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Snakefile
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configfile: "config.yaml"
sample = config["samples"]
reference = config["references"]
chr_list = list(range(1,30))
# Reduced paths
raw_data_path = config["resources"]["raw"]
fastp_output = config["fold_out"]["fastp"]
split_fastq = config["fold_out"]["split_fastq"]
alignment = config["fold_out"]["alignment"]
sorted_alignment = config["fold_out"]["sorted_alignment"]
dedup_alignment = config["fold_out"]["dedup_alignment"]
picard_metrics = config["fold_out"]["picard_metrics"]
reference_path = config["resources"]["reference"]
map_stats = config["fold_out"]["map_stats"]
# Tools
FASTP = config["tools"]["fastp"]
LOAD_PYTHON_374 = config["tools"]["load_python_374"]
FASTQ_SPLITTER = config["tools"]["fastq_splitter"]
LOAD_BWA = config["tools"]["load_bwa"]
LOAD_SAMTOOLS = config["tools"]["load_samtools"]
SAMBLASTER = config["tools"]["samblaster"]
SAMBAMBA = config["tools"]["sambamba"]
LOAD_PICARD_TOOLS = config["tools"]["load_picard_tools"]
LOAD_JAVA = config["tools"]["load_java_jdk"]
LOAD_R = config["tools"]["load_r"]
PICARD_TOOLS = config["tools"]["picard_tools"]
MQ_sam_flags_script = config["tools"]["sam_flags_script"]
rule all:
input:
stats_1=expand(sorted_alignment + "{reference}/{sample}/{sample}.stats",sample=sample,reference=reference),
stats_2=expand(dedup_alignment + "{reference}/{sample}/{sample}.stats",sample=sample,reference=reference),
index=expand(dedup_alignment + "{reference}/{sample}/{sample}.bam.bai",sample=sample,reference=reference),
metrics = expand(picard_metrics + "{reference}/{sample}/{sample}.alignment_summary_metrics",sample=sample,reference=reference),
stats = expand(picard_metrics + "{reference}/{sample}/{sample}_idxstats",sample=sample,reference=reference),
map_stats = expand(map_stats + "success/{reference}_{sample}_{chrm}.log",reference=reference,sample=sample,chrm=chr_list)
rule fastp:
input:
R1 = raw_data_path + "{sample}_R1.fastq.gz",
R2 = raw_data_path + "{sample}_R2.fastq.gz"
output:
R1_O = temp(fastp_output + "{sample}/{sample}_R1.fastq.gz"),
R2_O = temp(fastp_output + "{sample}/{sample}_R2.fastq.gz"),
R_HTML = fastp_output + "{sample}/{sample}_fastp.html",
R_JSON = fastp_output + "{sample}/{sample}_fastp.json"
params:
"-q 15 -u 40 -g"
shell:
FASTP + " -i {input.R1} -o {output.R1_O} -I {input.R2} -O {output.R2_O} -h {output.R_HTML} -j {output.R_JSON} {params} >/dev/null"
checkpoint split_fastq:
input:
R1 = fastp_output + "{sample}/{sample}_R1.fastq.gz",
R2 = fastp_output + "{sample}/{sample}_R2.fastq.gz"
output:
split_fastqs = temp(directory(split_fastq + "{sample}/"))
params:
prefix = split_fastq + "{sample}/{sample}_"
shell:
LOAD_PYTHON_374 + FASTQ_SPLITTER + " -o {params.prefix} {input.R1} {input.R2}"
rule alignment:
input:
R1 = split_fastq + "{sample}/{sample}_{flowcell}_{lane}_R1.fq.gz",
R2 = split_fastq + "{sample}/{sample}_{flowcell}_{lane}_R2.fq.gz",
ref = reference_path + "{reference}_ref.fa"
output:
BAM = temp(alignment + "{reference}/{sample}/{sample}_{flowcell}_{lane}.bam")
params:
rg="@RG\\tID:{flowcell}.{lane}\\tCN:TUM\\tLB:{sample}\\tPL:illumina\\tPU:{flowcell}:{lane}\\tSM:{sample}",
bwa_mem = "-M -t 12 -R"
shell:
LOAD_BWA + LOAD_SAMTOOLS +
"bwa mem {params.bwa_mem} '{params.rg}' {input.ref} {input.R1} {input.R2} | " + SAMBLASTER + " -M | samtools view -Sb - > {output.BAM}"
rule sambamba_sort:
input:
BAM = alignment + "{reference}/{sample}/{sample}_{flowcell}_{lane}.bam"
output:
sorted_BAM = temp(sorted_alignment + "{reference}/{sample}/{sample}_{flowcell}_{lane}.bam")
shell:
SAMBAMBA + " sort -t 6 --out {output.sorted_BAM} {input.BAM}"
def list_files(wildcards):
checkpoint_output = checkpoints.split_fastq.get(sample = wildcards.sample).output.split_fastqs
all_wildcards = glob_wildcards(os.path.join(checkpoint_output, "{sample}_{flowcell}_{lane}_R1.fq.gz"))
all_files = []
for sample, flowcell, lane in zip(all_wildcards.sample, all_wildcards.flowcell, all_wildcards.lane):
all_files.append(f"{sorted_alignment}" + "{reference}" + f"/{sample}/{sample}_{flowcell}_{lane}.bam")
return(all_files)
rule sambamba_merge:
input: list_files
output: temp(sorted_alignment + "{reference}/{sample}/{sample}.bam")
run:
if len(input) == 1:
shell("mv {input} {output} \n" + "mv {input}.bai {output}.bai")
else:
shell(SAMBAMBA + " merge -t 6 {output} {input}")
rule sambamba_flagstat_1:
input:
BAM = sorted_alignment + "{reference}/{sample}/{sample}.bam"
output:
Stats = sorted_alignment + "{reference}/{sample}/{sample}.stats"
params:
threads = "--nthreads 10"
shell:
SAMBAMBA + " flagstat -t 3 {params.threads} {input.BAM} > {output.Stats}"
rule mark_duplicates:
input:
sorted_alignment + "{reference}/{sample}/{sample}.bam"
output:
bam= dedup_alignment + "{reference}/{sample}/{sample}.bam",
metrics= dedup_alignment +"{reference}/{sample}/{sample}.metrics.txt"
shell:
LOAD_PICARD_TOOLS +
LOAD_JAVA +
PICARD_TOOLS + " MarkDuplicates I={input} O={output.bam} M={output.metrics}"
rule build_index:
input:
dedup_alignment + "{reference}/{sample}/{sample}.bam"
output:
dedup_alignment + "{reference}/{sample}/{sample}.bam.bai"
shell:
LOAD_PICARD_TOOLS +
LOAD_JAVA +
PICARD_TOOLS + " BuildBamIndex I={input} O={output}"
rule sambamba_flagstat_2:
input:
BAM = dedup_alignment + "{reference}/{sample}/{sample}.bam"
output:
Stats = dedup_alignment + "{reference}/{sample}/{sample}.stats"
params:
threads = "--nthreads 10"
shell:
SAMBAMBA + " flagstat -t 3 {params.threads} {input.BAM} > {output.Stats}"
rule picard_metrics:
input:
BAM = dedup_alignment + "{reference}/{sample}/{sample}.bam",
ref = reference_path + "{reference}_ref.fa"
output:
metrics = picard_metrics + "{reference}/{sample}/{sample}.alignment_summary_metrics"
params:
prefix = picard_metrics + "{reference}/{sample}/{sample}"
shell:
LOAD_PICARD_TOOLS +
LOAD_JAVA +
LOAD_R +
PICARD_TOOLS + " CollectMultipleMetrics I={input.BAM} O={params.prefix} R={input.ref}"
rule samtools_idxstats:
input:
BAM = dedup_alignment + "{reference}/{sample}/{sample}.bam"
output:
stats = picard_metrics + "{reference}/{sample}/{sample}_idxstats"
shell:
LOAD_SAMTOOLS + " samtools idxstats {input.BAM} > {output.stats}"
rule sam_flags:
input:
dedup_alignment + "{reference}/{sample}/{sample}.bam"
params:
map_stats + "MQ_sam_flag_reads.txt"
output:
map_stats + "success/{reference}_{sample}_{chrm}.log"
shell:
LOAD_SAMTOOLS + MQ_sam_flags_script + "{input} {output} {params} {wildcards.reference} {wildcards.sample} {wildcards.chrm}"