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snakefile
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import math
import subprocess
from snakemake.utils import R
from snakemake.utils import report
from os.path import split
#from pytools.persistent_dict import PersistentDict
#storage = PersistentDict("mystorage")
########################################
# DEFINITION OF DEFAULT INPUT SETTINGS
#
DEF_GENOME = "default_genome.fa"
DEF_ANALYSIS_TYPE = "classic"
DEF_DATA_TYPE = "paired"
DEF_ORGANISM_TYPE = "mammals"
DEF_TARGET = "report_all.html"
DEF_INPUT_DIR = "."
DEF_OUTPUT_DIR = "."
DEF_ANNOTATION = "default_annotation.gff3"
DEF_GENOME_INDEX = ""
DEF_SAM_HEADER = "default_SAM_header"
DEF_SE_DATA = "data"
DEF_PE1_DATA = "data1"
DEF_PE2_DATA = "data2"
DEF_ADAPTERS = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/adapters_merge.fa"
DEF_STAR_PARAMS = ""
DEF_STAR_ALIGN_INTRON_MAX = "0" # default value from STAR manual
DEF_STAR_ALIGN_MATES_GAP = "0" # default value from STAR manual
########################################
# DEFINITION OF INPUT SETTINGS NAMES
#
CONF_GENOME_TERM = "genome"
CONF_ANALYSIS_TYPE_TERM = "analysis_type"
CONF_DATA_TYPE_TERM = "data_type"
CONF_ORGANISM_TYPE_TERM = "organism_type"
CONF_TARGET_TERM = "target"
CONF_OUTPUT_DIR_TERM = "output_dir"
CONF_INPUT_DIR_TERM = "input_dir"
CONF_ANNOTATION_TERM = "annotation"
CONF_GENOME_INDEX_TERM = "genome_index"
CONF_SAM_HEADER_TERM = "SAM_header"
CONF_SE_DATA_TERM = "data"
CONF_PE1_DATA_TERM = "data1"
CONF_PE2_DATA_TERM = "data2"
CONF_STAR_PARAMS_TERM = "STAR_params"
CONF_STAR_ALIGN_INTRON_MAX_TERM = "STAR_alignIntronMax"
CONF_STAR_ALIGN_MATES_GAP_TERM = "STAR_alignMatesGapMax"
CONF_ADAPTERS_TERM = "adapters"
###########################################
# DEFINITION OF INPUT SETTINGS VARIABLES
#
#.split() is used if it's neccesary to use more than one option
GENOME = config[CONF_GENOME_TERM] if (CONF_GENOME_TERM in config) else DEF_GENOME
ANALYSIS_TYPE = config[CONF_ANALYSIS_TYPE_TERM].split() if (CONF_ANALYSIS_TYPE_TERM in config) else DEF_ANALYSIS_TYPE
DATA_TYPE = config[CONF_DATA_TYPE_TERM].split() if (CONF_DATA_TYPE_TERM in config) else DEF_DATA_TYPE
ORGANISM_TYPE = config[CONF_ORGANISM_TYPE_TERM] if (CONF_ORGANISM_TYPE_TERM in config) else DEF_ORGANISM_TYPE
TARGET = config[CONF_TARGET_TERM] if (CONF_TARGET_TERM in config) else DEF_TARGET
INPUT_DIR = config[CONF_INPUT_DIR_TERM] if (CONF_INPUT_DIR_TERM in config) else DEF_INPUT_DIR
OUTPUT_DIR = config[CONF_OUTPUT_DIR_TERM] if (CONF_OUTPUT_DIR_TERM in config) else DEF_OUTPUT_DIR
GENOME_INDEX = config[CONF_GENOME_INDEX_TERM] if (CONF_GENOME_INDEX_TERM in config) else DEF_GENOME_INDEX
SAM_HEADER = config[CONF_SAM_HEADER_TERM] if (CONF_SAM_HEADER_TERM in config) else DEF_SAM_HEADER
DATA1 = config[CONF_PE1_DATA_TERM] if (CONF_PE1_DATA_TERM in config) else DEF_PE1_DATA
DATA2 = config[CONF_PE2_DATA_TERM] if (CONF_PE2_DATA_TERM in config) else DEF_PE2_DATA
ADAPTERS = config[CONF_ADAPTERS_TERM] if (CONF_ADAPTERS_TERM in config) else DEF_ADAPTERS
if (config[CONF_DATA_TYPE_TERM] == "single") :
DATA = config[CONF_SE_DATA_TERM] if (CONF_SE_DATA_TERM in config) else DEF_SE_DATA
SAMPLES = DATA.strip(".gz").strip(".fq|.fastq")
else:
DATA = DATA1.split()+DATA2.split()
SAMPLES = DATA1.strip(".gz").strip(".fq|.fastq").split()+DATA2.strip(".gz").strip(".fq|.fastq").split()
#if not os.path.exists(OUTPUT_DIR):
# os.makedirs(OUTPUT_DIR)
# GENOME or ORGANISM dependent settings
#
if GENOME == "arabidopsis":
RAW_GENOME_FILE = ""
RAW_ANNOTATION = config[CONF_ANNOTATION_TERM] if (CONF_ANNOTATION_TERM in config) else ""
ORGANISM_TYPE = "plants"
STAR_INTRON_MAX = "10000" # 10k
STAR_MATES_GAP = "10000"
elif GENOME == "human":
RAW_GENOME_FILE = ""
RAW_ANNOTATION = config[CONF_ANNOTATION_TERM] if (CONF_ANNOTATION_TERM in config) else ""
ORGANISM_TYPE = "mammals"
STAR_INTRON_MAX = "100000" # 100k
STAR_MATES_GAP = "100000"
elif GENOME == "sifilis":
RAW_GENOME_FILE = ""
RAW_ANNOTATION = config[CONF_ANNOTATION_TERM] if (CONF_ANNOTATION_TERM in config) else ""
ORGANISM_TYPE = "bacteria"
STAR_INTRON_MAX = "1" # indicates no splicing
STAR_MATES_GAP = "1000"
else:
### TODO: check for existence of genome_file and annotation_file because in this case they are both needed
RAW_GENOME_FILE = GENOME
RAW_ANNOTATION = config[CONF_ANNOTATION_TERM] if (CONF_ANNOTATION_TERM in config) else DEF_ANNOTATION
ORGANISM_TYPE = "undefined"
STAR_INTRON_MAX = DEF_STAR_ALIGN_INTRON_MAX
STAR_MATES_GAP = DEF_STAR_ALIGN_MATES_GAP
GENOME_FILE = split(RAW_GENOME_FILE)[1].strip(".gz")
ANNOTATION = split(RAW_ANNOTATION)[1].strip(".gz")
STAR_INTRON_MAX = config[CONF_STAR_ALIGN_INTRON_MAX_TERM] if (CONF_STAR_ALIGN_INTRON_MAX_TERM in config) else STAR_INTRON_MAX
STAR_MATES_GAP = config[CONF_STAR_ALIGN_MATES_GAP_TERM] if (CONF_STAR_ALIGN_MATES_GAP_TERM in config) else STAR_MATES_GAP
STAR_PARAMS = config[CONF_STAR_PARAMS_TERM] if (CONF_STAR_PARAMS_TERM in config) else DEF_STAR_PARAMS
STRANDS = ["forward","reverse"]
####################################
# DEFINITION OF TOOLS
#
WRAPPERS_PATH = "file:/mnt/nfs/shared/999993-Bioda/scripts/martin/test/wrappers/"
FASTQC = "fastqc"
ADAPTERS = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/adapters_merge.fa"
REAPER_SRC = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/reaper-15-065/src"
TRIMMOMATIC = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/trimmomatic-master/classes/trimmomatic.jar"
STAR = "STAR"
SAMTOOLS = "samtools"
RSEM_PATH = ""
BBMAP = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/bbmap"
FEATURE_COUNTS = "featureCounts"
UCSC_SCRIPTS = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools"
GTF_TO_BED12 = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/gtf2bed12.py"
PICARD = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/picard-tools-1.119"
PICARD_JAR = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/picard-tools-1.119/picard-1.119.jar"
PRESEQ = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/preseq_v2.0.2/preseq" #"/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/preseq_v2.0.1/preseq"
RSEQC = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/RSeQC-2.6.4/build/scripts-2.7"
DUPRADAR = "/mnt/nfs/shared/999993-Bioda/scripts/martin/test/Tools/dupRadar.R"
PHRED_FILTER = 5 # Trim the 3' end of read if four consequent bases have average base quality smaller than this value
LEN_FILTER = 20 # Filter sequences shorter than this value after quality trimming
RD_LENGTH = 75 # Trim all sequences to this maximal length - depends on experiment design and I like to trim them to specified length
####################################
# DEFINITION OF FINAL RULES
#
rule all:
input: expand("{dir}/report_all_test.html", dir=OUTPUT_DIR)
rule final_report:
input: expand("{dir}/1st_qc/{data}_fastqc.html", dir=OUTPUT_DIR, data=SAMPLES),
expand("{dir}/minion/{data}.minion.compare",data=SAMPLES,dir=OUTPUT_DIR) if config[CONF_DATA_TYPE_TERM] == "single" else expand("{dir}/minion/{data}.minion.compare",data=DATA2.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR),
expand("{dir}/trimming",dir=OUTPUT_DIR),
expand("{dir}/2nd_qc",dir=OUTPUT_DIR),
expand("{dir}/aligned/Log.final.out",dir=OUTPUT_DIR),
expand("{dir}/RSEM/RSEM_calc_expr.pdf",dir=OUTPUT_DIR),
expand("{dir}/FeatureCounts/feature_counts.txt",dir=OUTPUT_DIR),
expand("{dir}/mapped_QC/{data}.rRNA.intervalListBody.txt", dir=OUTPUT_DIR, data=ANNOTATION.rstrip(".gtf")),
expand("{dir}/strandness.txt", dir=OUTPUT_DIR),
expand("{dir}/preseq/Aligned.sortedByCoord.estimates.txt",dir=OUTPUT_DIR),
expand("{dir}/mapped_QC/featureCounts.quantSeq.rev.biotype_counts.txt",dir=OUTPUT_DIR),
expand("{dir}/mapped_QC/Aligned.sortedByCoord.dupRadar_dupMatrix.txt",dir=OUTPUT_DIR),
expand("{dir}/RSeQC/Aligned.sortedByCoord.RPKM_saturation.rawCount.xls",dir=OUTPUT_DIR),
expand("{dir}/RSeQC/Aligned.sortedByCoord.read_duplication.seq.DupRate.xls",dir=OUTPUT_DIR),
expand("{dir}/RSeQC/Aligned.sortedByCoord.infer_experiment.txt",dir=OUTPUT_DIR),
expand("{dir}/RSeQC/Aligned.sortedByCoord.bam_stat.txt",dir=OUTPUT_DIR),
expand("{dir}/RSeQC/Aligned.sortedByCoord.junction_annotation.junction.xls",dir=OUTPUT_DIR),
expand("{dir}/RSeQC/Aligned.sortedByCoord.junction_saturation.junctionSaturation_plot.r",dir=OUTPUT_DIR)
output: expand("{dir}/report_all_test.html", dir=OUTPUT_DIR)
shell: "touch {output}"
rule step_zero_report:
input: expand("{dir}/1st_qc/{data}_fastqc.html", dir=OUTPUT_DIR, data=SAMPLES),
expand("{dir}/minion/{data}.minion.compare",data=SAMPLES,dir=OUTPUT_DIR) if config[CONF_DATA_TYPE_TERM] == "single" else expand("{dir}/minion/{data}.minion.compare",data=DATA2.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR)
output: "report_step_zero.html"
shell: "touch {output}"
####################################
# DEFINITION OF ZERO-STEP RULES (COPY OF DATA, FIRST QC, ADAPTERS CHECK)
#
### TODO: add check between genome and annotation (e.g., chromosome names)
if config[CONF_DATA_TYPE_TERM] == "single":
rule copy_data:
input: reads = expand("{dir}/{data}",data=DATA,dir=INPUT_DIR),
genome = expand("{data}", data=RAW_GENOME_FILE),
ref = expand("{data}", data=RAW_ANNOTATION)
output: reads = expand("{dir}/data/reads/{data}",data=DATA,dir=OUTPUT_DIR),
genome = expand("{dir}/data/genome/{data}", data=GENOME_FILE, dir=OUTPUT_DIR),
ref = expand("{dir}/data/reference/{data}", data=ANNOTATION, dir=OUTPUT_DIR)
params: genome = expand("{dir}/data/genome/{data}", data=split(RAW_GENOME_FILE)[1], dir=OUTPUT_DIR),
ref = expand("{dir}/data/reference/{data}", data=split(RAW_ANNOTATION)[1], dir=OUTPUT_DIR)
run:
shell("""
ln {input.reads} {output.reads}
cp {input.genome} {params.genome} && unpigz {params.genome} || echo $? > /dev/null
cp {input.ref} {params.ref} && unpigz {params.ref} || echo $? > /dev/null
""")
else:
rule copy_data:
input: reads1 = expand("{dir}/{data}",data=DATA1,dir=INPUT_DIR),
reads2 = expand("{dir}/{data}",data=DATA2,dir=INPUT_DIR),
genome = expand("{data}", data=RAW_GENOME_FILE),
ref = expand("{data}", data=RAW_ANNOTATION)
output: reads1 = expand("{dir}/data/reads/{data1}", data1=DATA1, dir=OUTPUT_DIR),
reads2 = expand("{dir}/data/reads/{data2}", data2=DATA2, dir=OUTPUT_DIR),
genome = expand("{dir}/data/genome/{data}", data=GENOME_FILE, dir=OUTPUT_DIR),
ref = expand("{dir}/data/reference/{data}", data=ANNOTATION, dir=OUTPUT_DIR)
params: genome = expand("{dir}/data/genome/{data}", data=split(RAW_GENOME_FILE)[1], dir=OUTPUT_DIR),
ref = expand("{dir}/data/reference/{data}", data=split(RAW_ANNOTATION)[1], dir=OUTPUT_DIR)
run:
shell("""
ln {input.reads1} {output.reads1}
ln {input.reads2} {output.reads2}
cp {input.genome} {params.genome} && unpigz {params.genome} || echo $? > /dev/null
cp {input.ref} {params.ref} && unpigz {params.ref} || echo $? > /dev/null
""")
rule first_qc:
input: reads = expand("{dir}/data/reads/{data}",data=DATA,dir=OUTPUT_DIR) if config[CONF_DATA_TYPE_TERM] == "single" else expand(["{dir}/data/reads/{data1}","{dir}/data/reads/{data2}"],data1=DATA1,data2=DATA2,dir=OUTPUT_DIR)
output: html = expand("{dir}/1st_qc/{data}_fastqc.html", dir=OUTPUT_DIR, data=SAMPLES)
log: run = expand("{dir}/1st_qc/run_stats.log", dir=OUTPUT_DIR),
report = expand("{dir}/report.html", dir=OUTPUT_DIR)
params: extra = "--noextract --format fastq --nogroup",
prefix = expand("{dir}/1st_qc", dir=OUTPUT_DIR)
threads: 4
run:
shell("""
{FASTQC} -o {params.prefix}/ {params.extra} --threads {threads} {input.reads} > {log.run} 2>&1
""")
### REPORT PART ###
report("""
=======================
The title of the report
=======================
Write your report here, explaining your results. Don't fear to use math
it will be rendered correctly in any browser using MathJAX,
e.g. inline :math:`\sum_{{j \in E}} t_j \leq I`,
or even properly separated:
.. math::
|cq_{{0ctrl}}^i - cq_{{nt}}^i| > 0.5
Include your files using their keyword name and an underscore: reads_.
Original html report from FastQC is here: html_.
Access your global and local variables like within shell commands, e.g. INPUT_DIR={INPUT_DIR}.
""", *log.report, **input, **output)
if config[CONF_DATA_TYPE_TERM] == "single":
rule check_adapters:
input: reads = expand("{dir}/data/reads/{data}",data=DATA,dir=OUTPUT_DIR)
output: minion = expand("{dir}/minion/{data}.minion.fa",data=DATA.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR)
log: run = expand("{dir}/minion/{data}.log",data=DATA.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR),
report = expand("{dir}/report.html", dir=OUTPUT_DIR)
params: minion = "-show 3 -write-fasta"
run:
shell("""
{REAPER_SRC}/minion search-adapter -i {input.reads} {params.minion} {output.minion} > {log.run} 2>&1
""")
### REPORT PART ###
report("""
=====================
Minion part of report
=====================
Original output is minion_.
""", *log.report, **input, **output)
else:
rule check_adapters:
input: reads1 = expand("{dir}/data/reads/{data}",data=DATA1,dir=OUTPUT_DIR),
reads2 = expand("{dir}/data/reads/{data}",data=DATA2,dir=OUTPUT_DIR)
output: minion1 = expand("{dir}/minion/{data}.minion.fa",data=DATA1.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR),
minion2 = expand("{dir}/minion/{data}.minion.fa",data=DATA2.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR)
log: run1 = expand("{dir}/minion/{data}.log",data=DATA1.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR),
run2 = expand("{dir}/minion/{data}.log",data=DATA2.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR),
report = expand("{dir}/report.html", dir=OUTPUT_DIR)
threads: 1
params: minion = "-show 3 -write-fasta"
run:
shell("""
{REAPER_SRC}/minion search-adapter -i {input.reads1} {params.minion} {output.minion1} > {log.run1} 2>&1
{REAPER_SRC}/minion search-adapter -i {input.reads2} {params.minion} {output.minion2} > {log.run2} 2>&1
""")
### REPORT PART ###
report("""
=====================
Minion part of report
=====================
Original output is minion1_ and minion2_.
""", *log.report, **input, **output)
if config[CONF_DATA_TYPE_TERM] == "single":
rule check_adapters_swan:
input: minion = expand("{dir}/minion/{data}.minion.fa",data=DATA.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR)
output: swan = expand("{dir}/minion/{data}.minion.compare",data=DATA.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR)
shell: """
{REAPER_SRC}/swan -r {ADAPTERS} -q {input.minion} > {output.swan} 2>&1
"""
else:
rule check_adapters_swan:
input: minion1 = expand("{dir}/minion/{data}.minion.fa",data=DATA1.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR),
minion2 = expand("{dir}/minion/{data}.minion.fa",data=DATA2.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR)
output: swan1 = expand("{dir}/minion/{data}.minion.compare",data=DATA1.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR),
swan2 = expand("{dir}/minion/{data}.minion.compare",data=DATA2.strip(".gz").strip(".fq|.fastq"),dir=OUTPUT_DIR)
threads: 1
shell: """
{REAPER_SRC}/swan -r {ADAPTERS} -q {input.minion1} > {output.swan1} 2>&1
{REAPER_SRC}/swan -r {ADAPTERS} -q {input.minion2} > {output.swan2} 2>&1
"""
#########################################################
# DEFINITION OF FIRST-STEP RULES (TRIMMING, SECOND QC)
#
if config[CONF_ANALYSIS_TYPE_TERM] == "quant":
if config[CONF_DATA_TYPE_TERM] == "single":
rule trimming_by_bbmap:
input: reads = expand("{dir}/data/reads/{data}",data=DATA,dir=OUTPUT_DIR),
adapters = expand("{data}",data=ADAPTERS)
output: dir = expand("{dir}/trimming",dir=OUTPUT_DIR),
clean = expand(["{dir}/trimming/{data}.clean.fastq.gz"],data=DATA.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
waste = expand(["{dir}/trimming/{data}.trimmed.fastq.gz"],data=DATA.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR)
log: run = expand("{dir}/trimming/run_stats.log",dir=OUTPUT_DIR),
stats = expand("{dir}/trimming/cont_stats.log",dir=OUTPUT_DIR),
rpkm = expand("{dir}/trimming/rpkm_stats.log",dir=OUTPUT_DIR),
kmers = expand("{dir}/trimming/kmers_stats.log",dir=OUTPUT_DIR)
threads: 4
params: extra = "literal=GGGGGGGGG,AAAAAAAAA k=13 useshortkmers=t mink=5 qtrim=rl trimq=10 minlength=20" # -Xmx10g for JVM is possible
shell: """
{BBMAP}/bbduk.sh in={input.reads} out={output.clean} outm={output.waste} ref={input.adapters} stats={log.stats} rpkm={log.rpkm} dump={log.kmers} threads={threads} ktrim=r {params.extra} > {log.run} 2>&1
"""
else:
rule trimming_by_bbmap:
input: r1 = expand("{dir}/data/reads/{data1}",data1=DATA1,dir=OUTPUT_DIR),
r2 = expand("{dir}/data/reads/{data2}",data2=DATA2,dir=OUTPUT_DIR),
adapters = expand("{data}",data=ADAPTERS)
output: dir = expand("{dir}/trimming",dir=OUTPUT_DIR),
c1 = expand(["{dir}/trimming/{data1}.clean.fastq.gz"],data1=DATA1.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
c2 = expand(["{dir}/trimming/{data2}.clean.fastq.gz"],data2=DATA2.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
w1 = expand(["{dir}/trimming/{data1}.trimmed.fastq.gz"],data1=DATA1.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
w2 = expand(["{dir}/trimming/{data2}.trimmed.fastq.gz"],data2=DATA2.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
sing = expand(["{dir}/trimming/{data}.singleton.fastq.gz"],data=DATA1.strip(".fastq.gz|.fq.gz").strip("_1|_R1|_L1|1"),dir=OUTPUT_DIR)
log: run = expand("{dir}/trimming/run_stats.log",dir=OUTPUT_DIR),
stats = expand("{dir}/trimming/cont_stats.log",dir=OUTPUT_DIR),
rpkm = expand("{dir}/trimming/rpkm_stats.log",dir=OUTPUT_DIR),
kmers = expand("{dir}/trimming/kmers_stats.log",dir=OUTPUT_DIR)
threads: 4
params: extra = "literal=GGGGGGGGG,AAAAAAAAA k=13 useshortkmers=t mink=5 qtrim=rl trimq=10 minlength=20"
shell: """
{BBMAP}/bbduk.sh in={input.r1} in2={input.r2} out={output.c1} out2={output.c2} outm={output.w1} outm2={output.w2} outs={output.sing} ref={input.adapters} stats={log.stats} rpkm={log.rpkm} dump={log.kmers} threads={threads} ktrim=r {params.extra} > {log.run} 2>&1
"""
elif config[CONF_ANALYSIS_TYPE_TERM] == "classic":
if config[CONF_DATA_TYPE_TERM] == "single":
rule trimming_by_Trimmomatic:
input: reads = expand("{dir}/data/reads/{data}",data=DATA,dir=OUTPUT_DIR)
output: dir = expand("{dir}/trimming",dir=OUTPUT_DIR),
clean = expand(["{dir}/trimming/{data}.clean.fastq.gz"],data=DATA.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR)
log: run = expand("{dir}/trimming/run_stats.log",dir=OUTPUT_DIR)
threads: 4
resources: mem = 5
params: phred = "-phred33",
leading = "3",
trailing = "3",
crop = expand("{par}",par=RD_LENGTH),
minlen = expand("{par}",par=LEN_FILTER),
slid_w_1 = "4",
slid_w_2 = expand("{par}",par=PHRED_FILTER)
wrapper:
WRAPPERS_PATH+"wrapper_trim_se"
# shell: """
# java -Xmx{resources.mem}g -jar {TRIMMOMATIC} SE -threads {threads} {params.phred} {input.reads} {output.clean} LEADING:{params.leading} TRAILING:{params.trailing}
# CROP:{params.crop} SLIDINGWINDOW:{params.slid_w_1}:{params.slid_w_2} MINLEN:{params.minlen} > {log.run} 2>&1
# """
else:
rule trimming_by_Trimmomatic:
input: r1 = expand("{dir}/data/reads/{data1}",data1=DATA1,dir=OUTPUT_DIR),
r2 = expand("{dir}/data/reads/{data2}",data2=DATA2,dir=OUTPUT_DIR)
output: dir = expand("{dir}/trimming",dir=OUTPUT_DIR),
r1p = expand(["{dir}/trimming/{data1}.clean.fastq.gz"],data1=DATA1.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
r1u = expand(["{dir}/trimming/{data1}.unpaired.fastq.gz"],data1=DATA1.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
r2p = expand(["{dir}/trimming/{data2}.clean.fastq.gz"],data2=DATA2.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
r2u = expand(["{dir}/trimming/{data2}.unpaired.fastq.gz"],data2=DATA2.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR)
log: run = expand("{dir}/trimming/run_stats.log",dir=OUTPUT_DIR)
threads: 4
resources: mem = 5
params: phred = "-phred33",
leading = "3",
trailing = "3",
crop = expand("{par}",par=RD_LENGTH),
minlen = expand("{par}",par=LEN_FILTER),
slid_w_1 = "4",
slid_w_2 = expand("{par}",par=PHRED_FILTER)
shell: """
java -Xmx{resources.mem}g -jar {TRIMMOMATIC} PE -threads {threads} {params.phred} {input.r1} {input.r2} {output.r1p} {output.r1u} {output.r2p} {output.r2u} LEADING:{params.leading} TRAILING:{params.trailing} CROP:{params.crop} SLIDINGWINDOW:{params.slid_w_1}:{params.slid_w_2} MINLEN:{params.minlen} > {log.run} 2>&1
"""
rule second_qc:
input: reads = expand("{dir}/trimming/{data}.clean.fastq.gz",data=DATA.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR) if config[CONF_DATA_TYPE_TERM] == "single" else expand(["{dir}/trimming/{data1}.clean.fastq.gz","{dir}/trimming/{data2}.clean.fastq.gz"],data1=DATA1.strip(".fastq.gz|.fq.gz"),data2=DATA2.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR)
output: dir = "{OUTPUT_DIR}/2nd_qc"
log: run = "{OUTPUT_DIR}/2nd_qc/run_stats.log"
threads: 4
params: "--noextract --format fastq --nogroup"
shell: """
{FASTQC} -o {output.dir}/ {params} --threads {threads} {input.reads} > {log.run} 2>&1
"""
#########################################################
# DEFINITION OF MAPPING RULES
#
if GENOME_INDEX == DEF_GENOME_INDEX :
rule STAR_gen_index:
input: expand("{dir}/data/genome/{data}",data=GENOME_FILE,dir=OUTPUT_DIR),
ref = expand("{dir}/data/reference/{data}",data=ANNOTATION,dir=OUTPUT_DIR)
output: dir = expand("{dir}/genome_index/{data}",dir=OUTPUT_DIR,data=GENOME_FILE.rstrip(".fa|.fasta")),
prefix = expand("{dir}/genome_index/",dir=OUTPUT_DIR)
threads: 12
params: extra = ""
run:
help = subprocess.Popen("grep -v '>' " + input[0] + " | wc -m",shell=True,stdout=subprocess.PIPE).communicate()[0]
STAR_GENOME_BASES_LOG = min(14,math.floor(math.log(float(int(help)),2)/2-1))
shell("""
mkdir -p {output.dir}
{STAR} --runMode genomeGenerate --runThreadN {threads} --genomeDir {output.dir} --genomeFastaFiles {input[0]} --sjdbGTFfile {input.ref} --outFileNamePrefix {output.prefix} --genomeSAindexNbases {STAR_GENOME_BASES_LOG} {params.extra}
""")
import os
rule STAR_alignment:
input: reads = expand("{dir}/trimming/{data}.clean.fastq.gz",data=DATA.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR) if config[CONF_DATA_TYPE_TERM] == "single" else expand(["{dir}/trimming/{data1}.clean.fastq.gz","{dir}/trimming/{data2}.clean.fastq.gz"],data1=DATA1.strip(".fastq.gz|.fq.gz"),data2=DATA2.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
gen_index = expand("{data}",data=GENOME_INDEX) if GENOME_INDEX != DEF_GENOME_INDEX else expand("{dir}/genome_index/{data}",dir=OUTPUT_DIR,data=GENOME_FILE.rstrip(".fa|.fasta")),
ref = expand("{dir}/data/reference/{data}",data=ANNOTATION,dir=OUTPUT_DIR)
output: final_log = expand("{dir}/aligned/Log.final.out",dir=OUTPUT_DIR),
gen_bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
trans_bam_tmp = temp(expand("{dir}/aligned/Aligned.toTranscriptome.out.bam",dir=OUTPUT_DIR)),
trans_bam = expand("{dir}/aligned/Aligned.toTranscriptome.sortedByCoord.out.bam",dir=OUTPUT_DIR)
threads: 12
resources: mem=30
params: reads = expand("{dir}/trimming/{data}.clean.fastq.gz",data=DATA.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR) if config[CONF_DATA_TYPE_TERM] == "single" else expand("{dir}/trimming/{data1}.clean.fastq.gz {dir}/trimming/{data2}.clean.fastq.gz",data1=DATA1.strip(".fastq.gz|.fq.gz"),data2=DATA2.strip(".fastq.gz|.fq.gz"),dir=OUTPUT_DIR),
dir = expand("{dir}/aligned/",dir=OUTPUT_DIR),
read_command = "zcat", #might be conditional to input
sort_RAM = 10000000000, #could be also a config parameter
sam_type = "--outSAMtype BAM SortedByCoordinate",
quant = "--quantMode TranscriptomeSAM", # if config[CONF_ANALYSIS_TYPE_TERM] == "quant" else "",
splicing = expand("--alignIntronMax {val}",val=STAR_INTRON_MAX),
mates_gap = expand("--alignMatesGapMax {val}",val=STAR_MATES_GAP),
extra = expand("{line}",line=STAR_PARAMS)
shell: """
{STAR} --runMode alignReads --runThreadN {threads} --genomeDir {input.gen_index} --readFilesIn {params.reads} --readFilesCommand {params.read_command} --sjdbGTFfile {input.ref} --outFileNamePrefix {params.dir} {params.sam_type} --limitBAMsortRAM {params.sort_RAM} {params.splicing} {params.mates_gap} {params.quant} {params.extra}
{SAMTOOLS} sort -@ {threads} {output.trans_bam_tmp} -o {output.trans_bam}
"""
rule STAR_index:
input: final_log = expand("{dir}/aligned/Log.final.out",dir=OUTPUT_DIR),
gen_bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
trans_bam = expand("{dir}/aligned/Aligned.toTranscriptome.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: gen_bai = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam.bai",dir=OUTPUT_DIR),
trans_bai = expand("{dir}/aligned/Aligned.toTranscriptome.sortedByCoord.out.bam.bai",dir=OUTPUT_DIR)
threads: 12
shell: """
{SAMTOOLS} index -@ {threads} {input.gen_bam}
{SAMTOOLS} index -@ {threads} {input.trans_bam}
"""
## I want this to be optional
#rule SAM_to_BAM:
# input: "{OUTPUT_DIR}/aligned/{SAMPLE}.sam"
# output: "{OUTPUT_DIR}/aligned/{SAMPLE}.bam"
# threads: 4
# shell: """
# {SAMTOOLS} view -@ {threads} -b {input} > {output}
# rm {input}
# """
#######################################
# DEFINITION OF COUNTING RULES (RSEM)
#
rule RSEM_prep_ref:
input: ref = expand("{dir}/data/reference/{data}",data=ANNOTATION,dir=OUTPUT_DIR),
genome = expand("{dir}/data/genome/{data}",data=GENOME_FILE,dir=OUTPUT_DIR)
output: idx = expand("{dir}/RSEM/{data}.idx.fa",dir=OUTPUT_DIR,data=ANNOTATION.rstrip(".gtf|.gff|.gff3"))
log: run = expand("{dir}/RSEM/RSEM_prep_ref.log",dir=OUTPUT_DIR)
threads: 4
params: rsem_ref = expand("{dir}/RSEM/{data}",dir=OUTPUT_DIR,data=ANNOTATION.rstrip(".gtf|.gff|.gff3")),
use_ref = "--gtf" if ANNOTATION.endswith(".gtf") else "--gff3"
shell: """
{RSEM_PATH}rsem-prepare-reference --num-threads {threads} {params.use_ref} {input.ref} {input.genome} {params.rsem_ref} > {log.run} 2>&1
"""
rule RSEM_calc_expr:
input: bam = expand("{dir}/aligned/Aligned.toTranscriptome.sortedByCoord.out.bam",dir=OUTPUT_DIR),
idx = expand("{dir}/RSEM/{data}.idx.fa",dir=OUTPUT_DIR,data=ANNOTATION.rstrip(".gtf|.gff|.gff3"))
output: dir = expand("{dir}/RSEM",dir=OUTPUT_DIR),
help_bam = temp(expand("{dir}/RSEM/Aligned.toTranscriptome.converted_for_RSEM.bam",dir=OUTPUT_DIR)),
pdf = expand("{dir}/RSEM/RSEM_calc_expr.pdf",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSEM/RSEM_calc_expr.log",dir=OUTPUT_DIR),
convert = expand("{dir}/RSEM/RSEM_convert_input.log",dir=OUTPUT_DIR)
threads: 4
resources: mem = 10
params: paired = "" if config[CONF_DATA_TYPE_TERM] == "single" else "--paired-end",
extra = "--estimate-rspd --calc-ci --no-bam-output --seed 12345 --forward-prob 0",
bam = expand("{dir}/aligned/Aligned.toTranscriptome.sortedByCoord.out.bam",dir=OUTPUT_DIR),
ref = expand("{dir}/RSEM/{data}",dir=OUTPUT_DIR,data=ANNOTATION.rstrip(".gtf|.gff|.gff3")),
help_bam = temp(expand("{dir}/RSEM/Aligned.toTranscriptome.converted_for_RSEM",dir=OUTPUT_DIR)),
prefix = expand("{dir}/RSEM/RSEM_calc_expr",dir=OUTPUT_DIR)
run:
shell("""
#cat <( {SAMTOOLS} view -H {input.bam} ) <( {SAMTOOLS} view -@ {threads} {input.bam} | awk '{{printf "%s", $0 " "; getline; print}}' | sort -S {resources.mem}G -T ./ | tr ' ' '\\n' ) | {SAMTOOLS} view -@ {threads} -bS - > {output.help_bam}
{RSEM_PATH}convert-sam-for-rsem -p {threads} {input.bam} {params.help_bam} &> {log.convert}
{RSEM_PATH}rsem-calculate-expression --alignments {params.paired} {params.extra} -p {threads} --ci-memory {resources.mem}000 {output.help_bam} {params.ref} {params.prefix} >& {log.run}
{RSEM_PATH}rsem-plot-model {params.prefix} {output.pdf}
""")
#rule RSEM_gen_data_matrix:
###############################################
# DEFINITION OF COUNTING RULES (FeatureCounts)
#
rule FeatureCounts:
input: ref = expand("{dir}/data/reference/{data}",data=ANNOTATION,dir=OUTPUT_DIR),
bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: expand("{dir}/FeatureCounts/feature_counts.txt",dir=OUTPUT_DIR)
log: run = expand("{dir}/FeatureCounts/run_stats.log",dir=OUTPUT_DIR)
threads: 4
params: paired = "" if config[CONF_DATA_TYPE_TERM] == "single" else "-p"
shell: """
{FEATURE_COUNTS} -t exon -g gene_id {params.paired} -T {threads} -a {input.ref} -o {output} {input.bam} > {log.run} 2>&1
"""
###############################################
# DEFINITION OF MAPPING QC RULES
#
rule mapping_qc_preparation:
input: ref = expand("{dir}/data/reference/{data}",data=ANNOTATION,dir=OUTPUT_DIR),
bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: tmp = temp(expand("{dir}/mapped_QC/temp_{data}",data=ANNOTATION, dir=OUTPUT_DIR)),
bed12 = expand("{dir}/mapped_QC/{data}.genes.bed12", data=ANNOTATION.rstrip(".gtf"), dir=OUTPUT_DIR),
tmp2 = temp(expand("{dir}/mapped_QC/temp_{data}.refFlat",data=ANNOTATION, dir=OUTPUT_DIR)),
flat = expand("{dir}/mapped_QC/{data}.refFlat.txt", dir=OUTPUT_DIR, data=ANNOTATION.rstrip(".gtf")),
head = expand("{dir}/mapped_QC/{data}.header", dir=OUTPUT_DIR, data=ANNOTATION.rstrip(".gtf")),
list = expand("{dir}/mapped_QC/{data}.rRNA.intervalListBody.txt", dir=OUTPUT_DIR, data=ANNOTATION.rstrip(".gtf")),
rrna = expand("{dir}/mapped_QC/{data}.rRNA.gtf", dir=OUTPUT_DIR, data=ANNOTATION.rstrip(".gtf"))
shell: """
{UCSC_SCRIPTS}/gtfToGenePred -genePredExt -geneNameAsName2 {input.ref} {output.tmp}
awk '{{print $2"\t"$4"\t"$5"\t"$1"\t0\t"$3"\t"$6"\t"$7"\t0\t"$8"\t"$9"\t"$10}}' {output.tmp} > {output.bed12}
#### TODO: resolve why single-exon transcripts starting from 1st position make troubles
cat {output.bed12} | awk '$7 != 0 || $10 != 1' > {output.tmp} && cp {output.tmp} {output.bed12}
####################
{UCSC_SCRIPTS}/gtfToGenePred -genePredExt {input.ref} {output.tmp2}
paste <(cut -f 12 {output.tmp2}) <(cut -f 1-10 {output.tmp2}) > {output.flat}
####################
{SAMTOOLS} view -H {input.bam} > {output.head}
cat {output.head} > {output.list}
grep 'gene_biotype \"rRNA' {input.ref} > {output.rrna} || echo $? > /dev/null
cut -s -f 1,4,5,7,9 {output.rrna} >> {output.list}
"""
rule mapping_qc_picard:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
bai = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam.bai",dir=OUTPUT_DIR),
flat = expand("{dir}/mapped_QC/{data}.refFlat.txt", dir=OUTPUT_DIR, data=ANNOTATION.rstrip(".gtf")),
list = expand("{dir}/mapped_QC/{data}.rRNA.intervalListBody.txt", dir=OUTPUT_DIR, data=ANNOTATION.rstrip(".gtf"))
output: txt_fwd = expand("{dir}/picard/Aligned.sortedByCoord.output.RNA_Metrics.forward.txt", dir=OUTPUT_DIR),
txt_rev = expand("{dir}/picard/Aligned.sortedByCoord.output.RNA_Metrics.reverse.txt", dir=OUTPUT_DIR)
log: run = expand("{dir}/picard/run_stats.log", dir=OUTPUT_DIR)
params: pdf_for = expand("{dir}/picard/Aligned.sortedByCoord.npc.forward.pdf", dir=OUTPUT_DIR),
pdf_rev = expand("{dir}/picard/Aligned.sortedByCoord.npc.reverse.pdf", dir=OUTPUT_DIR)
threads: 4
resources: mem = 5
run:
shell("""
java -Xmx{resources.mem}g -jar {PICARD}/CollectRnaSeqMetrics.jar \
I={input.bam} \
O={output.txt_fwd} \
REF_FLAT={input.flat} \
STRAND=FIRST_READ_TRANSCRIPTION_STRAND \
RIBOSOMAL_INTERVALS={input.list} \
CHART={params.pdf_for} \
VALIDATION_STRINGENCY=LENIENT > {log.run} 2>&1
java -Xmx{resources.mem}g -jar {PICARD}/CollectRnaSeqMetrics.jar \
I={input.bam} \
O={output.txt_rev} \
REF_FLAT={input.flat} \
STRAND=SECOND_READ_TRANSCRIPTION_STRAND \
RIBOSOMAL_INTERVALS={input.list} \
CHART={params.pdf_rev} \
VALIDATION_STRINGENCY=LENIENT > {log.run} 2>&1
""")
rule mapping_qc_strandness:
input: txt_fwd = expand("{dir}/picard/Aligned.sortedByCoord.output.RNA_Metrics.forward.txt", dir=OUTPUT_DIR),
txt_rev = expand("{dir}/picard/Aligned.sortedByCoord.output.RNA_Metrics.reverse.txt", dir=OUTPUT_DIR)
output: expand("{dir}/strandness.txt", dir=OUTPUT_DIR)
run:
# #with open(output[0],"w") as out:
# with open(input.txt_fwd,"r") as file:
# for line in file:
# if not line.startswith("#") & re.match("\d",line):
# #FORWARD_PCT = float(line.split("\t")[17])
# #out.write(float(line.split("\t")[17]))
# storage.store("myvar", float(line.split("\t")[17]))
R("""
table <- read.table("{input.txt_fwd}", sep="\t", as.is=T, nrows = 2) # TODO: nrows is temporary solution, should be done better
fwd <- as.numeric(table[2,"V18"])
table <- read.table("{input.txt_rev}", sep="\t", as.is=T, nrows = 2) # TODO: nrows is temporary solution, should be done better
rev <- as.numeric(table[2,"V18"])
data <- "none"
if(fwd > rev) {{
if(fwd >= 0.75) data <- "forward"
else if(fwd < 0.6 && rev < 0.6) data <- "none"
else {{
data <- "none"
print("Warning: Strandness is not obviously recognizable (between 0.6 and 0.75 for forward sense), hence, data are supposed to be non-stranded!")
}}
}} else {{
if(rev >= 0.75) data <- "reverse"
else if(fwd < 0.6 && rev < 0.6) data <- "none"
else {{
data <- "none"
print("Warning: Strandness is not obviously recognizable (between 0.6 and 0.75 for reverse sense), hence, data are supposed to be non-stranded!")
}}
}}
write(data, file = '{output}')
""")
##### JUST FOR TESTING (DELETE FINALLY)
rule test:
input: expand("{dir}/strandness.txt", dir=OUTPUT_DIR)
output:
run:
with open(input[0],"r") as file:
data = file.read().strip()
#data = storage.fetch("myvar")
#print(data)
if data == 'forward':
shell(" echo {data} ")
elif data == 'reverse':
shell(" echo 'REVERSE' ")
else:
shell(" echo 'NONE' ")
## TODO: check the automatic setting of the insert gap size according to the mapper setting
rule mapping_qc_preseq:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: extrap = expand("{dir}/preseq/Aligned.sortedByCoord.yield_estimates.txt",dir=OUTPUT_DIR),
curve = expand("{dir}/preseq/Aligned.sortedByCoord.estimates.txt",dir=OUTPUT_DIR)
params: paired = "" if config[CONF_DATA_TYPE_TERM] == "single" else "-pe",
seglen = STAR_MATES_GAP if STAR_MATES_GAP != "0" else "1000000" #default is only 5000
shell: """
{PRESEQ} lc_extrap -B {params.paired} -seg_len {params.seglen} -o {output.extrap} {input.bam}
{PRESEQ} c_curve -B {params.paired} -seg_len {params.seglen} -o {output.curve} {input.bam}
"""
rule summary_biotypes: ################## TODO: CHECK WITH HONZA: -s 0/1/2 #####################
input: ref = expand("{dir}/data/reference/{data}",data=ANNOTATION,dir=OUTPUT_DIR),
bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: fwd = expand("{dir}/mapped_QC/featureCounts.quantSeq.fwd.biotype_counts.txt",dir=OUTPUT_DIR),
rev = expand("{dir}/mapped_QC/featureCounts.quantSeq.rev.biotype_counts.txt",dir=OUTPUT_DIR)
log: run = expand("{dir}/mapped_QC/featureCounts.run_stats.summary_biotypes.log",dir=OUTPUT_DIR)
threads: 4
params: fwd = expand("{dir}/mapped_QC/featureCounts.quantSeq.fwd.biotype",dir=OUTPUT_DIR),
rev = expand("{dir}/mapped_QC/featureCounts.quantSeq.rev.biotype",dir=OUTPUT_DIR),
paired = "" if config[CONF_DATA_TYPE_TERM] == "single" else "-p"
shell: """
echo '###### FORWARD #######' > {log.run}
{FEATURE_COUNTS} -t exon -g gene_biotype {params.paired} -s 1 -T {threads} -a {input.ref} -o {params.fwd} {input.bam} >> {log.run} 2>&1
cut -f 1,7- {params.fwd} > {output.fwd}
echo '###### REVERSE #######' >> {log.run}
{FEATURE_COUNTS} -t exon -g gene_biotype {params.paired} -s 2 -T {threads} -a {input.ref} -o {params.rev} {input.bam} >> {log.run} 2>&1
cut -f 1,7- {params.rev} > {output.rev}
"""
#### TODO: not working - needs to be resolved
rule RSeQC_read_distribution:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
bed = expand("{dir}/mapped_QC/{data}.genes.bed12", data=ANNOTATION.rstrip(".gtf"), dir=OUTPUT_DIR)
output: expand("{dir}/RSeQC/Aligned.sortedByCoord.read_distribution.txt",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSeQC/read_distribution.log",dir=OUTPUT_DIR)
shell: """
{RSEQC}/read_distribution.py -i {input.bam} -r {input.bed} > {output} 2> {log.run}
"""
rule RSeQC_junction_saturation:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
bed = expand("{dir}/mapped_QC/{data}.genes.bed12", data=ANNOTATION.rstrip(".gtf"), dir=OUTPUT_DIR)
output: expand("{dir}/RSeQC/Aligned.sortedByCoord.junction_saturation.junctionSaturation_plot.r",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSeQC/junction_saturation.log",dir=OUTPUT_DIR)
params: prefix = expand("{dir}/RSeQC/Aligned.sortedByCoord.junction_saturation",dir=OUTPUT_DIR)
shell: """
{RSEQC}/junction_saturation.py -i {input.bam} -r {input.bed} -o {params.prefix} > {log.run} 2>&1
"""
rule RSeQC_junction_annotation:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
bed = expand("{dir}/mapped_QC/{data}.genes.bed12", data=ANNOTATION.rstrip(".gtf"), dir=OUTPUT_DIR)
output: expand("{dir}/RSeQC/Aligned.sortedByCoord.junction_annotation.junction.xls",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSeQC/junction_annotation.log",dir=OUTPUT_DIR)
params: prefix = expand("{dir}/RSeQC/Aligned.sortedByCoord.junction_annotation",dir=OUTPUT_DIR)
shell: """
{RSEQC}/junction_annotation.py -i {input.bam} -r {input.bed} -o {params.prefix} > {log.run} 2>&1
"""
rule RSeQC_bam_stat:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: expand("{dir}/RSeQC/Aligned.sortedByCoord.bam_stat.txt",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSeQC/bam_stat.log",dir=OUTPUT_DIR)
shell: """
{RSEQC}/bam_stat.py -i {input.bam} > {output} 2> {log.run}
"""
rule RSeQC_infer_experiment:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
bed = expand("{dir}/mapped_QC/{data}.genes.bed12", data=ANNOTATION.rstrip(".gtf"), dir=OUTPUT_DIR)
output: expand("{dir}/RSeQC/Aligned.sortedByCoord.infer_experiment.txt",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSeQC/infer_experiment.log",dir=OUTPUT_DIR)
shell: """
{RSEQC}/infer_experiment.py -i {input.bam} -r {input.bed} > {output} 2> {log.run}
"""
rule RSeQC_read_duplication:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: expand("{dir}/RSeQC/Aligned.sortedByCoord.read_duplication.seq.DupRate.xls",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSeQC/read_duplication.log",dir=OUTPUT_DIR)
params: prefix = expand("{dir}/RSeQC/Aligned.sortedByCoord.read_duplication",dir=OUTPUT_DIR)
shell: """
{RSEQC}/read_duplication.py -i {input.bam} -o {params.prefix} > {log.run} 2>&1
"""
rule RSeQC_RPKM_saturation:
input: expand("{dir}/strandness.txt", dir=OUTPUT_DIR),
bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR),
bed = expand("{dir}/mapped_QC/{data}.genes.bed12", data=ANNOTATION.rstrip(".gtf"), dir=OUTPUT_DIR)
output: expand("{dir}/RSeQC/Aligned.sortedByCoord.RPKM_saturation.rawCount.xls",dir=OUTPUT_DIR)
log: run = expand("{dir}/RSeQC/RPKM_saturation.log",dir=OUTPUT_DIR)
params: prefix = expand("{dir}/RSeQC/Aligned.sortedByCoord.RPKM_saturation",dir=OUTPUT_DIR)
run:
with open(input[0],"r") as file:
data = file.read().strip()
#print(data)
if data == 'forward':
if config[CONF_DATA_TYPE_TERM] == "single":
shell(" {RSEQC}/RPKM_saturation.py -i {input.bam} -r {input.bed} -d '++,--' -o {params.prefix} > {log.run} 2>&1")
else:
shell(" {RSEQC}/RPKM_saturation.py -i {input.bam} -r {input.bed} -d '1++,1--,2+-,2-+' -o {params.prefix} > {log.run} 2>&1 ")
elif data == 'reverse':
if config[CONF_DATA_TYPE_TERM] == "single":
shell(" {RSEQC}/RPKM_saturation.py -i {input.bam} -r {input.bed} -d '+-,-+' -o {params.prefix} > {log.run} 2>&1 ")
else:
shell(" {RSEQC}/RPKM_saturation.py -i {input.bam} -r {input.bed} -d '1+-,1-+,2++,2--' -o {params.prefix} > {log.run} 2>&1 ")
else:
shell(" {RSEQC}/RPKM_saturation.py -i {input.bam} -r {input.bed} -o {params.prefix} > {log.run} 2>&1 ")
rule picard_mark_duplicates:
input: bam = expand("{dir}/aligned/Aligned.sortedByCoord.out.bam",dir=OUTPUT_DIR)
output: bam = expand("{dir}/mapped_QC/Aligned.sortedByCoord.markDups.bam",dir=OUTPUT_DIR),
mtx = expand("{dir}/mapped_QC/Aligned.sortedByCoord.markDups_metrics.txt",dir=OUTPUT_DIR)
log: run = expand("{dir}/mapped_QC/Aligned.sortedByCoord.markDups.log",dir=OUTPUT_DIR)
threads: 4
resources: mem = 5
shell: """
java -Xmx{resources.mem}g -jar {PICARD}/MarkDuplicates.jar \
INPUT={input.bam} \
OUTPUT={output.bam} \
METRICS_FILE={output.mtx} \
REMOVE_DUPLICATES=false \
ASSUME_SORTED=true \
PROGRAM_RECORD_ID=null \
VALIDATION_STRINGENCY=LENIENT > {log.run} 2>&1
"""
rule dupradar_count_duplicates:
input: expand("{dir}/strandness.txt", dir=OUTPUT_DIR),
bam = expand("{dir}/mapped_QC/Aligned.sortedByCoord.markDups.bam",dir=OUTPUT_DIR),
ref = expand("{dir}/data/reference/{data}",data=ANNOTATION,dir=OUTPUT_DIR)
output: expand("{dir}/mapped_QC/Aligned.sortedByCoord.dupRadar_dupMatrix.txt",dir=OUTPUT_DIR)
log: run = expand("{dir}/mapped_QC/Aligned.sortedByCoord.dupRadar.log",dir=OUTPUT_DIR)
params: prefix = expand("{dir}/mapped_QC/Aligned.sortedByCoord.dupRadar",dir=OUTPUT_DIR),
installation = ""
threads: 4
run:
with open(input[0],"r") as file:
data = file.read().strip()
#print(data)
if data == 'forward':
if config[CONF_DATA_TYPE_TERM] == "single":
shell(" {DUPRADAR} {input.bam} {input.ref} 'single' 1 {params.prefix} {threads} {params.installation} > {log.run} 2>&1 ")
else:
shell(" {DUPRADAR} {input.bam} {input.ref} 'paired' 1 {params.prefix} {threads} {params.installation} > {log.run} 2>&1 ")
elif data == 'reverse':
if config[CONF_DATA_TYPE_TERM] == "single":
shell(" {DUPRADAR} {input.bam} {input.ref} 'single' 2 {params.prefix} {threads} {params.installation} > {log.run} 2>&1 ")
else:
shell(" {DUPRADAR} {input.bam} {input.ref} 'paired' 2 {params.prefix} {threads} {params.installation} > {log.run} 2>&1 ")
else:
if config[CONF_DATA_TYPE_TERM] == "single":
shell(" {DUPRADAR} {input.bam} {input.ref} 'single' 0 {params.prefix} {threads} {params.installation} > {log.run} 2>&1 ")
else:
shell(" {DUPRADAR} {input.bam} {input.ref} 'paired' 0 {params.prefix} {threads} {params.installation} > {log.run} 2>&1 ")