rule remove_adapters_cutadapt: ''' Remove adapters ''' input: reads = os.path.join( config["output_dir"], "samples", "{sample}", "start", "{sample}.fq1.fastq.gz") output: reads = temp(os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.se.remove_adapters_mate1.fastq.gz")) params: adapters_3 = lambda wildcards: get_sample( 'fq1_3p', search_id='index', search_value=wildcards.sample), adapters_5 = lambda wildcards: get_sample( 'fq1_5p', search_id='index', search_value=wildcards.sample) singularity: "docker://zavolab/cutadapt:1.16-slim" threads: 8 log: stderr = os.path.join( config["log_dir"], "samples", "{sample}", "remove_adapters_cutadapt.se.stderr.log"), stdout = os.path.join( config["log_dir"], "samples", "{sample}", "remove_adapters_cutadapt.se.stdout.log") shell: "(cutadapt \ -j {threads} \ -m 10 \ -n 2 \ -a {params.adapters_3} \ -g {params.adapters_5} \ -o {output.reads} \ {input.reads}) \ 1> {log.stdout} 2> {log.stderr}" rule remove_polya_cutadapt: ''' Remove ployA tails ''' input: reads = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.se.remove_adapters_mate1.fastq.gz") output: reads = temp(os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.se.remove_polya_mate1.fastq.gz")) params: polya_3 = lambda wildcards: get_sample( 'fq1_polya_3p', search_id='index', search_value=wildcards.sample), polya_5 = lambda wildcards: get_sample( 'fq1_polya_5p', search_id='index', search_value=wildcards.sample) singularity: "docker://zavolab/cutadapt:1.16-slim" threads: 8 log: stderr = os.path.join( config["log_dir"], "samples", "{sample}", "remove_polya_cutadapt.se.stderr.log"), stdout = os.path.join( config["log_dir"], "samples", "{sample}", "remove_polya_cutadapt.se.stdout.log") shell: "(cutadapt \ -j {threads} \ -O 1 \ -m 10 \ -a {params.polya_3} \ -g {params.polya_5} \ -o {output.reads} \ {input.reads};) \ 1> {log.stdout} 2> {log.stderr}" rule map_genome_star: ''' Map to genome using STAR ''' input: index = lambda wildcards: os.path.join( config["star_indexes"], get_sample('organism', search_id='index', search_value=wildcards.sample), get_sample('index_size', search_id='index', search_value=wildcards.sample), "STAR_index", "chrNameLength.txt"), reads = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.se.remove_polya_mate1.fastq.gz") output: bam = os.path.join( config["output_dir"], "samples", "{sample}", "map_genome", "{sample}.se.Aligned.sortedByCoord.out.bam"), logfile = os.path.join( config["output_dir"], "samples", "{sample}", "map_genome", "{sample}.se.Log.final.out") shadow: "minimal" params: sample_id = "{sample}", index = lambda wildcards: os.path.abspath(os.path.join( config["star_indexes"], get_sample('organism', search_id='index', search_value=wildcards.sample), get_sample('index_size', search_id='index', search_value=wildcards.sample), "STAR_index")), outFileNamePrefix = os.path.join( config["output_dir"], "samples", "{sample}", "map_genome", "{sample}.se."), multimappers = lambda wildcards: get_sample( 'multimappers', search_id='index', search_value=wildcards.sample), soft_clip = lambda wildcards: get_sample( 'soft_clip', search_id='index', search_value=wildcards.sample), pass_mode = lambda wildcards: get_sample( 'pass_mode', search_id='index', search_value=wildcards.sample) singularity: "docker://zavolab/star:2.7.3a-slim" threads: 12 log: stderr = os.path.join( config["log_dir"], "samples", "{sample}", "map_genome_star.se.stderr.log") shell: "(STAR \ -- twopassMode {params.pass_mode} \ --runThreadN {threads} \ --genomeDir {params.index} \ --readFilesIn {input.reads} \ --readFilesCommand zcat \ --outFilterMultimapNmax {params.multimappers} \ --outFilterMultimapScoreRange 0 \ --outFileNamePrefix {params.outFileNamePrefix} \ --outSAMattributes All \ --outStd BAM_SortedByCoordinate \ --outSAMtype BAM SortedByCoordinate \ --outFilterType BySJout \ --outSAMattrRGline ID:rnaseq_pipeline SM:{params.sample_id} \ --alignEndsType {params.soft_clip} > {output.bam};) \ 2> {log.stderr}" rule quantification_salmon: ''' Quantification at transcript and gene level using Salmon ''' input: reads = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.se.remove_polya_mate1.fastq.gz"), index = lambda wildcards: os.path.join( config["salmon_indexes"], get_sample( 'organism', search_id='index', search_value=wildcards.sample), get_sample( 'kmer', search_id='index', search_value=wildcards.sample), "salmon.idx"), gtf = lambda wildcards: os.path.abspath(get_sample( 'gtf', search_id='index', search_value=wildcards.sample)) output: gn_estimates = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.salmon.se", "quant.genes.sf"), tr_estimates = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.salmon.se", "quant.sf"), meta_info = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.salmon.se", "aux_info", "meta_info.json"), flenDist = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.salmon.se", "libParams", "flenDist.txt") shadow: "minimal" params: output_dir = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.salmon.se"), libType = lambda wildcards: get_sample( 'libtype', search_id='index', search_value=wildcards.sample), fraglen = lambda wildcards: get_sample( 'mean', search_id='index', search_value=wildcards.sample), fragsd = lambda wildcards: get_sample( 'sd', search_id='index', search_value=wildcards.sample) log: stderr = os.path.join( config["log_dir"], "samples", "{sample}", "quantification_salmon.se.stderr.log"), stdout = os.path.join( config["log_dir"], "samples", "{sample}", "quantification_salmon.se.stdout.log") threads: 12 singularity: "docker://zavolab/salmon:1.1.0-slim" shell: "(salmon quant \ --libType {params.libType} \ --seqBias \ --validateMappings \ --threads {threads} \ --fldMean {params.fraglen} \ --fldSD {params.fragsd} \ --index {input.index} \ --geneMap {input.gtf} \ --unmatedReads {input.reads} \ -o {params.output_dir};) \ 1> {log.stdout} 2> {log.stderr}" rule genome_quantification_kallisto: ''' Quantification at transcript and gene level using Kallisto ''' input: reads = os.path.join( config["output_dir"], "samples", "{sample}", "{sample}.se.remove_polya_mate1.fastq.gz"), index = lambda wildcards: os.path.join( config["kallisto_indexes"], get_sample( 'organism', search_id='index', search_value=wildcards.sample), "kallisto.idx") output: pseudoalignment = os.path.join( config["output_dir"], "samples", "{sample}", "quant_kallisto", "{sample}.se.kallisto.pseudo.sam"), abundances = os.path.join( config["output_dir"], "samples", "{sample}", "quant_kallisto", "abundance.h5") shadow: "minimal" params: output_dir = os.path.join( config["output_dir"], "samples", "{sample}", "quant_kallisto"), fraglen = lambda wildcards: get_sample( 'mean', search_id='index', search_value=wildcards.sample), fragsd = lambda wildcards: get_sample( 'sd', search_id='index', search_value=wildcards.sample), directionality = lambda wildcards: get_sample( 'kallisto_directionality', search_id='index', search_value=wildcards.sample) threads: 8 log: stderr = os.path.join( config["log_dir"], "samples", "{sample}", "genome_quantification_kallisto.se.stderr.log") singularity: "docker://zavolab/kallisto:0.46.1-slim" shell: "(kallisto quant \ -i {input.index} \ -o {params.output_dir} \ --single \ -l {params.fraglen} \ -s {params.fragsd} \ --pseudobam \ -t {threads} \ {params.directionality}-stranded \ {input.reads} > {output.pseudoalignment};) \ 2> {log.stderr}"