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rule remove_adapters_cutadapt:
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'''
Remove adapters
'''
reads = os.path.join(
config["output_dir"],
"samples",
"{sample}",
"start",
"{sample}.fq1.fastq.gz")
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reads = os.path.join(
config["output_dir"],
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"{sample}",
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adapters_3 = lambda wildcards:
samples_table.loc[wildcards.sample, 'fq1_3p'],
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adapters_5 = lambda wildcards:
samples_table.loc[wildcards.sample, 'fq1_5p']
singularity:
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stderr = os.path.join(
config["log_dir"],
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"{sample}",
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stdout = os.path.join(
config["log_dir"],
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"{sample}",
shell:
"(cutadapt \
-e 0.1 \
-j {threads} \
-m 10 \
-a {params.adapters_3} \
-g {params.adapters_5} \
-o {output.reads} \
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1> {log.stdout} 2> {log.stderr}"
rule remove_polya_cutadapt:
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'''
Remove ployA tails
'''
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reads = os.path.join(
config["output_dir"],
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"{sample}",
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reads = os.path.join(
config["output_dir"],
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"{sample}",
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polya_3 = lambda wildcards:
samples_table.loc[wildcards.sample, "fq1_polya_3p"],
polya_5 = lambda wildcards:
samples_table.loc[wildcards.sample, "fq1_polya_5p"]
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singularity:
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stderr = os.path.join(
config["log_dir"],
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"{sample}",
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stdout = os.path.join(
config["log_dir"],
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"{sample}",
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shell:
"(cutadapt \
-j {threads} \
-e 0.1 \
-O 1 \
-m 10 \
-a {params.polya_3} \
-o {output.reads} \
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1> {log.stdout} 2> {log.stderr}"
rule map_genome_star:
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'''
Map to genome using STAR
'''
input:
index = lambda wildcards:
os.path.join(
config["star_indexes"],
str(samples_table.loc[wildcards.sample, "organism"]),
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str(samples_table.loc[wildcards.sample, "index_size"]),
"STAR_index",
"chrNameLength.txt"),
reads = os.path.join(
config["output_dir"],
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"{sample}",
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bam = os.path.join(
config["output_dir"],
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"{sample}",
"map_genome",
"{sample}.se.Aligned.sortedByCoord.out.bam"),
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logfile = os.path.join(
config["output_dir"],
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"{sample}",
"map_genome",
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params:
sample_id = "{sample}",
index = lambda wildcards:
os.path.join(
config["star_indexes"],
str(samples_table.loc[wildcards.sample, "organism"]),
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str(samples_table.loc[wildcards.sample, "index_size"]),
"STAR_index"),
outFileNamePrefix = os.path.join(
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config["output_dir"],
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"{sample}",
"map_genome",
multimappers = lambda wildcards:
samples_table.loc[wildcards.sample, "multimappers"],
soft_clip = lambda wildcards:
samples_table.loc[wildcards.sample, "soft_clip"],
pass_mode = lambda wildcards:
samples_table.loc[wildcards.sample, "pass_mode"],
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singularity:
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threads: 12
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stderr = os.path.join(
config["log_dir"],
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"{sample}",
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shell:
"(STAR \
--runMode alignReads \
-- twopassMode {params.pass_mode} \
--runThreadN {threads} \
--genomeDir {params.index} \
--readFilesIn {input.reads} \
--readFilesCommand zcat \
--outSAMunmapped None \
--outFilterMultimapNmax {params.multimappers} \
--outFileNamePrefix {params.outFileNamePrefix} \
--outSAMattributes All \
--outStd BAM_SortedByCoordinate \
--outSAMtype BAM SortedByCoordinate \
--outFilterMismatchNoverLmax 0.04 \
--outFilterScoreMinOverLread 0.3 \
--outFilterMatchNminOverLread 0.3 \
--outFilterType BySJout \
--outReadsUnmapped None \
--outSAMattrRGline ID:rnaseq_pipeline SM:{params.sample_id} \
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--alignEndsType {params.soft_clip} > {output.bam};) \
2> {log.stderr}"
rule quantification_salmon:
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'''
Quantification at transcript and gene level using Salmon
'''
input:
reads = os.path.join(
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config["output_dir"],
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"{sample}",
index = lambda wildcards:
os.path.join(
config["salmon_indexes"],
str(samples_table.loc[wildcards.sample, "organism"]),
str(samples_table.loc[wildcards.sample, "kmer"]),
"salmon.idx"),
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gtf = lambda wildcards:
samples_table.loc[wildcards.sample, "gtf"]
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output:
gn_estimates = os.path.join(
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config["output_dir"],
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"{sample}",
"quant.genes.sf"),
tr_estimates = os.path.join(
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config["output_dir"],
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"{sample}",
"quant.sf")
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params:
output_dir = os.path.join(
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config["output_dir"],
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"{sample}",
libType = lambda wildcards:
samples_table.loc[wildcards.sample, "libtype"]
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stderr = os.path.join(
config["log_dir"],
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"{sample}",
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stdout = os.path.join(
config["log_dir"],
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"{sample}",
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threads: 12
singularity:
"docker://zavolab/salmon:1.1.0-slim"
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shell:
"(salmon quant \
--libType {params.libType} \
--seqBias \
--validateMappings \
--threads {threads} \
--writeUnmappedNames \
--index {input.index} \
--geneMap {input.gtf} \
--unmatedReads {input.reads} \
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-o {params.output_dir};) \
1> {log.stdout} 2> {log.stderr}"
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rule genome_quantification_kallisto:
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'''
Quantification at transcript and gene level using Kallisto
'''
input:
reads = os.path.join(
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config["output_dir"],
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"{sample}",
index = lambda wildcards:
os.path.join(
config["kallisto_indexes"],
samples_table.loc[wildcards.sample, "organism"],
"kallisto.idx")
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output:
pseudoalignment = os.path.join(
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config["output_dir"],
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"{sample}",
"quant_kallisto",
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params:
output_dir = os.path.join(
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config["output_dir"],
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"{sample}",
"quant_kallisto"),
fraglen = lambda wildcards:
samples_table.loc[wildcards.sample, 'mean'],
fragsd = lambda wildcards:
samples_table.loc[wildcards.sample, 'sd'],
directionality = lambda wildcards:
samples_table.loc[wildcards.sample, 'kallisto_directionality']
threads: 8
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stderr = os.path.join(
config["log_dir"],
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"{sample}",
"genome_quantification_kallisto.se.stderr.log")
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singularity:
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shell:
"(kallisto quant \
-i {input.index} \
-o {params.output_dir} \
--single \
-l {params.fraglen} \
-s {params.fragsd} \
--pseudobam \
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{input.reads} > {output.pseudoalignment};) \