diff --git a/process_data/single_end.snakefile b/process_data/single_end.snakefile
new file mode 100644
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+
+rule fastqc:
+ ''' A quality control tool for high throughput sequence data. '''
+ input:
+ reads = os.path.join(get_fq_1("{sample}"))
+ output:
+ outdir = directory(os.path.join(config["output_dir"], "single_end", "{sample}", "fastqc"))
+ singularity:
+ "docker://zavolab/fastqc:0.11.8"
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "fastqc.log")
+ shell:
+ "(mkdir -p {output.outdir}; \
+ fastqc \
+ --outdir {output.outdir} \
+ {input.reads}) &> {log}"
+
+
+rule htseq_qa:
+ ''' Assess the technical quality of a run. '''
+ input:
+ reads = os.path.join(get_fq_1("{sample}"))
+ output:
+ qual_pdf = os.path.join(config["output_dir"], "single_end", "{sample}", "htseq_quality.pdf")
+ singularity:
+ "docker://zavolab/python_htseq:3.6.5_0.10.0"
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "htseq_qa.log")
+ shell:
+ "(htseq-qa \
+ -t fastq \
+ -o {output.qual_pdf} \
+ {input.reads} ) &> {log}"
+
+
+rule remove_adapters_cutadapt:
+ ''' Remove adapters '''
+ input:
+ reads = os.path.join(get_fq_1("{sample}"))
+ output:
+ reads = os.path.join(config["output_dir"], "single_end", "{sample}", "{sample}.remove_adapters.fastq.gz")
+ params:
+ adapters_3 = lambda wildcards:
+ sample_table.loc[wildcards.sample, 'fq1_3p']
+ adapters_5 = lambda wildcards:
+ sample_table.loc[wildcards.sample, 'fq1_5p']
+
+ singularity:
+ "docker://zavolab/cutadapt:1.16"
+ threads: 8
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "remove_adapters_cutadapt.log")
+ shell:
+ "cutadapt \
+ -e 0.1 \
+ -O 1 \
+ -j {threads} \
+ -m 10 \
+ -n 3 \
+ -a {params.adapters_3} \
+ -g {params.adapters_5} \
+ -o {output.reads} \
+ {input.reads}) &> {log}"
+
+
+rule remove_polya_cutadapt:
+ ''' Remove ployA tails'''
+ input:
+ reads = os.path.join(get_fq_1("{sample}"))
+ output:
+ reads = os.path.join(config["output_dir"], "single_end", "{sample}", "{sample}.remove_polya.fastq.gz")
+ params:
+ polya_3 =
+ adapters_3 = lambda wildcards:
+ sample_table.loc[wildcards.sample, "fq1_polya"]
+ singularity:
+ "docker://zavolab/cutadapt:1.16"
+ threads: 8
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "remove_polya_cutadapt.log")
+ shell:
+ "(cutadapt \
+ --match-read-wildcards \
+ -j {threads} \
+ -n 2 \
+ -e 0.1 \
+ -O 1 \
+ -q 6 \
+ -m 10 \
+ -a {params.polya_3} \
+ -o {output.reads} \
+ {input.reads}) &> {log}"
+
+
+rule create_index_star:
+ ''' Create index using STAR. '''
+ input:
+ genome = sample_table.loc["{sample}", "genome"],
+ gtf = sample_table.loc["{sample}", "gtf"]
+ output:
+ chromosome_info = os.path.join(
+ config["star_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ sample_table.loc["{sample}", "index_size"],
+ "STAR_index","chrNameLength.txt"),
+ chromosome_names = os.path.join(
+ config["star_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ sample_table.loc["{sample}", "index_size"],
+ "STAR_index", "chrName.txt")
+ params:
+ output_dir = lambda wildcards:
+ os.path.join(
+ config["star_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ sample_table.loc[wildcards.sample, "index_size"],
+ "STAR_index"),
+ outFileNamePrefix = lambda wildcards:
+ os.path.join(
+ config["star_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ sample_table.loc[wildcards.sample, "index_size"],
+ "STAR_index/STAR_"),
+ sjdbOverhang = lambda wildcards:
+ sample_table.loc[wildcards.sample, "index_size"]
+ singularity:
+ "docker://zavolab/star:2.6.0a"
+ threads: 12
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "create_index_star.log")
+ shell:
+ "(mkdir -p {params.output_dir}; \
+ chmod -R 777 {params.output_dir}; \
+ STAR \
+ --runMode genomeGenerate \
+ --sjdbOverhang {params.sjdbOverhang} \
+ --genomeDir {params.output_dir} \
+ --genomeFastaFiles {input.genome} \
+ --runThreadN {threads} \
+ --outFileNamePrefix {params.outFileNamePrefix} \
+ --sjdbGTFfile {input.gtf}) &> {log}"
+
+
+rule map_genome_star:
+ ''' Map to genome using STAR. '''
+ input:
+ index = os.path.join(
+ config["star_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ sample_table.loc["{sample}", "index_size"],
+ "STAR_index","chrNameLength.txt"),
+ reads = os.path.join(config["output_dir"], "single_end", "{sample}", "{sample}.remove_polya.fastq.gz")
+ output:
+ bam = os.path.join(config["output_dir"], "single_end",
+ "{sample}",
+ "map_genome",
+ "{sample}_Aligned.sortedByCoord.out.bam"),
+ logfile = os.path.join(config["output_dir"], "single_end",
+ "{sample}",
+ "map_genome",
+ "{sample}_Log.final.out")
+ params:
+ sample_id = "{sample}"
+ index = lambda wildcards:
+ os.path.join(
+ config["star_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ sample_table.loc[wildcards.sample, "index_size"],
+ "STAR_index"),
+ outFileNamePrefix = lambda wildcards:
+ os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}", "map_genome", "{sample}_"),
+ multimappers = lambda wildcards:
+ sample_table.loc[wildcards.sample, "multimappers"],
+ soft_clip = lambda wildcards:
+ sample_table.loc[wildcards.sample, "soft_clip"],
+ pass_mode = lambda wildcards:
+ sample_table.loc[wildcards.sample, "pass_mode"],
+ singularity:
+ "docker://zavolab/star:2.6.0a"
+ threads: 12
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "map_genome_star.log")
+ shell:
+ "(STAR \
+ --runMode alignReads \
+ -- twopassMode {params.pass_mode} \
+ --runThreadN {threads} \
+ --genomeDir {params.index} \
+ --readFilesIn {input.reads} \
+ --readFilesCommand zcat \
+ --outSAMunmapped None \
+ --outFilterMultimapNmax {params.multimappers} \
+ --outFilterMultimapScoreRange 1 \
+ --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:rcrunch SM:{params.sample_id} \
+ --alignEndsType {params.soft_clip}} > {output.bam};) &> {log}"
+
+
+rule index_genomic_alignment_samtools:
+ '''Index genome bamfile using samtools.'''
+ input:
+ bam = os.path.join(config["output_dir"],
+ "single_end",
+ "{sample}",
+ "map_genome",
+ "{sample}_Aligned.sortedByCoord.out.bam")
+ output:
+ bai = os.path.join(config["output_dir"],
+ "single_end",
+ "{sample}",
+ "map_genome",
+ "{sample}_Aligned.sortedByCoord.out.bam.bai")
+ singularity:
+ "docker://zavolab/samtools:1.8"
+ threads: 1
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "index_genomic_alignment_samtools.log")
+ shell:
+ "(samtools index {input.bam} {output.bai};) &> {log}"
+
+
+rule create_index_salmon:
+ ''' Create index for Salmon quantification. '''
+ input:
+ transcriptome = sample_table.loc[wildcards.sample, 'tr_fasta_filtered']
+ output:
+ index = os.path.join(
+ config["salmon_indexes"],
+ sample_table["{sample}", 'organism'],
+ "salmon.idx")
+ params:
+ kmerLen = lambda wildcards:
+ sample_table.loc[wildcards.sample, 'kmer']
+ singularity:
+ "docker://zavolab/salmon:0.11.0"
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "create_index_salmon.log")
+ threads: 8
+ shell:
+ "(salmon index \
+ --t {input.transcriptome} \
+ --i {output.index} \
+ --k {params.kmerLen} \
+ --threads {threads}) &> {log}"
+
+
+rule quantification_salmon:
+ ''' Quantification at transcript and gene level using Salmon. '''
+ input:
+ reads = os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}",
+ "{sample}.remove_polya.fastq.gz"),
+ index = os.path.join(
+ config["salmon_indexes"],
+ sample_table["{sample}", 'organism'],
+ "salmon.idx"),
+ gtf = sample_table.loc["{sample}", "gtf_filtered"]
+ output:
+ gn_estimates = os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}",
+ "salmon_quant",
+ "quant.genes.sf"),
+ tr_estimates = os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}",
+ "salmon_quant",
+ "quant.sf")
+ params:
+ output_dir = os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}",
+ "salmon_quant"),
+ libType = lambda wildcards:
+ sample_table.loc[wildcards.sample, "libtype"]
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "quantification_salmon.log")
+ threads: 12
+ conda:
+ "envs/salmon.yaml"
+ shell:
+ "(salmon quant \
+ --libType {params.libType} \
+ --seqBias \
+ --validateMappings \
+ --threads {threads} \
+ --writeUnmappedNames \
+ --index {input.index} \
+ --geneMap {input.gtf} \
+ --unmatedReads {input.reads} \
+ -o {params.output_dir}) &> {log}"
+
+
+
+rule create_index_kallisto:
+ ''' Create index for running Kallisto. '''
+ input:
+ transcriptome = sample_table.loc["{sample}", 'tr_fasta_filtered']
+ output:
+ index = os.path.join(
+ config["kallisto_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ "kallisto.idx")
+ params:
+ output_dir = lambda wildcards:
+ os.path.join(
+ config["kallisto_indexes"],
+ sample_table.loc["{sample}", "organism"]),
+ log:
+ os.path.join(config["local_log"], "single_end", "{sample}", "create_index_kallisto.log")
+ singularity:
+ "docker://zavolab/kallisto:0.9"
+ shell:
+ "(mkdir -p {params.output_dir}; \
+ chmod -R 777 {params.output_dir}; \
+ kallisto index -i {output.index} {input.transcriptome}) &> {log}"
+
+rule genome_quantification_kallisto:
+ ''' Quantification at transcript and gene level using Kallisto. '''
+ input:
+ reads = os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}",
+ "{sample}.remove_polya.fastq.gz"),
+ index = os.path.join(
+ config["kallisto_indexes"],
+ sample_table.loc["{sample}", "organism"],
+ "kallisto.idx")
+ output:
+ pseudoalignment = os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}",
+ "{sample}.kallisto.pseudo.sam")
+ params:
+ output_dir = lambda wildcards:
+ os.path.join(
+ config["output_dir"],
+ "single_end",
+ "{sample}",
+ "quant_kallisto"),
+ fraglen = lambda wildcards: sample_table.loc[wildcards.sample, 'mean'],
+ fragsd = lambda wildcards: sample_table.loc[wildcards.sample, 'sd'],
+ directionality = lambda wildcards: sample_table.loc[wildcards.sample, 'kallisto_directionality']
+ threads: 8
+ log:
+ os.path.join(config["local_log"],"kallisto_align_{sample}.log")
+ singularity:
+ "docker://zavolab/kallisto:0.9"
+ shell:
+ "(kallisto quant \
+ -i {input.index} \
+ -o {params.output_dir} \
+ --single \
+ -l {params.fraglen} \
+ -s {params.fragsd} \
+ --pseudobam \
+ --{params.directionality}-stranded \
+ {input.reads} > {output.pseudoalignment}) &> {log}"
+
+
\ No newline at end of file