# reads processed: 14464188 # reads with at least one alignment: 11910701 (82.35%) # reads that failed to align: 2553487 (17.65%) Reported 64617671 alignments ShortStack version 4.0.3 Beginning run Options: { 'adapter': None, 'align_only': False, 'autotrim': False, 'autotrim_key': 'TCGGACCAGGCTTCATTCCCC', 'bamfile': None, 'dicermax': 24, 'dicermin': 21, 'dn_mirna': True, 'genomefile': '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/data/Porites_evermanni_v1.fa', 'known_miRNAs': '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/data/mirbase-mature-v22.1.fa', 'locifile': None, 'locus': None, 'mincov': 1, 'mmap': 'u', 'nohp': False, 'outdir': '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out', 'pad': 200, 'readfile': [ '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.1-Peve-sRNAseq-trimming-R1-only/trimmed-reads/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.fq.gz'], 'show_secondaries': False, 'strand_cutoff': 0.8, 'threads': 20} Required executable RNAfold : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/RNAfold Required executable strucVis : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/strucVis Required executable bowtie : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/bowtie Required executable bowtie-build : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/bowtie-build Required executable ShortTracks : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/ShortTracks Required executable wigToBigWig : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/wigToBigWig Required executable samtools : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/samtools Beginning alignment phase Fri 22 Mar 2024 16:06:00 -0700 PDT Aligning /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.1-Peve-sRNAseq-trimming-R1-only/trimmed-reads/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.fq.gz First pass alignment with bowtie using 20 threads Second pass - placing multimappers using 20 threads to process 15 chunks [bam_sort_core] merging from 0 files and 20 in-memory blocks... ShortTracks version 1.1 Required executable samtools : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/samtools Required executable wigToBigWig : /home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/wigToBigWig Preparing commands, counting overall reads Getting raw depths samtools view -F 272 -e "qlen==21" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_p_depths.txt samtools view -F 256 -f 16 -e "qlen==21" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_m_depths.txt samtools view -F 272 -e "qlen==22" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_p_depths.txt samtools view -F 256 -f 16 -e "qlen==22" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_m_depths.txt samtools view -F 272 -e "qlen==23 || qlen==24" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_p_depths.txt samtools view -F 256 -f 16 -e "qlen==23 || qlen==24" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_m_depths.txt samtools view -F 272 -e "qlen < 21 || qlen > 24" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_p_depths.txt samtools view -F 256 -f 16 -e "qlen < 21 || qlen > 24" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_m_depths.txt Writing wiggle files /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_m.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_m.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_m.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_m.wig Writing bigwig files wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_21_m.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_22_m.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_23-24_m.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.1-Peve-sRNAseq-ShortStack-R1-reads/ShortStack_out/sRNA-POR-82-S1-TP2_R1_001.fastp-R1-31bp-auto_adapters-polyG_other_m.bw Converting to sorted bam format Uniquely mapped (U) reads: 6187101/14464188 (42.8%) Multi-mapped reads placed (P) with guidance: 4068202/14464188 (28.1%) Multi-mapped reads randomly (R) placed: 1530247/14464188 (10.6%) Very highly (H) multi-mapped reads (>=50 hits): 125151/14464188 (0.9%) Not mapped (N) reads (no hits): 2553487/14464188 (17.7%) Creating browser tracks by readlength and strand using ShortTracks Fri 22 Mar 2024 16:16:27 -0700 PDT Defining small RNA clusters de novo With 14464188 total reads and mincov of 1 reads per million, the min read depth is 14 Fri 22 Mar 2024 16:17:03 -0700 PDT Analyzing cluster properties using 20 threads # reads processed: 48885 # reads with at least one alignment: 84 (0.17%) # reads that failed to align: 48801 (99.83%) Reported 2502 alignments [bam_sort_core] merging from 0 files and 20 in-memory blocks... Fri 22 Mar 2024 16:17:36 -0700 PDT Completed Fri 22 Mar 2024 16:17:36 -0700 PDT Searching for valid microRNA loci Aligning known_miRNAs sequences to genome Screening of possible microRNAs from user provided known_miRNAs Screening of possible de novo microRNAs multiprocessing.pool.RemoteTraceback: """ Traceback (most recent call last): File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/lib/python3.10/multiprocessing/pool.py", line 125, in worker result = (True, func(*args, **kwds)) File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/lib/python3.10/multiprocessing/pool.py", line 51, in starmapstar return list(itertools.starmap(args[0], args[1])) File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/ShortStack", line 2391, in mir_analysis new_locus, s_start, s_end = analyze_fold(mir_locus, dotbracket, bedfields, merged_bam, args) File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/ShortStack", line 2954, in analyze_fold new_dotbracket = foldlines[2].rstrip().split(' ')[0] IndexError: list index out of range """ The above exception was the direct cause of the following exception: Traceback (most recent call last): File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/ShortStack", line 3619, in mir_qdata = mirna(args, merged_bam, fai, pmir_bedfile, read_count) File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/bin/ShortStack", line 2255, in mirna denovo_mloci1 = pool.starmap(mir_analysis, File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/lib/python3.10/multiprocessing/pool.py", line 375, in starmap return self._map_async(func, iterable, starmapstar, chunksize).get() File "/home/sam/programs/mambaforge/envs/ShortStack-4.0.3_env/lib/python3.10/multiprocessing/pool.py", line 774, in get raise self._value IndexError: list index out of range real 11m59.366s user 85m47.874s sys 37m10.732s