__ = "[email protected]"
__license__ = "MIT"

from snakemake.shell import shell
from snakemake.utils import makedirs

log = snakemake.log_fmt_shell(stdout=True, stderr=True)

extra = snakemake.params.get("extra", "")
sjdb_overhang = snakemake.params.get("sjdbOverhang", "100")

gtf = snakemake.input.get("gtf")
if gtf is not None:
    gtf = "--sjdbGTFfile " + gtf
    sjdb_overhang = "--sjdbOverhang " + sjdb_overhang
else:
    gtf = sjdb_overhang = ""

makedirs(snakemake.output)

shell(
    "STAR "  # Tool
    "--runMode genomeGenerate "  # Indexation mode
    "{extra} "  # Optional parameters
    "--runThreadN {snakemake.threads} "  # Number of threads
    "--genomeDir {snakemake.output} "  # Path to output
    "--genomeFastaFiles {snakemake.input.fasta} "  # Path to fasta files
    "{sjdb_overhang} "  # Read-len - 1
    "{gtf} "  # Highly recommended GTF
    "{log}"  # Logging
)
Python Snakemake STAR From line 3 of index/wrapper.py
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/plycrsk/rna-seq-star-deseq2
Name: rna-seq-star-deseq2
Version: v1.2.1
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Copyright: Public Domain
License: MIT License
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public

ARMOR ( A utomated R eproducible MO dular R NA-seq) is a Snakemake workflow , aimed at performing a ty...
JSON User metadata Expression analysis Quality control report bedGraphToBigWig BEDTools Cutadapt FastQC MultiQC Quant Salmon SAMtools Snakemake STAR Trim_Galore RNA-Seq
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