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slurm-19779092.out
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[-] Unloading python 3.7 ...
[+] Loading python 3.7 ...
[-] Unloading snakemake 5.24.1
[+] Loading snakemake 5.24.1
[+] Loading singularity 3.8.0 on cn2291
Building DAG of jobs...
Using shell: /usr/bin/bash
Provided cluster nodes: 500
Job counts:
count jobs
1 all
16 call_mutations
1 create_nfragments_table
16 create_toSNPcalling_BAM
16 cutadapt
16 fastq_screen
1 get_fastq_nreads
16 get_fastuniq_readids
16 get_nfragments_json
32 get_split_reads
1 gtf2bed12
16 hisat
16 htseq
16 split_bam_by_mutation
16 star
196
[Thu Jul 22 22:23:06 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_4SU_labeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_4SU_labeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_4SU_labeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_4SU_labeled.R2.trim.fastq.gz
jobid: 7
wildcards: sample=WT4_4SU_labeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_4SU_labeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_4SU_labeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_4SU_labeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_4SU_labeled.R2.fastq.gz
Submitted job 7 with external jobid '19779332'.
[Thu Jul 22 22:23:09 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_1.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_1.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz
jobid: 9
wildcards: sample=SRA_labeled_1
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_1.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_1.R2.fastq.gz
Submitted job 9 with external jobid '19779334'.
[Thu Jul 22 22:23:11 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_2.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_2.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz
jobid: 10
wildcards: sample=SRA_labeled_2
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_2.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_labeled_2.R2.fastq.gz
Submitted job 10 with external jobid '19779336'.
[Thu Jul 22 22:23:19 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_2.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_2.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz
jobid: 12
wildcards: sample=SRA_unlabeled_2
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_2.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_2.R2.fastq.gz
Submitted job 12 with external jobid '19779339'.
[Thu Jul 22 22:23:22 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep2_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep2_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep2_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep2_unlabeled.R2.trim.fastq.gz
jobid: 14
wildcards: sample=KO1_techrep2_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep2_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep2_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep2_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep2_unlabeled.R2.fastq.gz
Submitted job 14 with external jobid '19779342'.
[Thu Jul 22 22:23:27 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_4SU_labeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_4SU_labeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_4SU_labeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_4SU_labeled.R2.trim.fastq.gz
jobid: 1
wildcards: sample=KO1_4SU_labeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_4SU_labeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_4SU_labeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_4SU_labeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_4SU_labeled.R2.fastq.gz
Submitted job 1 with external jobid '19779344'.
[Thu Jul 22 22:23:31 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep2_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep2_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep2_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep2_unlabeled.R2.trim.fastq.gz
jobid: 16
wildcards: sample=WT4_techrep2_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep2_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep2_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep2_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep2_unlabeled.R2.fastq.gz
Submitted job 16 with external jobid '19779452'.
[Thu Jul 22 22:23:38 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_unlabeled.R2.trim.fastq.gz
jobid: 4
wildcards: sample=KO8_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_unlabeled.R2.fastq.gz
Submitted job 4 with external jobid '19779542'.
[Thu Jul 22 22:23:49 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep1_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep1_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep1_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep1_unlabeled.R2.trim.fastq.gz
jobid: 15
wildcards: sample=WT4_techrep1_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep1_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_techrep1_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep1_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_techrep1_unlabeled.R2.fastq.gz
Submitted job 15 with external jobid '19779544'.
[Thu Jul 22 22:23:52 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_unlabeled.R2.trim.fastq.gz
jobid: 6
wildcards: sample=WT3_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_unlabeled.R2.fastq.gz
Submitted job 6 with external jobid '19779545'.
[Thu Jul 22 22:23:57 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_unlabeled.R2.trim.fastq.gz
jobid: 8
wildcards: sample=WT4_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT4_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT4_unlabeled.R2.fastq.gz
Submitted job 8 with external jobid '19779547'.
[Thu Jul 22 22:24:01 2021]
rule gtf2bed12:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.v25.plus_rDNA.plus_5S.gtf
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/mm10.bed12
jobid: 195
bn=$(basename /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.v25.plus_rDNA.plus_5S.gtf)
gtfToGenePred /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.v25.plus_rDNA.plus_5S.gtf /dev/shm/${bn%.*}.genepred
genePredToBed /dev/shm/${bn%.*}.genepred /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/mm10.bed12
Submitted job 195 with external jobid '19779549'.
[Thu Jul 22 22:24:03 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_1.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_1.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz
jobid: 11
wildcards: sample=SRA_unlabeled_1
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_1.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/SRA_unlabeled_1.R2.fastq.gz
Submitted job 11 with external jobid '19779550'.
[Thu Jul 22 22:24:06 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep1_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep1_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep1_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep1_unlabeled.R2.trim.fastq.gz
jobid: 13
wildcards: sample=KO1_techrep1_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep1_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_techrep1_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep1_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_techrep1_unlabeled.R2.fastq.gz
Submitted job 13 with external jobid '19779552'.
[Thu Jul 22 22:24:08 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_unlabeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_unlabeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_unlabeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_unlabeled.R2.trim.fastq.gz
jobid: 2
wildcards: sample=KO1_unlabeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_unlabeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO1_unlabeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_unlabeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO1_unlabeled.R2.fastq.gz
Submitted job 2 with external jobid '19779554'.
[Thu Jul 22 22:24:12 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_4SU_labeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_4SU_labeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_4SU_labeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_4SU_labeled.R2.trim.fastq.gz
jobid: 3
wildcards: sample=KO8_4SU_labeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_4SU_labeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/KO8_4SU_labeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_4SU_labeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/KO8_4SU_labeled.R2.fastq.gz
Submitted job 3 with external jobid '19779561'.
[Thu Jul 22 22:24:19 2021]
rule cutadapt:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_4SU_labeled.R1.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_4SU_labeled.R2.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_4SU_labeled.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_4SU_labeled.R2.trim.fastq.gz
jobid: 5
wildcards: sample=WT3_4SU_labeled
threads: 56
cutadapt --pair-filter=any --nextseq-trim=2 --trim-n -n 5 -O 5 -q 10,10 -m 35:35 -b file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -B file:/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/TruSeq_and_nextera_adapters.consolidated.fa -j 56 -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_4SU_labeled.R1.trim.fastq.gz -p /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/WT3_4SU_labeled.R2.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_4SU_labeled.R1.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/fastqs/WT3_4SU_labeled.R2.fastq.gz
Submitted job 5 with external jobid '19779563'.
[Thu Jul 22 22:27:14 2021]
Finished job 195.
1 of 196 steps (0.51%) done
[Thu Jul 22 22:35:17 2021]
Finished job 12.
2 of 196 steps (1%) done
[Thu Jul 22 22:35:17 2021]
rule star:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.Aligned.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.Aligned.toTranscriptome.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.minus.bam.flagstat
jobid: 158
wildcards: sample=SRA_unlabeled_2
threads: 56
tmpdir=/lscratch/$SLURM_JOB_ID
cd /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star
# Align with STAR and remove secondary/supplementary alignments
readlength=$(
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz | awk -v maxlen=100 'NR%4==2 {if (length($1) > maxlen+0) maxlen=length($1)}; END {print maxlen-1}'
)
STAR --genomeDir /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/STAR_index_mm10_plus_rDNA_plus_5S_id90_masked --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outFilterType BySJout --readFilesIn /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz --readFilesCommand zcat --runThreadN 56 --outFileNamePrefix SRA_unlabeled_2. --twopassMode Basic --sjdbGTFfile /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.v25.plus_rDNA.plus_5S.gtf --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM Unsorted --alignEndsProtrude 10 ConcordantPair --peOverlapNbasesMin 10 --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp_SRA_unlabeled_2 --sjdbOverhang ${readlength}
rm -rf SRA_unlabeled_2._STAR*
samtools flagstat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.Aligned.out.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam.flagstat
# The above STAR command will generate WT4_techrep2_unlabeled.Aligned.out.bam and WT4_techrep2_unlabeled.Aligned.toTranscriptome.out.bam
samtools view -@56 -b -F 4 -F 256 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.Aligned.out.bam > ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
samtools sort -@56 --output-fmt BAM -o ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.bam ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.tmp.bam && rm -f ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.tmp.bam
samtools flagstat -@56 ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.post_secondary_supplementary_filter.bam.flagstat
rm -f ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.tmp.bam
samtools index -@56 ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.bam
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_unlabeled_2.post_secondary_supplementary_filter.bam -o ${tmpdir}/SRA_unlabeled_2.post_insertion_filter.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=${tmpdir}/SRA_unlabeled_2.post_insertion_filter.bam O=${tmpdir}/SRA_unlabeled_2.postfixmate.bam ASSUME_SORTED=false CREATE_INDEX=true SORT_ORDER=coordinate
samtools flagstat -@56 ${tmpdir}/SRA_unlabeled_2.postfixmate.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_unlabeled_2.postfixmate.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam -q 3 -n 1000
# Index and collect stats
samtools index -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam > ${tmpdir}/SRA_unlabeled_2
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam > ${tmpdir}/SRA_unlabeled_2.F1.bam
samtools view -@56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam > ${tmpdir}/SRA_unlabeled_2.F2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_unlabeled_2.F.bam ${tmpdir}/SRA_unlabeled_2.F1.bam ${tmpdir}/SRA_unlabeled_2.F2.bam
#rm -f ${tmpdir}/SRA_unlabeled_2.F1.bam ${tmpdir}/SRA_unlabeled_2.F2.bam
samtools view -@56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam > ${tmpdir}/SRA_unlabeled_2.R1.bam
samtools view -@56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.bam > ${tmpdir}/SRA_unlabeled_2.R2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_unlabeled_2.R.bam ${tmpdir}/SRA_unlabeled_2.R1.bam ${tmpdir}/SRA_unlabeled_2.R2.bam
#rm -f ${tmpdir}/SRA_unlabeled_2.R1.bam ${tmpdir}/SRA_unlabeled_2.R2.bam
# Sort and collect stats for plus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.plus.bam ${tmpdir}/SRA_unlabeled_2.F.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.minus.bam ${tmpdir}/SRA_unlabeled_2.R.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_2.minus.bam.flagstat
# cleanup
rm -rf ${tmpdir}/SRA_unlabeled_2.*
rm -rf _STARtmp
Submitted job 158 with external jobid '19780419'.
[Thu Jul 22 22:35:22 2021]
rule hisat:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.minus.bam.flagstat
jobid: 61
wildcards: sample=SRA_unlabeled_2
threads: 56
# Align with HISAT and remove secondary/supplementary alignments
hisat2 -x /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.plus_rDNA_plus_5S.id90_masked -1 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz -2 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz --rna-strandness RF --known-splicesite-infile /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/mm10.splicesites.txt --summary-file /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.hisat2.summary.log --threads 56 --rg-id SRA_unlabeled_2 --rg SM:SRA_unlabeled_2 --un-gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.unmapped.fastq.gz --mp 4,2 | samtools view -@56 -bS -F 4 -F 256 - > /dev/shm/SRA_unlabeled_2.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_unlabeled_2.post_secondary_supplementary_filter.bam /dev/shm/SRA_unlabeled_2.post_secondary_supplementary_filter.tmp.bam && rm -f /dev/shm/SRA_unlabeled_2.post_secondary_supplementary_filter.tmp.bam
sambamba flagstat --nthreads=56 /dev/shm/SRA_unlabeled_2.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.post_secondary_supplementary_filter.bam.flagstat
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_unlabeled_2.post_secondary_supplementary_filter.bam -o /dev/shm/SRA_unlabeled_2.post_insertion_filter.tmp.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
sambamba sort -n --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_unlabeled_2.post_insertion_filter.bam /dev/shm/SRA_unlabeled_2.post_insertion_filter.tmp.bam && rm -f /dev/shm/SRA_unlabeled_2.post_insertion_filter.tmp.bam
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=/dev/shm/SRA_unlabeled_2.post_insertion_filter.bam O=/dev/stdout ASSUME_SORTED=true QUIET=true | sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_unlabeled_2.postsambambasort.bam /dev/stdin
sambamba flagstat --nthreads=56 /dev/shm/SRA_unlabeled_2.postsambambasort.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_unlabeled_2.postsambambasort.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam -q 3 -n 1000
# Index and collect stats
sambamba index --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam.flagstat
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@ 56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam > /dev/shm/SRA_unlabeled_2.F1.bam
samtools view -@ 56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam > /dev/shm/SRA_unlabeled_2.F2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_unlabeled_2.F.bam /dev/shm/SRA_unlabeled_2.F1.bam /dev/shm/SRA_unlabeled_2.F2.bam && rm -f /dev/shm/SRA_unlabeled_2.F1.bam /dev/shm/SRA_unlabeled_2.F2.bam
samtools view -@ 56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam > /dev/shm/SRA_unlabeled_2.R1.bam
samtools view -@ 56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.bam > /dev/shm/SRA_unlabeled_2.R2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_unlabeled_2.R.bam /dev/shm/SRA_unlabeled_2.R1.bam /dev/shm/SRA_unlabeled_2.R2.bam && rm -f /dev/shm/SRA_unlabeled_2.R1.bam /dev/shm/SRA_unlabeled_2.R2.bam
# Sort and collect stats for plus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.plus.bam /dev/shm/SRA_unlabeled_2.F.bam && rm -f /dev/shm/SRA_unlabeled_2.F.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.minus.bam /dev/shm/SRA_unlabeled_2.R.bam && rm -f /dev/shm/SRA_unlabeled_2.R.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_2.minus.bam.flagstat
# cleanup
rm -rf /dev/shm/SRA_unlabeled_2.*
Submitted job 61 with external jobid '19780503'.
[Thu Jul 22 22:35:25 2021]
rule get_fastuniq_readids:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_2.R1.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_2.R2.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_2.fastuniq.readids
jobid: 28
wildcards: sample=SRA_unlabeled_2
threads: 56
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R1.fastq
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R2.fastq
echo "/lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R1.fastq" > /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.list
echo "/lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R2.fastq" >> /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.list
/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastuniq -i /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.list -t q -o /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R1.trim.fastuniq.fastq -p /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R2.trim.fastuniq.fastq -c 0
awk '{if (! ((FNR + 3) % 4)) { print(substr($1,2))}}' /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R1.trim.fastuniq.fastq > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_2.fastuniq.readids
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R1.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R1.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_2.R1.trim.fastuniq.fastq.gz
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R2.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_unlabeled_2.R2.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_2.R2.trim.fastuniq.fastq.gz
echo "DONE!"
Submitted job 28 with external jobid '19780556'.
[Thu Jul 22 22:35:31 2021]
rule fastq_screen:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_unlabeled_2.R1.trim_screen.txt, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_unlabeled_2.R2.trim_screen.txt
jobid: 45
wildcards: sample=SRA_unlabeled_2
threads: 56
fastq_screen --conf /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastq_screen.conf --outdir "/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen" --threads 56 --subset 1000000 --aligner bowtie2 --force /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_2.R2.trim.fastq.gz
Submitted job 45 with external jobid '19780558'.
[Thu Jul 22 22:37:29 2021]
Finished job 11.
3 of 196 steps (2%) done
[Thu Jul 22 22:37:29 2021]
rule hisat:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.minus.bam.flagstat
jobid: 60
wildcards: sample=SRA_unlabeled_1
threads: 56
# Align with HISAT and remove secondary/supplementary alignments
hisat2 -x /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.plus_rDNA_plus_5S.id90_masked -1 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz -2 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz --rna-strandness RF --known-splicesite-infile /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/mm10.splicesites.txt --summary-file /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.hisat2.summary.log --threads 56 --rg-id SRA_unlabeled_1 --rg SM:SRA_unlabeled_1 --un-gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.unmapped.fastq.gz --mp 4,2 | samtools view -@56 -bS -F 4 -F 256 - > /dev/shm/SRA_unlabeled_1.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_unlabeled_1.post_secondary_supplementary_filter.bam /dev/shm/SRA_unlabeled_1.post_secondary_supplementary_filter.tmp.bam && rm -f /dev/shm/SRA_unlabeled_1.post_secondary_supplementary_filter.tmp.bam
sambamba flagstat --nthreads=56 /dev/shm/SRA_unlabeled_1.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.post_secondary_supplementary_filter.bam.flagstat
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_unlabeled_1.post_secondary_supplementary_filter.bam -o /dev/shm/SRA_unlabeled_1.post_insertion_filter.tmp.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
sambamba sort -n --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_unlabeled_1.post_insertion_filter.bam /dev/shm/SRA_unlabeled_1.post_insertion_filter.tmp.bam && rm -f /dev/shm/SRA_unlabeled_1.post_insertion_filter.tmp.bam
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=/dev/shm/SRA_unlabeled_1.post_insertion_filter.bam O=/dev/stdout ASSUME_SORTED=true QUIET=true | sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_unlabeled_1.postsambambasort.bam /dev/stdin
sambamba flagstat --nthreads=56 /dev/shm/SRA_unlabeled_1.postsambambasort.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_unlabeled_1.postsambambasort.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam -q 3 -n 1000
# Index and collect stats
sambamba index --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam.flagstat
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@ 56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam > /dev/shm/SRA_unlabeled_1.F1.bam
samtools view -@ 56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam > /dev/shm/SRA_unlabeled_1.F2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_unlabeled_1.F.bam /dev/shm/SRA_unlabeled_1.F1.bam /dev/shm/SRA_unlabeled_1.F2.bam && rm -f /dev/shm/SRA_unlabeled_1.F1.bam /dev/shm/SRA_unlabeled_1.F2.bam
samtools view -@ 56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam > /dev/shm/SRA_unlabeled_1.R1.bam
samtools view -@ 56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.bam > /dev/shm/SRA_unlabeled_1.R2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_unlabeled_1.R.bam /dev/shm/SRA_unlabeled_1.R1.bam /dev/shm/SRA_unlabeled_1.R2.bam && rm -f /dev/shm/SRA_unlabeled_1.R1.bam /dev/shm/SRA_unlabeled_1.R2.bam
# Sort and collect stats for plus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.plus.bam /dev/shm/SRA_unlabeled_1.F.bam && rm -f /dev/shm/SRA_unlabeled_1.F.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.minus.bam /dev/shm/SRA_unlabeled_1.R.bam && rm -f /dev/shm/SRA_unlabeled_1.R.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_unlabeled_1.minus.bam.flagstat
# cleanup
rm -rf /dev/shm/SRA_unlabeled_1.*
Submitted job 60 with external jobid '19780630'.
[Thu Jul 22 22:37:31 2021]
rule get_fastuniq_readids:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_1.R1.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_1.R2.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_1.fastuniq.readids
jobid: 27
wildcards: sample=SRA_unlabeled_1
threads: 56
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R1.fastq
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R2.fastq
echo "/lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R1.fastq" > /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.list
echo "/lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R2.fastq" >> /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.list
/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastuniq -i /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.list -t q -o /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R1.trim.fastuniq.fastq -p /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R2.trim.fastuniq.fastq -c 0
awk '{if (! ((FNR + 3) % 4)) { print(substr($1,2))}}' /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R1.trim.fastuniq.fastq > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_1.fastuniq.readids
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R1.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R1.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_1.R1.trim.fastuniq.fastq.gz
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R2.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_unlabeled_1.R2.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_unlabeled_1.R2.trim.fastuniq.fastq.gz
echo "DONE!"
Submitted job 27 with external jobid '19780633'.
[Thu Jul 22 22:37:33 2021]
rule star:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.Aligned.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.Aligned.toTranscriptome.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.minus.bam.flagstat
jobid: 157
wildcards: sample=SRA_unlabeled_1
threads: 56
tmpdir=/lscratch/$SLURM_JOB_ID
cd /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star
# Align with STAR and remove secondary/supplementary alignments
readlength=$(
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz | awk -v maxlen=100 'NR%4==2 {if (length($1) > maxlen+0) maxlen=length($1)}; END {print maxlen-1}'
)
STAR --genomeDir /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/STAR_index_mm10_plus_rDNA_plus_5S_id90_masked --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outFilterType BySJout --readFilesIn /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz --readFilesCommand zcat --runThreadN 56 --outFileNamePrefix SRA_unlabeled_1. --twopassMode Basic --sjdbGTFfile /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.v25.plus_rDNA.plus_5S.gtf --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM Unsorted --alignEndsProtrude 10 ConcordantPair --peOverlapNbasesMin 10 --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp_SRA_unlabeled_1 --sjdbOverhang ${readlength}
rm -rf SRA_unlabeled_1._STAR*
samtools flagstat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.Aligned.out.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam.flagstat
# The above STAR command will generate WT4_techrep2_unlabeled.Aligned.out.bam and WT4_techrep2_unlabeled.Aligned.toTranscriptome.out.bam
samtools view -@56 -b -F 4 -F 256 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.Aligned.out.bam > ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
samtools sort -@56 --output-fmt BAM -o ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.bam ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.tmp.bam && rm -f ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.tmp.bam
samtools flagstat -@56 ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.post_secondary_supplementary_filter.bam.flagstat
rm -f ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.tmp.bam
samtools index -@56 ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.bam
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_unlabeled_1.post_secondary_supplementary_filter.bam -o ${tmpdir}/SRA_unlabeled_1.post_insertion_filter.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=${tmpdir}/SRA_unlabeled_1.post_insertion_filter.bam O=${tmpdir}/SRA_unlabeled_1.postfixmate.bam ASSUME_SORTED=false CREATE_INDEX=true SORT_ORDER=coordinate
samtools flagstat -@56 ${tmpdir}/SRA_unlabeled_1.postfixmate.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_unlabeled_1.postfixmate.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam -q 3 -n 1000
# Index and collect stats
samtools index -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam > ${tmpdir}/SRA_unlabeled_1
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam > ${tmpdir}/SRA_unlabeled_1.F1.bam
samtools view -@56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam > ${tmpdir}/SRA_unlabeled_1.F2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_unlabeled_1.F.bam ${tmpdir}/SRA_unlabeled_1.F1.bam ${tmpdir}/SRA_unlabeled_1.F2.bam
#rm -f ${tmpdir}/SRA_unlabeled_1.F1.bam ${tmpdir}/SRA_unlabeled_1.F2.bam
samtools view -@56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam > ${tmpdir}/SRA_unlabeled_1.R1.bam
samtools view -@56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.bam > ${tmpdir}/SRA_unlabeled_1.R2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_unlabeled_1.R.bam ${tmpdir}/SRA_unlabeled_1.R1.bam ${tmpdir}/SRA_unlabeled_1.R2.bam
#rm -f ${tmpdir}/SRA_unlabeled_1.R1.bam ${tmpdir}/SRA_unlabeled_1.R2.bam
# Sort and collect stats for plus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.plus.bam ${tmpdir}/SRA_unlabeled_1.F.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.minus.bam ${tmpdir}/SRA_unlabeled_1.R.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_unlabeled_1.minus.bam.flagstat
# cleanup
rm -rf ${tmpdir}/SRA_unlabeled_1.*
rm -rf _STARtmp
Submitted job 157 with external jobid '19780636'.
[Thu Jul 22 22:37:34 2021]
rule fastq_screen:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_unlabeled_1.R1.trim_screen.txt, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_unlabeled_1.R2.trim_screen.txt
jobid: 44
wildcards: sample=SRA_unlabeled_1
threads: 56
fastq_screen --conf /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastq_screen.conf --outdir "/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen" --threads 56 --subset 1000000 --aligner bowtie2 --force /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_unlabeled_1.R2.trim.fastq.gz
Submitted job 44 with external jobid '19780638'.
[Thu Jul 22 22:37:52 2021]
Finished job 9.
4 of 196 steps (2%) done
[Thu Jul 22 22:37:52 2021]
rule get_fastuniq_readids:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_1.R1.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_1.R2.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_1.fastuniq.readids
jobid: 25
wildcards: sample=SRA_labeled_1
threads: 56
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_labeled_1.R1.fastq
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_labeled_1.R2.fastq
echo "/lscratch/${SLURM_JOBID}/SRA_labeled_1.R1.fastq" > /lscratch/${SLURM_JOBID}/SRA_labeled_1.list
echo "/lscratch/${SLURM_JOBID}/SRA_labeled_1.R2.fastq" >> /lscratch/${SLURM_JOBID}/SRA_labeled_1.list
/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastuniq -i /lscratch/${SLURM_JOBID}/SRA_labeled_1.list -t q -o /lscratch/${SLURM_JOBID}/SRA_labeled_1.R1.trim.fastuniq.fastq -p /lscratch/${SLURM_JOBID}/SRA_labeled_1.R2.trim.fastuniq.fastq -c 0
awk '{if (! ((FNR + 3) % 4)) { print(substr($1,2))}}' /lscratch/${SLURM_JOBID}/SRA_labeled_1.R1.trim.fastuniq.fastq > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_1.fastuniq.readids
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_labeled_1.R1.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_labeled_1.R1.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_1.R1.trim.fastuniq.fastq.gz
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_labeled_1.R2.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_labeled_1.R2.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_1.R2.trim.fastuniq.fastq.gz
echo "DONE!"
Submitted job 25 with external jobid '19780656'.
[Thu Jul 22 22:37:54 2021]
rule fastq_screen:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_labeled_1.R1.trim_screen.txt, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_labeled_1.R2.trim_screen.txt
jobid: 42
wildcards: sample=SRA_labeled_1
threads: 56
fastq_screen --conf /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastq_screen.conf --outdir "/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen" --threads 56 --subset 1000000 --aligner bowtie2 --force /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz
Submitted job 42 with external jobid '19780658'.
[Thu Jul 22 22:37:55 2021]
rule star:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.Aligned.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.Aligned.toTranscriptome.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.minus.bam.flagstat
jobid: 155
wildcards: sample=SRA_labeled_1
threads: 56
tmpdir=/lscratch/$SLURM_JOB_ID
cd /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star
# Align with STAR and remove secondary/supplementary alignments
readlength=$(
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz | awk -v maxlen=100 'NR%4==2 {if (length($1) > maxlen+0) maxlen=length($1)}; END {print maxlen-1}'
)
STAR --genomeDir /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/STAR_index_mm10_plus_rDNA_plus_5S_id90_masked --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outFilterType BySJout --readFilesIn /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz --readFilesCommand zcat --runThreadN 56 --outFileNamePrefix SRA_labeled_1. --twopassMode Basic --sjdbGTFfile /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.v25.plus_rDNA.plus_5S.gtf --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM Unsorted --alignEndsProtrude 10 ConcordantPair --peOverlapNbasesMin 10 --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp_SRA_labeled_1 --sjdbOverhang ${readlength}
rm -rf SRA_labeled_1._STAR*
samtools flagstat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.Aligned.out.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam.flagstat
# The above STAR command will generate WT4_techrep2_unlabeled.Aligned.out.bam and WT4_techrep2_unlabeled.Aligned.toTranscriptome.out.bam
samtools view -@56 -b -F 4 -F 256 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.Aligned.out.bam > ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
samtools sort -@56 --output-fmt BAM -o ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.bam ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.tmp.bam && rm -f ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.tmp.bam
samtools flagstat -@56 ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.post_secondary_supplementary_filter.bam.flagstat
rm -f ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.tmp.bam
samtools index -@56 ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.bam
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_labeled_1.post_secondary_supplementary_filter.bam -o ${tmpdir}/SRA_labeled_1.post_insertion_filter.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=${tmpdir}/SRA_labeled_1.post_insertion_filter.bam O=${tmpdir}/SRA_labeled_1.postfixmate.bam ASSUME_SORTED=false CREATE_INDEX=true SORT_ORDER=coordinate
samtools flagstat -@56 ${tmpdir}/SRA_labeled_1.postfixmate.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_labeled_1.postfixmate.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam -q 3 -n 1000
# Index and collect stats
samtools index -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam > ${tmpdir}/SRA_labeled_1
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam > ${tmpdir}/SRA_labeled_1.F1.bam
samtools view -@56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam > ${tmpdir}/SRA_labeled_1.F2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_labeled_1.F.bam ${tmpdir}/SRA_labeled_1.F1.bam ${tmpdir}/SRA_labeled_1.F2.bam
#rm -f ${tmpdir}/SRA_labeled_1.F1.bam ${tmpdir}/SRA_labeled_1.F2.bam
samtools view -@56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam > ${tmpdir}/SRA_labeled_1.R1.bam
samtools view -@56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.bam > ${tmpdir}/SRA_labeled_1.R2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_labeled_1.R.bam ${tmpdir}/SRA_labeled_1.R1.bam ${tmpdir}/SRA_labeled_1.R2.bam
#rm -f ${tmpdir}/SRA_labeled_1.R1.bam ${tmpdir}/SRA_labeled_1.R2.bam
# Sort and collect stats for plus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.plus.bam ${tmpdir}/SRA_labeled_1.F.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.minus.bam ${tmpdir}/SRA_labeled_1.R.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_1.minus.bam.flagstat
# cleanup
rm -rf ${tmpdir}/SRA_labeled_1.*
rm -rf _STARtmp
Submitted job 155 with external jobid '19780660'.
[Thu Jul 22 22:37:56 2021]
rule hisat:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.minus.bam.flagstat
jobid: 58
wildcards: sample=SRA_labeled_1
threads: 56
# Align with HISAT and remove secondary/supplementary alignments
hisat2 -x /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.plus_rDNA_plus_5S.id90_masked -1 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R1.trim.fastq.gz -2 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_1.R2.trim.fastq.gz --rna-strandness RF --known-splicesite-infile /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/mm10.splicesites.txt --summary-file /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.hisat2.summary.log --threads 56 --rg-id SRA_labeled_1 --rg SM:SRA_labeled_1 --un-gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.unmapped.fastq.gz --mp 4,2 | samtools view -@56 -bS -F 4 -F 256 - > /dev/shm/SRA_labeled_1.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_labeled_1.post_secondary_supplementary_filter.bam /dev/shm/SRA_labeled_1.post_secondary_supplementary_filter.tmp.bam && rm -f /dev/shm/SRA_labeled_1.post_secondary_supplementary_filter.tmp.bam
sambamba flagstat --nthreads=56 /dev/shm/SRA_labeled_1.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.post_secondary_supplementary_filter.bam.flagstat
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_labeled_1.post_secondary_supplementary_filter.bam -o /dev/shm/SRA_labeled_1.post_insertion_filter.tmp.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
sambamba sort -n --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_labeled_1.post_insertion_filter.bam /dev/shm/SRA_labeled_1.post_insertion_filter.tmp.bam && rm -f /dev/shm/SRA_labeled_1.post_insertion_filter.tmp.bam
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=/dev/shm/SRA_labeled_1.post_insertion_filter.bam O=/dev/stdout ASSUME_SORTED=true QUIET=true | sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_labeled_1.postsambambasort.bam /dev/stdin
sambamba flagstat --nthreads=56 /dev/shm/SRA_labeled_1.postsambambasort.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_labeled_1.postsambambasort.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam -q 3 -n 1000
# Index and collect stats
sambamba index --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam.flagstat
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@ 56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam > /dev/shm/SRA_labeled_1.F1.bam
samtools view -@ 56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam > /dev/shm/SRA_labeled_1.F2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_labeled_1.F.bam /dev/shm/SRA_labeled_1.F1.bam /dev/shm/SRA_labeled_1.F2.bam && rm -f /dev/shm/SRA_labeled_1.F1.bam /dev/shm/SRA_labeled_1.F2.bam
samtools view -@ 56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam > /dev/shm/SRA_labeled_1.R1.bam
samtools view -@ 56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.bam > /dev/shm/SRA_labeled_1.R2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_labeled_1.R.bam /dev/shm/SRA_labeled_1.R1.bam /dev/shm/SRA_labeled_1.R2.bam && rm -f /dev/shm/SRA_labeled_1.R1.bam /dev/shm/SRA_labeled_1.R2.bam
# Sort and collect stats for plus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.plus.bam /dev/shm/SRA_labeled_1.F.bam && rm -f /dev/shm/SRA_labeled_1.F.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.minus.bam /dev/shm/SRA_labeled_1.R.bam && rm -f /dev/shm/SRA_labeled_1.R.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_1.minus.bam.flagstat
# cleanup
rm -rf /dev/shm/SRA_labeled_1.*
Submitted job 58 with external jobid '19780662'.
[Thu Jul 22 22:40:53 2021]
Finished job 10.
5 of 196 steps (3%) done
[Thu Jul 22 22:40:53 2021]
rule get_fastuniq_readids:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_2.R1.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_2.R2.trim.fastuniq.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_2.fastuniq.readids
jobid: 26
wildcards: sample=SRA_labeled_2
threads: 56
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_labeled_2.R1.fastq
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz > /lscratch/${SLURM_JOBID}/SRA_labeled_2.R2.fastq
echo "/lscratch/${SLURM_JOBID}/SRA_labeled_2.R1.fastq" > /lscratch/${SLURM_JOBID}/SRA_labeled_2.list
echo "/lscratch/${SLURM_JOBID}/SRA_labeled_2.R2.fastq" >> /lscratch/${SLURM_JOBID}/SRA_labeled_2.list
/gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastuniq -i /lscratch/${SLURM_JOBID}/SRA_labeled_2.list -t q -o /lscratch/${SLURM_JOBID}/SRA_labeled_2.R1.trim.fastuniq.fastq -p /lscratch/${SLURM_JOBID}/SRA_labeled_2.R2.trim.fastuniq.fastq -c 0
awk '{if (! ((FNR + 3) % 4)) { print(substr($1,2))}}' /lscratch/${SLURM_JOBID}/SRA_labeled_2.R1.trim.fastuniq.fastq > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_2.fastuniq.readids
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_labeled_2.R1.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_labeled_2.R1.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_2.R1.trim.fastuniq.fastq.gz
pigz -p4 /lscratch/${SLURM_JOBID}/SRA_labeled_2.R2.trim.fastuniq.fastq && mv /lscratch/${SLURM_JOBID}/SRA_labeled_2.R2.trim.fastuniq.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/fastuniq/SRA_labeled_2.R2.trim.fastuniq.fastq.gz
echo "DONE!"
Submitted job 26 with external jobid '19780951'.
[Thu Jul 22 22:40:54 2021]
rule fastq_screen:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_labeled_2.R1.trim_screen.txt, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen/SRA_labeled_2.R2.trim_screen.txt
jobid: 43
wildcards: sample=SRA_labeled_2
threads: 56
fastq_screen --conf /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/fastq_screen.conf --outdir "/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/qc/fastqscreen" --threads 56 --subset 1000000 --aligner bowtie2 --force /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz
Submitted job 43 with external jobid '19780953'.
[Thu Jul 22 22:40:56 2021]
rule hisat:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.minus.bam.flagstat
jobid: 59
wildcards: sample=SRA_labeled_2
threads: 56
# Align with HISAT and remove secondary/supplementary alignments
hisat2 -x /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.plus_rDNA_plus_5S.id90_masked -1 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz -2 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz --rna-strandness RF --known-splicesite-infile /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/resources/mm10.splicesites.txt --summary-file /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.hisat2.summary.log --threads 56 --rg-id SRA_labeled_2 --rg SM:SRA_labeled_2 --un-gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.unmapped.fastq.gz --mp 4,2 | samtools view -@56 -bS -F 4 -F 256 - > /dev/shm/SRA_labeled_2.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_labeled_2.post_secondary_supplementary_filter.bam /dev/shm/SRA_labeled_2.post_secondary_supplementary_filter.tmp.bam && rm -f /dev/shm/SRA_labeled_2.post_secondary_supplementary_filter.tmp.bam
sambamba flagstat --nthreads=56 /dev/shm/SRA_labeled_2.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.post_secondary_supplementary_filter.bam.flagstat
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_labeled_2.post_secondary_supplementary_filter.bam -o /dev/shm/SRA_labeled_2.post_insertion_filter.tmp.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
sambamba sort -n --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_labeled_2.post_insertion_filter.bam /dev/shm/SRA_labeled_2.post_insertion_filter.tmp.bam && rm -f /dev/shm/SRA_labeled_2.post_insertion_filter.tmp.bam
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=/dev/shm/SRA_labeled_2.post_insertion_filter.bam O=/dev/stdout ASSUME_SORTED=true QUIET=true | sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/dev/shm/SRA_labeled_2.postsambambasort.bam /dev/stdin
sambamba flagstat --nthreads=56 /dev/shm/SRA_labeled_2.postsambambasort.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i /dev/shm/SRA_labeled_2.postsambambasort.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam -q 3 -n 1000
# Index and collect stats
sambamba index --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam.flagstat
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@ 56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam > /dev/shm/SRA_labeled_2.F1.bam
samtools view -@ 56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam > /dev/shm/SRA_labeled_2.F2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_labeled_2.F.bam /dev/shm/SRA_labeled_2.F1.bam /dev/shm/SRA_labeled_2.F2.bam && rm -f /dev/shm/SRA_labeled_2.F1.bam /dev/shm/SRA_labeled_2.F2.bam
samtools view -@ 56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam > /dev/shm/SRA_labeled_2.R1.bam
samtools view -@ 56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.bam > /dev/shm/SRA_labeled_2.R2.bam
samtools merge -c -f -p -@ 56 /dev/shm/SRA_labeled_2.R.bam /dev/shm/SRA_labeled_2.R1.bam /dev/shm/SRA_labeled_2.R2.bam && rm -f /dev/shm/SRA_labeled_2.R1.bam /dev/shm/SRA_labeled_2.R2.bam
# Sort and collect stats for plus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.plus.bam /dev/shm/SRA_labeled_2.F.bam && rm -f /dev/shm/SRA_labeled_2.F.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
sambamba sort --memory-limit=200G --tmpdir=/dev/shm --nthreads=56 --out=/data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.minus.bam /dev/shm/SRA_labeled_2.R.bam && rm -f /dev/shm/SRA_labeled_2.R.bam
sambamba flagstat --nthreads=56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/hisat2/SRA_labeled_2.minus.bam.flagstat
# cleanup
rm -rf /dev/shm/SRA_labeled_2.*
Submitted job 59 with external jobid '19780955'.
[Thu Jul 22 22:40:57 2021]
rule star:
input: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz
output: /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.Aligned.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.Aligned.toTranscriptome.out.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.plus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.minus.bam, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.post_secondary_supplementary_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.post_insertion_filter.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.plus.bam.flagstat, /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.minus.bam.flagstat
jobid: 156
wildcards: sample=SRA_labeled_2
threads: 56
tmpdir=/lscratch/$SLURM_JOB_ID
cd /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star
# Align with STAR and remove secondary/supplementary alignments
readlength=$(
zcat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz | awk -v maxlen=100 'NR%4==2 {if (length($1) > maxlen+0) maxlen=length($1)}; END {print maxlen-1}'
)
STAR --genomeDir /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/STAR_index_mm10_plus_rDNA_plus_5S_id90_masked --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outFilterType BySJout --readFilesIn /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R1.trim.fastq.gz /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/trim/SRA_labeled_2.R2.trim.fastq.gz --readFilesCommand zcat --runThreadN 56 --outFileNamePrefix SRA_labeled_2. --twopassMode Basic --sjdbGTFfile /data/RBL_NCI/Wolin/mESC_slam_analysis/resources/mm10/mm10.v25.plus_rDNA.plus_5S.gtf --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM Unsorted --alignEndsProtrude 10 ConcordantPair --peOverlapNbasesMin 10 --outTmpDir=/lscratch/$SLURM_JOB_ID/STARtmp_SRA_labeled_2 --sjdbOverhang ${readlength}
rm -rf SRA_labeled_2._STAR*
samtools flagstat /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.Aligned.out.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam.flagstat
# The above STAR command will generate WT4_techrep2_unlabeled.Aligned.out.bam and WT4_techrep2_unlabeled.Aligned.toTranscriptome.out.bam
samtools view -@56 -b -F 4 -F 256 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.Aligned.out.bam > ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.tmp.bam
# Sort the "post_secondary_supplementary_filter" BAM and collect some stats
samtools sort -@56 --output-fmt BAM -o ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.bam ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.tmp.bam && rm -f ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.tmp.bam
samtools flagstat -@56 ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.post_secondary_supplementary_filter.bam.flagstat
rm -f ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.tmp.bam
samtools index -@56 ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.bam
# Apply the "number of insertions in read" filter
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_labeled_2.post_secondary_supplementary_filter.bam -o ${tmpdir}/SRA_labeled_2.post_insertion_filter.bam -q 0 -n 1
# Sort, FixMateInfo, collect stats
java -Xmx200g -jar $PICARDJARPATH/picard.jar FixMateInformation I=${tmpdir}/SRA_labeled_2.post_insertion_filter.bam O=${tmpdir}/SRA_labeled_2.postfixmate.bam ASSUME_SORTED=false CREATE_INDEX=true SORT_ORDER=coordinate
samtools flagstat -@56 ${tmpdir}/SRA_labeled_2.postfixmate.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.post_insertion_filter.bam.flagstat
# Apply the MAPQ filter and remove "widowed" reads
python /gpfs/gsfs11/users/RBL_NCI/Wolin/mESC_slam_analysis/workflow/4SUseq/workflow/scripts/filter_bam.py -i ${tmpdir}/SRA_labeled_2.postfixmate.bam -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam -q 3 -n 1000
# Index and collect stats
samtools index -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam > ${tmpdir}/SRA_labeled_2
# Split reads into :
# 1. Reads originating from + strand fragments --> these will go for T-to-C mutation detection
# R2
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R1
# 2. Reads originating from - strand fragments --> these will go for A-to-G mutation detection
# R1
# ------>
# 5'-------------------------------------------------3'
# 3'-------------------------------------------------5'
# <------
# R2
samtools view -@56 -b -f 128 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam > ${tmpdir}/SRA_labeled_2.F1.bam
samtools view -@56 -b -f 80 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam > ${tmpdir}/SRA_labeled_2.F2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_labeled_2.F.bam ${tmpdir}/SRA_labeled_2.F1.bam ${tmpdir}/SRA_labeled_2.F2.bam
#rm -f ${tmpdir}/SRA_labeled_2.F1.bam ${tmpdir}/SRA_labeled_2.F2.bam
samtools view -@56 -b -f 144 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam > ${tmpdir}/SRA_labeled_2.R1.bam
samtools view -@56 -b -f 64 -F 16 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.bam > ${tmpdir}/SRA_labeled_2.R2.bam
samtools merge -c -f -p -@56 ${tmpdir}/SRA_labeled_2.R.bam ${tmpdir}/SRA_labeled_2.R1.bam ${tmpdir}/SRA_labeled_2.R2.bam
#rm -f ${tmpdir}/SRA_labeled_2.R1.bam ${tmpdir}/SRA_labeled_2.R2.bam
# Sort and collect stats for plus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.plus.bam ${tmpdir}/SRA_labeled_2.F.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.plus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.plus.bam.flagstat
# Sort and collect stats for minus strand BAM
samtools sort -@56 --output-fmt BAM -o /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.minus.bam ${tmpdir}/SRA_labeled_2.R.bam
samtools flagstat -@56 /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.minus.bam > /data/RBL_NCI/Wolin/mESC_slam_analysis/find_mutations_072221/star/SRA_labeled_2.minus.bam.flagstat
# cleanup
rm -rf ${tmpdir}/SRA_labeled_2.*
rm -rf _STARtmp