# reads processed: 12238952 # reads with at least one alignment: 9008382 (73.60%) # reads that failed to align: 3230570 (26.40%) Reported 36091450 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/cnidarian-mirbase-mature-v22.1.fasta', 'locifile': None, 'locus': None, 'mincov': 1, 'mmap': 'u', 'nohp': False, 'outdir': '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out', 'pad': 200, 'readfile': [ '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.2-Peve-sRNAseq-trimming-31bp-fastp-merged/trimmed-reads/POR-73-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.2-Peve-sRNAseq-trimming-31bp-fastp-merged/trimmed-reads/POR-79-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.2-Peve-sRNAseq-trimming-31bp-fastp-merged/trimmed-reads/POR-82-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz'], 'show_secondaries': False, 'strand_cutoff': 0.8, 'threads': 40} 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 Thu 09 May 2024 10:11:56 -0700 PDT Aligning /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.2-Peve-sRNAseq-trimming-31bp-fastp-merged/trimmed-reads/POR-73-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 13 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 12259576 # reads with at least one alignment: 9725164 (79.33%) # reads that failed to align: 2534412 (20.67%) Reported 56953362 alignments Converting to sorted bam format Uniquely mapped (U) reads: 3761839/12238952 (30.7%) Multi-mapped reads placed (P) with guidance: 2450384/12238952 (20.0%) Multi-mapped reads randomly (R) placed: 2714795/12238952 (22.2%) Very highly (H) multi-mapped reads (>=50 hits): 81364/12238952 (0.7%) Not mapped (N) reads (no hits): 3230570/12238952 (26.4%) Thu 09 May 2024 10:17:11 -0700 PDT Aligning /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.2-Peve-sRNAseq-trimming-31bp-fastp-merged/trimmed-reads/POR-79-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 13 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 13933812 # reads with at least one alignment: 11339144 (81.38%) # reads that failed to align: 2594668 (18.62%) Reported 60241680 alignments Converting to sorted bam format Uniquely mapped (U) reads: 4937927/12259576 (40.3%) Multi-mapped reads placed (P) with guidance: 3494007/12259576 (28.5%) Multi-mapped reads randomly (R) placed: 1181589/12259576 (9.6%) Very highly (H) multi-mapped reads (>=50 hits): 111641/12259576 (0.9%) Not mapped (N) reads (no hits): 2534412/12259576 (20.7%) Thu 09 May 2024 10:24:50 -0700 PDT Aligning /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/06.2-Peve-sRNAseq-trimming-31bp-fastp-merged/trimmed-reads/POR-82-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 14 chunks [bam_sort_core] merging from 0 files and 40 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 256 -r POR-73-S1-TP2-fastp-adapters-polyG-31bp-merged -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-73-S1-TP2-fastp-adapters-polyG-31bp-merged_depths.txt samtools view -F 256 -r POR-79-S1-TP2-fastp-adapters-polyG-31bp-merged -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-79-S1-TP2-fastp-adapters-polyG-31bp-merged_depths.txt samtools view -F 256 -r POR-82-S1-TP2-fastp-adapters-polyG-31bp-merged -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-82-S1-TP2-fastp-adapters-polyG-31bp-merged_depths.txt Writing wiggle files /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-73-S1-TP2-fastp-adapters-polyG-31bp-merged.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-79-S1-TP2-fastp-adapters-polyG-31bp-merged.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-82-S1-TP2-fastp-adapters-polyG-31bp-merged.wig Writing bigwig files wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-73-S1-TP2-fastp-adapters-polyG-31bp-merged.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-73-S1-TP2-fastp-adapters-polyG-31bp-merged.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-79-S1-TP2-fastp-adapters-polyG-31bp-merged.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-79-S1-TP2-fastp-adapters-polyG-31bp-merged.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-82-S1-TP2-fastp-adapters-polyG-31bp-merged.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_POR-82-S1-TP2-fastp-adapters-polyG-31bp-merged.bw 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/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_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/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_21_m_depths.txt samtools view -F 272 -e "qlen==22" -h /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_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/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_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/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_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/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_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/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_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/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments.bam | samtools depth - > /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_other_m_depths.txt Writing wiggle files /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_21_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_21_m.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_22_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_22_m.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_23-24_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_23-24_m.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_other_p.wig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_other_m.wig Writing bigwig files wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_21_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_21_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_21_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_21_m.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_22_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_22_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_22_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_22_m.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_23-24_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_23-24_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_23-24_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_23-24_m.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_other_p.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_other_p.bw wigToBigWig /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_other_m.wig chromSizes.txt /home/shared/8TB_HDD_01/sam/gitrepos/deep-dive/E-Peve/output/08.2-Peve-sRNAseq-ShortStack-31bp-fastp-merged/ShortStack_out/merged_alignments_other_m.bw Converting to sorted bam format Uniquely mapped (U) reads: 6089177/13933812 (43.7%) Multi-mapped reads placed (P) with guidance: 3666025/13933812 (26.3%) Multi-mapped reads randomly (R) placed: 1477830/13933812 (10.6%) Very highly (H) multi-mapped reads (>=50 hits): 106112/13933812 (0.8%) Not mapped (N) reads (no hits): 2594668/13933812 (18.6%) Thu 09 May 2024 10:33:03 -0700 PDT Merging and indexing alignments Creating browser tracks by readgroup using ShortTracks Creating browser tracks by readlength and strand using ShortTracks Thu 09 May 2024 10:42:11 -0700 PDT Defining small RNA clusters de novo With 38432340 total reads and mincov of 1 reads per million, the min read depth is 38 Thu 09 May 2024 10:42:48 -0700 PDT Analyzing cluster properties using 40 threads # reads processed: 49415 # reads with at least one alignment: 133 (0.27%) # reads that failed to align: 49282 (99.73%) Reported 2562 alignments [bam_sort_core] merging from 0 files and 40 in-memory blocks... Thu 09 May 2024 10:43:39 -0700 PDT Completed Thu 09 May 2024 10:43:39 -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 Thu 09 May 2024 10:44:04 -0700 PDT Analyzing cluster properties using 40 threads Thu 09 May 2024 10:44:07 -0700 PDT Completed Writing final files Found a total of 46 MIRNA loci Non-MIRNA loci by DicerCall: N 16529 22 59 23 47 21 31 24 24 Creating visualizations of microRNA loci with strucVis <<< WARNING >>> Do not rely on these results alone to annotate new MIRNA loci! The false positive rate for de novo MIRNA identification is low, but NOT ZERO Insepct each mirna locus, especially the strucVis output, and see https://doi.org/10.1105/tpc.17.00851 , https://doi.org/10.1093/nar/gky1141 Thu 09 May 2024 10:44:28 -0700 PDT Run Completed! real 32m32.272s user 441m7.302s sys 194m17.834s