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TMPDIR = "/localscratch/msauria"
SYSTEM="linux.x86_64"
MAXTHREADS=100
MAXMEM=1024
ANNOTATION_FILE="data/t2t_cenAnnotation.v2.021621.bed"
GENOMES = ["chm13v1", "GRCh38p13"]
GENOME_SIZE = {"chm13v1": "3.03e9", "GRCh38p13": "2.79e9"}
SAMPLEFILE="data/encode_samples.txt"
KMERS=["50", "75", "80", "85", "90", "95", "100"]
CHROMS=['chr1', 'chr2', 'chr3', 'chr4', 'chr5', 'chr6', 'chr7', 'chr8', 'chr9', 'chr10',
'chr11', 'chr12', 'chr13', 'chr14', 'chr15', 'chr16', 'chr17', 'chr18', 'chr19', 'chr20',
'chr21', 'chr22', 'chrX']
CONCAT_DICT = {}
EXPERIMENT_DICT = {}
lib_count = {}
data = []
for line in open(SAMPLEFILE):
if line.startswith('#'):
continue
line = line.rstrip().split(',')
data.append(line)
lib_count.setdefault(line[1], 0)
lib_count[line[1]] += 1
EXPERIMENT_DICT.setdefault(line[0], [])
EXPERIMENT_DICT[line[0]].append(line[1])
for libID, count in lib_count.items():
if count > 1:
CONCAT_DICT[libID] = [[], []]
ARRAYS=set()
for line in open(ANNOTATION_FILE):
a = line.split()[3].split('_')[0].upper()
if a != "CENSAT":
ARRAYS.add(a)
ARRAYS=list(ARRAYS)
ARRAYS.sort()
CONTROL_EXPERIMENT_DICT = {}
NONCONTROL_EXPERIMENT_DICT = {}
NONCONTROL_LIBRARIES = set()
CONTROL_LIBRARIES = set()
SLINK_DICT = {}
EXPERIMENT_CONTROL_DICT = {}
EXPERIMENT_CONTROL_LIB_DICT = {}
LABEL_DICT = {}
LABEL_CONTROL_DICT = {}
broad = set(['H3K9me3', 'H3K27me3', 'H3K36me3'])
BROAD_TRACKS = set()
NARROW_TRACKS = set()
READS = []
CTs = set()
MARK_DICT = {}
MARKS = set()
EXP2LABEL_DICT = {}
for line in data:
expID, libID, read1, read2, control, target, ct = line[:7]
MARKS.add(target)
ct = ct.replace(' ', '_').replace('(', '').replace(')', '')
CTs.add(ct)
label = "{}_{}".format(ct, target)
if libID in CONCAT_DICT:
CONCAT_DICT[libID][0].append(read1)
CONCAT_DICT[libID][1].append(read2)
else:
SLINK_DICT[libID] = [read1, read2]
READS += [read1, read2]
if control == 'NA':
CONTROL_EXPERIMENT_DICT.setdefault(expID, [])
CONTROL_EXPERIMENT_DICT[expID].append(libID)
CONTROL_LIBRARIES.add(libID)
else:
if target in broad:
BROAD_TRACKS.add(label)
else:
NARROW_TRACKS.add(label)
NONCONTROL_EXPERIMENT_DICT.setdefault(expID, [])
NONCONTROL_EXPERIMENT_DICT[expID].append(libID)
EXPERIMENT_CONTROL_LIB_DICT[expID] = EXPERIMENT_DICT[control]
EXPERIMENT_CONTROL_DICT[expID] = control
LABEL_CONTROL_DICT[label] = "{}_Control".format(ct)
MARK_DICT.setdefault(ct, set())
MARK_DICT[ct].add(target)
LABEL_DICT[label] = expID
EXP2LABEL_DICT[expID] = label
for key in MARK_DICT.keys():
MARK_DICT[key] = list(MARK_DICT[key])
EXPERIMENTS = list(EXPERIMENT_DICT.keys())
LIBRARIES = list(CONCAT_DICT.keys()) + list(SLINK_DICT.keys())
CONTROL_LIBRARIES = list(CONTROL_LIBRARIES)
TRACKS=list(LABEL_DICT.keys())
TRACKS.sort()
NONCONTROL_TRACKS = []
CONTROL_TRACKS = []
for label in LABEL_DICT.keys():
if not label.endswith("Control"):
NONCONTROL_TRACKS.append(label)
else:
CONTROL_TRACKS.append(label)
NONCONTROL_TRACKS.sort()
CONTROL_TRACKS.sort()
MARKS = list(MARKS)
CTs = list(CTs)
CONTROL_EXPERIMENTS = list(CONTROL_EXPERIMENT_DICT.keys())
BROAD_TRACKS = list(BROAD_TRACKS)
NARROW_TRACKS = list(NARROW_TRACKS)
CT_MARK_SET = {}
for ct, marks in MARK_DICT.items():
CT_MARK_SET[ct] = "|".join(marks)
CONTROL_EXPERIMENT_SET = "|".join(CONTROL_EXPERIMENTS)
NONCONTROL_EXPERIMENT_SET = "|".join(list(NONCONTROL_EXPERIMENT_DICT.keys()))
EXPERIMENT_SET = "|".join(EXPERIMENTS)
TRACK_SET = "|".join(TRACKS)
NONCONTROL_TRACK_SET = "|".join(NONCONTROL_TRACKS)
CONTROL_TRACK_SET = "|".join(CONTROL_TRACKS)
LIBRARY_SET = "|".join(LIBRARIES)
CONTROL_LIBRARY_SET = "|".join(CONTROL_LIBRARIES)
CONCAT_SET = "|".join(list(CONCAT_DICT.keys()))
SLINK_SET = "|".join(list(SLINK_DICT.keys()))
BROAD_TRACK_SET = "|".join(BROAD_TRACKS)
NARROW_TRACK_SET = "|".join(NARROW_TRACKS)
GENOME_SET = "|".join(GENOMES)
CHROM_SET="|".join(CHROMS)
KMER_SET="|".join(KMERS)
ARRAY_SET = "|".join(ARRAYS)
MARK_SET = "|".join(MARKS)
CT_SET = "|".join(CTs)
READ_SET = "|".join(READS)
rule all:
input:
expand("results/{file}",
file=["chm13v1_array_enrichments.tsv",
"macs2_peak_overlap.txt",
"control_mapping_stats.txt",
"mapping_stats.txt",
"chm13v1_array_bounds.csv",
"chm13v1_count_depletions.tsv",
"GRCh38p13_count_depletions.tsv"]),
######## Get software #########
rule download_software:
output:
"bin/{sw}"
wildcard_constraints:
sw="liftOver|bedToBigBed|wigToBigWig|bigBedToBed|bigBedToBed"
params:
sw="{sw}",
system=SYSTEM
log:
"logs/Encode/download/{sw}.log"
shell:
"""
wget http://hgdownload.soe.ucsc.edu/admin/exe/{params.system}/{params.sw} -O {output}
chmod a+rx {output}
"""
rule build_KMC:
output:
"bin/kmc",
"bin/kmc_genome_counts"
log:
"logs/build_kmc.log"
conda:
"envs/kmc.yaml"
shell:
"""
git clone https://github.com/msauria/KMC.git
git checkout kmer_mapping
cd KMC && make
cp KMC/bin/kmc bin/
chmod a+rx bin/kmc
cp KMC/bin/kmc_genome_counts bin/
chmod a+rx bin/kmc_genome_counts
"""
######## Get chm13 data #########
rule preprocess_annotations:
output:
"data/t2t_cenSatAnnotation.bed",
"data/t2t_cenSatAnnotation2.bed"
params:
anno=ANNOTATION_FILE
log:
"logs/preprocess/cenSatAnnotation.log"
shell:
"""
grep -v "censat" {params.anno} > {output[1]}
awk 'BEGIN{{ OFS="\t" }} {{ split($4,a,"_"); $4=toupper(a[1]); print $0 }}' {output[1]} > {output[0]}
"""
rule download_liftover:
output:
"data/hg38.t2t-chm13-v1.0.over.chain.gz"
log:
"logs/download/liftover.log"
shell:
"""
wget -O {output} http://t2t.gi.ucsc.edu/chm13/hub/t2t-chm13-v1.0/hg38Lastz/hg38.t2t-chm13-v1.0.over.chain.gz
"""
######## Get genomic data #########
rule get_GRCh38_fasta:
output:
fa="fasta/GRCh38p13.fasta",
sizes="fasta/GRCh38p13.chrom.sizes"
log:
"logs/fasta/GRCh38p13_download.log"
shell:
"""
wget -O {output.fa}.gz http://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz
gzip -d -c {output.fa}.gz > {output.fa}
wget -O {output.sizes} http://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.chrom.sizes
"""
rule get_chm13v1_fasta:
output:
fa="fasta/chm13v1.fasta",
sizes="fasta/chm13v1.chrom.sizes"
log:
"logs/fasta/chm13v1_download.log"
shell:
"""
wget -O {output.fa}.gz http://t2t.gi.ucsc.edu/chm13/hub/t2t-chm13-v1.0/genome/t2t-chm13-v1.0.fa.gz
gzip -d -c {output.fa}.gz > {output.fa}
cat {output.fa} | awk 'BEGIN{{ OFS=""; ORS=""; tab="\t"; nl="\n"; tl=0 }}\
{{ if ( NR == 1 ){{ split($1,A,">"); print A[2],tab; }} \
else {{ if ( $1 ~ /^>/ ){{ split($1,A,">"); print tl,nl,A[2],tab; tl = 0;}} \
else {{ tl = tl + length; }} }} }}\
END{{ print tl,nl }}' | sort -r -k2,2n > {output.sizes}
"""
rule get_chm13v1_segdup:
output:
"data/chm13v1_segdup.bed"
log:
"logs/data/chm13v1_segdup_download.log"
conda:
"envs/bedtools.yaml"
shell:
"""
wget -O {output}.bb http://t2t.gi.ucsc.edu/chm13/hub/t2t-chm13-v1.0/sedefSegDups/chm13.draft_v1.0_plus38Y.SDs.bed.bb
bin/bigBedToBed {output}.bb {output}.tmp
awk 'BEGIN{{OFS="\t"}}{{ if($3-$2 >= 1000) print $1,$2,$3,$4}}' {output}.tmp > {output}.tmp1
sort -k1,1 -k2,2n {output}.tmp1 > {output}.tmp2
bedtools merge -d 1 -i {output}.tmp2 > {output}
rm {output}.tmp* {output}.bb
"""
rule get_chm13v1_repeat:
input:
segdup="data/chm13v1_segdup.bed",
sizes="fasta/chm13v1.chrom.sizes"
output:
rpt="data/chm13v1_repeat.bed",
LINE="data/chm13v1_LINE.bed",
sat="data/chm13v1_satellite.bed",
rpt_full="data/chm13v1_repeatall.bed",
nonrep="data/chm13v1_nonrep.bed"
log:
"logs/data/chm13v1_repeat_download.log"
conda:
"envs/bedtools.yaml"
shell:
"""
wget -O {output.rpt}.bb http://t2t.gi.ucsc.edu/chm13/hub/t2t-chm13-v1.0/rmskV2/rmskV2.bigBed
bin/bigBedToBed {output.rpt}.bb {output.rpt}.tmp1
awk 'BEGIN{{OFS="\t"}}{{ print $1,$2,$3,$10,$11 }}' {output.rpt}.tmp1 > {output.rpt_full}
grep LINE {output.rpt_full} > {output.rpt}.tmp2
grep SINE {output.rpt_full} >> {output.rpt}.tmp2
sort -k1,1 -k2,2n {output.rpt}.tmp2 > {output.rpt}.tmp3
bedtools merge -d 1 -i {output.rpt}.tmp3 > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {input.segdup} > {output.LINE}
grep Satellite {output.rpt_full} > {output.rpt}.tmp2
sort -k1,1 -k2,2n {output.rpt}.tmp2 > {output.rpt}.tmp3
bedtools merge -d 1 -i {output.rpt}.tmp3 > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {input.segdup} > {output.rpt}.tmp5
bedtools subtract -a {output.rpt}.tmp5 -b {output.LINE} > {output.sat}
grep -v LINE {output.rpt_full} | grep -v SINE | grep -v Satellite > {output.rpt}.tmp2
sort -k1,1 -k2,2n {output.rpt}.tmp2 > {output.rpt}.tmp3
bedtools merge -d 1 -i {output.rpt}.tmp3 > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {input.segdup} > {output.rpt}.tmp5
bedtools subtract -a {output.rpt}.tmp5 -b {output.LINE} > {output.rpt}.tmp6
bedtools subtract -a {output.rpt}.tmp6 -b {output.sat} > {output.rpt}
awk 'BEGIN{{OFS="\t"; A="0"}}{{ print $1,A,$2 }}' {input.sizes} > {output.rpt}.tmp1
bedtools subtract -a {output.rpt}.tmp1 -b {input.segdup} > {output.rpt}.tmp2
bedtools subtract -a {output.rpt}.tmp2 -b {output.rpt} > {output.rpt}.tmp3
bedtools subtract -a {output.rpt}.tmp3 -b {output.LINE} > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {output.sat} > {output.nonrep}
rm {output.rpt}.tmp* {output.rpt}.bb
"""
rule get_GRCh38_segdup:
output:
"data/GRCh38p13_segdup.bed"
log:
"logs/data/GRCh38p13_segdup_download.log"
conda:
"envs/bedtools.yaml"
shell:
"""
wget -O {output}.gz https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/genomicSuperDups.txt.gz
gzip -d -c {output}.gz > {output}.tmp
awk 'BEGIN{{OFS="\t"}}{{ if($3-$2 >= 1000) print $2,$3,$4,$5 }}' {output}.tmp > {output}.tmp1
sort -k1,1 -k2,2n {output}.tmp1 > {output}.tmp2
bedtools merge -d 1 -i {output}.tmp2 > {output}
rm {output}.tmp*
"""
rule get_GRCh38_repeat_mask:
input:
segdup="data/GRCh38p13_segdup.bed",
sizes="fasta/GRCh38p13.chrom.sizes"
output:
rpt="data/GRCh38p13_repeat.bed",
LINE="data/GRCh38p13_LINE.bed",
sat="data/GRCh38p13_satellite.bed",
rpt_full="data/GRCh38p13_repeatall.bed",
nonrep="data/GRCh38p13_nonrep.bed"
log:
"logs/data/GRCh38p13_repeat_download.log"
conda:
"envs/bedtools.yaml"
shell:
"""
wget -O {output.rpt}.gz https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/rmsk.txt.gz
gzip -d -c {output.rpt}.gz > {output.rpt}.tmp
awk 'BEGIN{{OFS="\t"}}{{ print $6,$7,$8,$11,$12,$13 }}' {output.rpt}.tmp > {output.rpt_full}
grep LINE {output.rpt_full} > {output.rpt}.tmp2
grep SINE {output.rpt_full} >> {output.rpt}.tmp2
sort -k1,1 -k2,2n {output.rpt}.tmp2 > {output.rpt}.tmp3
bedtools merge -d 1 -i {output.rpt}.tmp3 > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {input.segdup} > {output.LINE}
grep Satellite {output.rpt_full} > {output.rpt}.tmp2
sort -k1,1 -k2,2n {output.rpt}.tmp2 > {output.rpt}.tmp3
bedtools merge -d 1 -i {output.rpt}.tmp3 > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {input.segdup} > {output.rpt}.tmp5
bedtools subtract -a {output.rpt}.tmp5 -b {output.LINE} > {output.sat}
grep -v LINE {output.rpt_full} | grep -v SINE | grep -v Satellite > {output.rpt}.tmp2
sort -k1,1 -k2,2n {output.rpt}.tmp2 > {output.rpt}.tmp3
bedtools merge -d 1 -i {output.rpt}.tmp3 > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {input.segdup} > {output.rpt}.tmp5
bedtools subtract -a {output.rpt}.tmp5 -b {output.LINE} > {output.rpt}.tmp6
bedtools subtract -a {output.rpt}.tmp6 -b {output.sat} > {output.rpt}
awk 'BEGIN{{OFS="\t"; A="0"}}{{ print $1,A,$2 }}' {input.sizes} > {output.rpt}.tmp1
bedtools subtract -a {output.rpt}.tmp1 -b {input.segdup} > {output.rpt}.tmp2
bedtools subtract -a {output.rpt}.tmp2 -b {output.rpt} > {output.rpt}.tmp3
bedtools subtract -a {output.rpt}.tmp3 -b {output.LINE} > {output.rpt}.tmp4
bedtools subtract -a {output.rpt}.tmp4 -b {output.sat} > {output.nonrep}
rm {output.rpt}.tmp* {output.rpt}.gz
"""
######## Get Encode data #########
rule download_data:
output:
"fastq/{readID}.fastq.gz"
params:
readID="{readID}"
wildcard_constraints:
readID=READ_SET
log:
"logs/download/{readID}.log"
shell:
"""
wget https://www.encodeproject.org/files/{params.readID}/@@download/{params.readID}.fastq.gz -O {output}
"""
######## Concatenate fastq files #########
rule concat:
input:
lambda wildcards: expand("fastq/{read}.fastq.gz",
read=CONCAT_DICT[wildcards.libID][int(wildcards.side)-1])
output:
"concat/{libID}.{side}.fastq.gz"
wildcard_constraints:
libID=CONCAT_SET,
genome=GENOME_SET,
side="1|2"
log:
"logs/concat/{libID}.{side}.log"
shell:
"""
zcat {input} | gzip > {output}
"""
rule symlink:
input:
lambda wildcards: expand("fastq/{read}.fastq.gz",
read=SLINK_DICT[wildcards.libID])
output:
"concat/{libID}.1.fastq.gz",
"concat/{libID}.2.fastq.gz"
wildcard_constraints:
libID=SLINK_SET,
genome=GENOME_SET
log:
"logs/symlink/{libID}.log"
shell:
"""
ln -s ${{PWD}}/{input[0]} {output[0]}
ln -s ${{PWD}}/{input[1]} {output[1]}
"""
rule count_fastq:
input:
"concat/{libID}.1.fastq.gz"
output:
"concat/{libID}.fastq.count"
wildcard_constraints:
libID=LIBRARY_SET
log:
"logs/count/{libID}.log"
shell:
"""
echo "$(expr $(eval zcat {input} | wc -l) / 4)" > {output}
"""
######## Bowtie2 Alignment #########
rule bowtie2_build:
input:
"fasta/{genome}.fasta"
output:
multiext(
"bt2_indices/{genome}",
".1.bt2", ".2.bt2", ".3.bt2", ".4.bt2", ".rev.1.bt2", ".rev.2.bt2"
)
params:
extra="" # optional parameters
threads: 8
log:
"logs/bowtie2_build.{genome}.log"
wrapper:
"v0.69.0/bio/bowtie2/build"
rule bowtie2:
input:
sample=["concat/{libID}.1.fastq.gz", "concat/{libID}.2.fastq.gz"],
index="bt2_indices/{genome}.1.bt2"
output:
"mapped/{libID}.{genome}.bam"
params:
index="bt2_indices/{genome}", # prefix of reference genome index (built with bowtie2-build)
extra="--no-discordant --no-mixed --very-sensitive \
--no-unal --omit-sec-seq --xeq --reorder" # optional parameters
threads: 100 # Use at least two threads
wildcard_constraints:
genome=GENOME_SET,
libID=LIBRARY_SET
log:
"logs/bowtie2/{libID}.{genome}.log"
wrapper:
"v0.69.0/bio/bowtie2/align"
######## Create KMC DBs #########
rule create_kmc_db:
input:
fa="fasta/{genome}.fasta",
kmc="bin/kmc"
output:
"KMC_db/{genome}_{kmer}.kmc_pre",
"KMC_db/{genome}_{kmer}.kmc_suf"
params:
tmpdir=TMPDIR,
kmer="{kmer}",
maxmem=MAXMEM,
prefix="KMC_db/{genome}_{kmer}"
threads:
MAXTHREADS
wildcard_constraints:
kmer=KMER_SET,
genome=GENOME_SET
log:
"logs/kmc/{genome}_{kmer}.log"
conda:
"envs/kmc.yaml"
shell:
"""
{input.kmc} -k{params.kmer} -m{params.maxmem} -fm -ci2 -t{threads} \
{input.fa} {params.prefix} {params.tmpdir}
"""
######## Create Kmer BigWigs #########
rule create_kmer_wiggle:
input:
fa="fasta/{genome}.fasta",
db="KMC_db/{genome}_{kmer}.kmc_pre",
kmc="bin/kmc_genome_counts"
output:
temp("Kmer_BigWigs/{genome}_{kmer}mer.wig")
params:
prefix="KMC_db/{genome}_{kmer}",
chroms=CHROMS,
tmpdir=TMPDIR
wildcard_constraints:
genome=GENOME_SET,
kmer=KMER_SET
threads:
len(CHROMS)
log:
"logs/wiggle/{genome}_{kmer}.log"
conda:
"envs/kmc.yaml"
shell:
"""
tmpdir=$(mktemp -d -p {params.tmpdir});
for C in {params.chroms}; do \
{input.kmc} -ch$C {params.prefix} {input.fa} > $tmpdir/$C.wig & \
done; wait
wigs=""
for C in {params.chroms}; do wigs="$wigs $tmpdir/$C.wig"; done;
cat $wigs > {output}
rm -r $tmpdir
"""
rule create_kmer_bigwig:
input:
wig="Kmer_BigWigs/{genome}_{kmer}mer.wig",
wigtobigwig="bin/wigToBigWig",
sizes="fasta/{genome}.chrom.sizes"
output:
"Kmer_BigWigs/{genome}_{kmer}mer.bw"
wildcard_constraints:
genome=GENOME_SET,
kmer=KMER_SET
log:
"logs/bigwigs/kmer_{genome}_{kmer}.log"
shell:
"""
{input.wigtobigwig} -clip {input.wig} {input.sizes} {output}
"""
######## BAM filtering #########
rule samtools_flag_mapped:
input:
"mapped/{libID}.{genome}.bam"
output:
"mapped/{libID}.{genome}.stats"
wildcard_constraints:
genome=GENOME_SET,
libID=LIBRARY_SET
log:
"logs/samtools/stats/mapped.{libID}.{genome}.log"
wrapper:
"0.68.0/bio/samtools/flagstat"
rule samtools_flag_exp:
input:
"{folder}/{expID}.{genome}.bam"
output:
"{folder}/{expID}.{genome}.stats"
wildcard_constraints:
genome=GENOME_SET,
expID=EXPERIMENT_SET
log:
"logs/samtools/stats/{folder}.{expID}.{genome}.log"
wrapper:
"0.68.0/bio/samtools/flagstat"
rule samtools_index:
input:
"{folder}/{expID}.{genome}.bam"
output:
"{folder}/{expID}.{genome}.bam.bai"
wildcard_constraints:
genome=GENOME_SET,
expID=EXPERIMENT_SET
log:
"logs/samtools/index/{folder}.{expID}.{genome}.log"
wrapper:
"0.68.0/bio/samtools/index"
rule samtools_filter:
input:
"mapped/{libID}.{genome}.bam"
output:
"filtered/{libID}.{genome}.bam",
"filtered/{libID}.{genome}.stats"
wildcard_constraints:
genome=GENOME_SET,
libID=LIBRARY_SET
log:
"logs/samtools/stats/mapped.{libID}.{genome}.log"
conda:
"envs/samtools.yaml"
shell:
"""
samtools view -b -F 1804 -f 2 -q 2 {input[0]} > {output[0]}
samtools flagstats {output[0]} > {output[1]}
"""
# 1804 = 1024+512+256+8+4
# Remove flag bits 4, 8, 256, 512, and 1024 (unmapped segment, unmapped next segment, secondary, failed filter, duplicate)
# Require flag bit 2 (both sides properly aligned)
rule remove_duplicates:
input:
"filtered/{libID}.{genome}.bam"
output:
bam="dedup/{libID}.{genome}.bam",
metrics="dedup/{libID}.{genome}.metrics.txt"
params:
"VALIDATION_STRINGENCY=LENIENT ASSUME_SORT_ORDER=queryname REMOVE_DUPLICATES=true"
# optional specification of memory usage of the JVM that snakemake will respect with global
# resource restrictions (https://snakemake.readthedocs.io/en/latest/snakefiles/rules.html#resources)
# and which can be used to request RAM during cluster job submission as `{resources.mem_mb}`:
# https://snakemake.readthedocs.io/en/latest/executing/cluster.html#job-properties
wildcard_constraints:
libID=LIBRARY_SET,
genome=GENOME_SET
resources:
mem_mb=MAXMEM
log:
"logs/picard/dedup/{libID}.{genome}.log"
wrapper:
"0.68.0/bio/picard/markduplicates"
rule concat_bam:
input:
lambda wildcards: expand("{{folder}}/{libID}.{{genome}}.bam",
libID=EXPERIMENT_DICT[wildcards.expID])
output:
"{folder}_concat/{expID}.{genome}.bam"
params:
prefix="{folder}_concat/{expID}.{genome}"
wildcard_constraints:
expID=EXPERIMENT_SET,
genome=GENOME_SET,
folder="dedup|filtered|mapped"
threads:
8
log:
"logs/samtools/merge/{folder}.{expID}.{genome}.log"
conda:
"envs/samtools.yaml"
shell:
"""
INPUTS=({input})
if [ ${{#INPUTS[*]}} \> 1 ]; then \
samtools merge -n --threads {threads} {params.prefix}.tmp {input}
else \
cp {input[0]} {params.prefix}.tmp
fi;
samtools sort -@ {threads} -o {output} {params.prefix}.tmp
rm {params.prefix}.tmp
"""
rule filter_by_kmers_presort:
input:
"dedup_concat/{expID}.{genome}.bam"
output:
temp("dedup_kmer/{expID}.{genome}.namesorted.bam")
wildcard_constraints:
genome=GENOME_SET,
expID=EXPERIMENT_SET
params:
tmp_dir=TMPDIR,
extra="-n"
threads: 8
log:
"logs/samtools/sort/dedup_concat.{expID}.{genome}.log"
wrapper:
"0.68.0/bio/samtools/sort"
rule filter_by_kmers:
input:
bam="dedup_kmer/{expID}.{genome}.namesorted.bam",
bigwigs=expand("Kmer_BigWigs/{{genome}}_{kmer}mer.bw", kmer=KMERS)
output:
temp("dedup_kmer/{expID}.{genome}.filtered.bam")
params:
kmers=KMERS,
genome="{genome}",
prefix="{expID}.{genome}",
kmer_prefix="Kmer_BigWigs/{genome}",
tmpdir=TMPDIR
wildcard_constraints:
genome=GENOME_SET,
expID=EXPERIMENT_SET
threads: 5
log:
"logs/kmer_filter/{expID}.{genome}.log"
conda:
"envs/deepsam.yaml"
shell:
"""
tmpdir=$(mktemp -d -p {params.tmpdir})
kmers=({params.kmers})
SIZES=${{kmers[0]}}
cp {params.kmer_prefix}_${{kmers[0]}}mer.bw $tmpdir
for I in $(seq 1 $(expr ${{#kmers[*]}} - 1)); do
SIZES="${{SIZES}},${{kmers[$I]}}"
cp {params.kmer_prefix}_${{kmers[$I]}}mer.bw $tmpdir
done
cp {input.bam} ${{tmpdir}}/{params.prefix}.namesorted.bam
bin/filter_by_unique_kmers.py ${{tmpdir}}/{params.prefix}.namesorted.bam "${{tmpdir}}/{params.genome}_*mer.bw" \
$SIZES ${{tmpdir}}/{params.prefix}.tmp
mv ${{tmpdir}}/{params.prefix}.tmp {output}
rm -r $tmpdir
"""
rule filter_by_kmers_postsort:
input:
"dedup_kmer/{expID}.{genome}.filtered.bam"
output:
"dedup_kmer/{expID}.{genome}.bam"
wildcard_constraints:
genome=GENOME_SET,
expID=EXPERIMENT_SET
params:
tmp_dir=TMPDIR,
extra=""
threads: 8
log:
"logs/samtools/sort/dedup_kmer.{expID}.{genome}.log"
wrapper:
"0.68.0/bio/samtools/sort"
######## Encode BigWigs #########
rule bam_coverage:
input:
bam=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam",
expID=LABEL_DICT[wildcards.label]),
index=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam.bai",
expID=LABEL_DICT[wildcards.label])
output:
"Encode_BigWigs/{label}.{genome}.bw"
wildcard_constraints:
label=TRACK_SET,
genome=GENOME_SET
threads:
16
log:
"logs/deeptools/bamCoverage/{label}.{genome}.log"
conda:
"envs/deeptools.yaml"
shell:
"""
bamCoverage -b {input.bam} -o {output} -p {threads} -bs 1
"""
rule dedup_bam_coverage:
input:
bam=lambda wildcards: expand("dedup_concat/{expID}.{{genome}}.bam",
expID=LABEL_DICT[wildcards.label]),
index=lambda wildcards: expand("dedup_concat/{expID}.{{genome}}.bam.bai",
expID=LABEL_DICT[wildcards.label])
output:
"Dedup_BigWigs/{label}.{genome}.bw"
wildcard_constraints:
label=CONTROL_TRACK_SET,
genome=GENOME_SET
threads:
16
log:
"logs/deeptools/bamCoverage/dedup_{label}.{genome}.log"
conda:
"envs/deeptools.yaml"
shell:
"""
bamCoverage -b {input.bam} -o {output} -p {threads} -bs 1
"""
rule bigwig_compare:
input:
treat="Encode_BigWigs/{label}.{genome}.bw",
control=lambda wildcards: expand("bigwigs/{control}.{{genome}}.bw",
control=LABEL_CONTROL_DICT[wildcards.label])
output:
"Encode_BigWigs/{label}.{genome}_normed.bw"
wildcard_constraints:
label=NONCONTROL_TRACK_SET,
genome=GENOME_SET
threads:
16
log:
"logs/deeptools/bigwigCompare/{label}.{genome}.log"
conda:
"envs/deeptools.yaml"
shell:
"""
bigwigCompare -p {threads} -bs 50 -b1 {input.treat} -b2 {input.control} \
-o {output} --skipZeroOverZero -of bigwig
"""
######## Macs2 #########
rule macs2_callpeak:
input:
treatment=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam",
expID=LABEL_DICT[wildcards.label]),
control=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam",
expID=EXPERIMENT_CONTROL_DICT[LABEL_DICT[wildcards.label]]),
treatment_index=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam.bai",
expID=LABEL_DICT[wildcards.label]),
control_index=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam.bai",
expID=EXPERIMENT_CONTROL_DICT[LABEL_DICT[wildcards.label]])
output:
multiext("macs2/{label}.{genome}",
"_peaks.xls",
"_peaks.narrowPeak")
params:
lambda wildcards: expand("-f BAMPE -g {size}", size=GENOME_SIZE[wildcards.genome])[0]
wildcard_constraints:
label=NARROW_TRACK_SET,
genome=GENOME_SET
log:
"logs/macs2/{label}.{genome}.log"
wrapper:
"0.66.0/bio/macs2/callpeak"
rule macs2_callpeak_broad:
input:
treatment=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam",
expID=LABEL_DICT[wildcards.label]),
control=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam",
expID=EXPERIMENT_CONTROL_DICT[LABEL_DICT[wildcards.label]]),
treatment_index=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam.bai",
expID=LABEL_DICT[wildcards.label]),
control_index=lambda wildcards: expand("dedup_kmer/{expID}.{{genome}}.bam.bai",
expID=EXPERIMENT_CONTROL_DICT[LABEL_DICT[wildcards.label]])
output:
multiext("macs2/{label}.{genome}",
"_peaks.xls",
"_peaks.broadPeak")
params:
lambda wildcards: expand("-f BAMPE -g {size}", size=GENOME_SIZE[wildcards.genome])[0]
wildcard_constraints:
label=BROAD_TRACK_SET,
genome=GENOME_SET
log:
"logs/macs2/{label}.{genome}.log"
wrapper:
"0.66.0/bio/macs2/callpeak"
rule bedToBigBed:
input:
peaks="macs2/{label}.{genome}_peaks.narrowPeak",
sizes="fasta/{genome}.chrom.sizes",
bedtobigbed="bin/bedToBigBed"
output:
"Encode_BigBeds/{label}.{genome}_peaks.bb"
params:
tmp="Encode_BigBeds/{label}.{genome}.tmp"
wildcard_constraints:
label=NARROW_TRACK_SET,
genome=GENOME_SET
log:
"logs/bigbeds/{label}.{genome}.log"
shell:
"""
awk -v OFS="\t" '{{$5=$5>1000?1000:$5}} {{print}}' {input.peaks} > {params.tmp};
{input.bedtobigbed} -type=bed6+4 {params.tmp} {input.sizes} {output};
rm -f {params.tmp}
"""
rule bedToBigBed_broad:
input:
peaks="macs2/{label}.{genome}_peaks.broadPeak",
sizes="fasta/{genome}.chrom.sizes",
bedtobigbed="bin/bedToBigBed"
output:
"Encode_BigBeds/{label}.{genome}_peaks.bb"
params:
tmp="Encode_BigBeds/{label}.{genome}.tmp"
wildcard_constraints:
label=BROAD_TRACK_SET,
genome=GENOME_SET
log:
"logs/bigbeds_{label}.{genome}.log"
shell:
"""
awk -v OFS="\t" '{{$5=$5>1000?1000:$5}} {{print}}' {input.peaks} > {params.tmp};
{input.bedtobigbed} -type=bed6+3 {params.tmp} {input.sizes} {output};
rm -f {params.tmp}
"""
rule liftOver_peaks:
input:
peaks="macs2/{track}.GRCh38p13_peaks.narrowPeak",
liftover="bin/liftOver",
bedtobigbed="bin/bedToBigBed",
sizes="fasta/chm13v1.chrom.sizes",
chain="data/hg38.t2t-chm13-v1.0.over.chain.gz"
output:
lifted="macs2/{track}.GRCh38p13-chm13v1_peaks.narrowPeak",
unlifted="macs2/{track}.GRCh38p13-chm13v1_peaks.unlifted",
bb="Encode_BigBeds/{track}_GRCh38p13LO_peaks.bb"
params:
track="{track}"
wildcard_constraints:
track=NARROW_TRACK_SET
log:
"logs/liftover/{track}.log"
shell:
"""
{input.liftover} -minMatch=0.2 -bedPlus=3 {input.peaks} {input.chain} \
tmp_{params.track} {output.unlifted}
sort -k1,1 -k2,2n tmp_{params.track} > tmp2_{params.track}
awk 'BEGIN{{ OFS="\t" }} NR==FNR {{ sizes[$1]=$2; next }} \
{{ if (sizes[$1] > $3) print $0; else if (sizes[$1] > $2){{ $3=sizes[$1]; print $0; }} }}' \
{input.sizes} tmp2_{params.track} > {output.lifted}
{input.bedtobigbed} -type=bed3+7 {output.lifted} {input.sizes} {output.bb}
rm tmp_{params.track} tmp2_{params.track}
"""
rule liftOver_peaks_broad:
input:
peaks="macs2/{track}.GRCh38p13_peaks.broadPeak",
liftover="bin/liftOver",
bedtobigbed="bin/bedToBigBed",
sizes="fasta/chm13v1.chrom.sizes",
chain="data/hg38.t2t-chm13-v1.0.over.chain.gz"
output:
lifted="macs2/{track}.GRCh38p13-chm13v1_peaks.broadPeak",
unlifted="macs2/{track}.GRCh38p13-chm13v1_peaks.unlifted",
bb="Encode_BigBeds/{track}_GRCh38p13LO_peaks.bb",
params:
track="{track}"
wildcard_constraints:
track=BROAD_TRACK_SET
log:
"logs/liftover/{track}.log"
shell:
"""
{input.liftover} -minMatch=0.2 -bedPlus=3 {input.peaks} {input.chain} \
tmp_{params.track} {output.unlifted}
sort -k1,1 -k2,2n tmp_{params.track} > tmp2_{params.track}
awk 'BEGIN{{ OFS="\t" }} NR==FNR {{ sizes[$1]=$2; next }} \
{{ if (sizes[$1] > $3) print $0; else if (sizes[$1] > $2){{ $3=sizes[$1]; print $0; }} }}' \
{input.sizes} tmp2_{params.track} > {output.lifted}
{input.bedtobigbed} -type=bed3+6 {output.lifted} {input.sizes} {output.bb}
rm tmp_{params.track} tmp2_{params.track}
"""
######## Analysis #########
rule get_control_mapping_stats:
input:
expand("{folder}/{libID}.{genome}.stats",
folder=['mapped','filtered'],
genome=GENOMES, libID=CONTROL_LIBRARIES),
expand("dedup_concat/{expID}.{genome}.stats",
genome=GENOMES, expID=CONTROL_EXPERIMENTS),
expand("dedup_kmer/{expID}.{genome}.stats",
genome=GENOMES, expID=CONTROL_EXPERIMENTS)
output:
"results/control_mapping_stats.txt"
params:
genomes=GENOMES,
folders=['mapped','filtered','dedup', 'kmer'],
expID=CONTROL_EXPERIMENTS,
samples=SAMPLEFILE
log:
"logs/analysis/control_mapping_stats.log"
shell:
"""
genomes=({params.genomes})
folders=({params.folders})
expID=({params.expID})
temp="expID,group,mark"
for G in ${{genomes[*]}}; do
temp="${{temp}},${{G}}"
done
echo -e "$temp" > {output}
for E in ${{expID[*]}}; do
total=$(eval grep ${{E}} {params.samples} | grep -v ",${{E}}," | \
awk 'BEGIN{{ FS=","; ORS=""; COUNT=0 }}{{ COUNT=COUNT+$9 }}END{{ print COUNT }}')
echo "${{E}},total,${{total}},${{total}}" >> {output}
LIBS=($(eval grep ${{E}} {params.samples} | grep -v ",${{E}}," | \
awk 'BEGIN{{ FS=","; OFS=""; ORS=" " }}{{ print $2 }}'))
for F in mapped filtered; do
temp="${{E}},${{F}}"