Single end sequencing analysis subpipeline

process_data/single_end.snakefile

+
+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}"
+
+		
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