# reads processed: 2825871 # reads with at least one alignment: 2115333 (74.86%) # reads that failed to align: 710538 (25.14%) Reported 9597321 alignments ShortStack version 4.1.0 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_02/shedurkin/deep-dive-expression/D-Apul/data/Apulchra-genome.fa', 'known_miRNAs': '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/data/cnidarian-mirbase-mature-v22.1.fasta', 'locifile': None, 'locus': None, 'make_bigwigs': False, 'mincov': 1, 'mmap': 'u', 'nohp': False, 'outdir': '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/output/11-Apul-sRNA-ShortStack-pulchra_genome/ShortStack_out', 'pad': 200, 'readfile': [ '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/data/sRNA-trimmed-reads/sRNA-ACR-140-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/data/sRNA-trimmed-reads/sRNA-ACR-145-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/data/sRNA-trimmed-reads/sRNA-ACR-150-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/data/sRNA-trimmed-reads/sRNA-ACR-173-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/data/sRNA-trimmed-reads/sRNA-ACR-178-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz'], 'strand_cutoff': 0.8, 'threads': 40} Required executable RNAfold : /home/sam/programs/mambaforge/envs/ShortStack-4.1.0_env/bin/RNAfold Required executable strucVis : /home/sam/programs/mambaforge/envs/ShortStack-4.1.0_env/bin/strucVis Required executable bowtie : /home/sam/programs/mambaforge/envs/ShortStack-4.1.0_env/bin/bowtie Required executable bowtie-build : /home/sam/programs/mambaforge/envs/ShortStack-4.1.0_env/bin/bowtie-build Required executable samtools : /home/sam/programs/mambaforge/envs/ShortStack-4.1.0_env/bin/samtools Tue 08 Oct 2024 15:40:21 -0700 PDT Condensing reads Beginning alignment phase Tue 08 Oct 2024 15:45:23 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/output/11-Apul-sRNA-ShortStack-pulchra_genome/ShortStack_out/sRNA-ACR-140-S1-TP2-fastp-adapters-polyG-31bp-merged_condensed.fa First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 3 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 3107660 # reads with at least one alignment: 2279588 (73.35%) # reads that failed to align: 828072 (26.65%) Reported 10805580 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 963631/2825871 (34.1%) reads: 5008387/15076180 (33.2%) Multi-mapped placed with guidance (P): sequences: 672045/2825871 (23.8%) reads: 4199666/15076180 (27.9%) Multi-mapped randomly placed (R): sequences: 300433/2825871 (10.6%) reads: 1020321/15076180 (6.8%) Very highly multi-mapped (>=20 hits)(H): sequences: 179224/2825871 (6.3%) reads: 1826082/15076180 (12.1%) Not mapped (no hits)(N): sequences: 710538/2825871 (25.1%) reads: 3021724/15076180 (20.0%) Tue 08 Oct 2024 15:46:53 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/output/11-Apul-sRNA-ShortStack-pulchra_genome/ShortStack_out/sRNA-ACR-145-S1-TP2-fastp-adapters-polyG-31bp-merged_condensed.fa First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 4 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 3060154 # reads with at least one alignment: 2121516 (69.33%) # reads that failed to align: 938638 (30.67%) Reported 9591122 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 1000866/3107660 (32.2%) reads: 4987496/17265150 (28.9%) Multi-mapped placed with guidance (P): sequences: 760563/3107660 (24.5%) reads: 4660106/17265150 (27.0%) Multi-mapped randomly placed (R): sequences: 307899/3107660 (9.9%) reads: 1064178/17265150 (6.2%) Very highly multi-mapped (>=20 hits)(H): sequences: 210260/3107660 (6.8%) reads: 2897341/17265150 (16.8%) Not mapped (no hits)(N): sequences: 828072/3107660 (26.6%) reads: 3656029/17265150 (21.2%) Tue 08 Oct 2024 15:48:27 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/output/11-Apul-sRNA-ShortStack-pulchra_genome/ShortStack_out/sRNA-ACR-150-S1-TP2-fastp-adapters-polyG-31bp-merged_condensed.fa First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 4 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 2628116 # reads with at least one alignment: 1791942 (68.18%) # reads that failed to align: 836174 (31.82%) Reported 8809299 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 976624/3060154 (31.9%) reads: 6448061/18395068 (35.1%) Multi-mapped placed with guidance (P): sequences: 667898/3060154 (21.8%) reads: 4809906/18395068 (26.1%) Multi-mapped randomly placed (R): sequences: 290687/3060154 (9.5%) reads: 1101854/18395068 (6.0%) Very highly multi-mapped (>=20 hits)(H): sequences: 186307/3060154 (6.1%) reads: 1952550/18395068 (10.6%) Not mapped (no hits)(N): sequences: 938638/3060154 (30.7%) reads: 4082697/18395068 (22.2%) Tue 08 Oct 2024 15:49:52 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/output/11-Apul-sRNA-ShortStack-pulchra_genome/ShortStack_out/sRNA-ACR-173-S1-TP2-fastp-adapters-polyG-31bp-merged_condensed.fa First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 3 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 2451637 # reads with at least one alignment: 1713282 (69.88%) # reads that failed to align: 738355 (30.12%) Reported 8701768 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 788295/2628116 (30.0%) reads: 5076151/16332641 (31.1%) Multi-mapped placed with guidance (P): sequences: 558968/2628116 (21.3%) reads: 4409741/16332641 (27.0%) Multi-mapped randomly placed (R): sequences: 256045/2628116 (9.7%) reads: 1039978/16332641 (6.4%) Very highly multi-mapped (>=20 hits)(H): sequences: 188634/2628116 (7.2%) reads: 2227892/16332641 (13.6%) Not mapped (no hits)(N): sequences: 836174/2628116 (31.8%) reads: 3578879/16332641 (21.9%) Tue 08 Oct 2024 15:51:11 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/D-Apul/output/11-Apul-sRNA-ShortStack-pulchra_genome/ShortStack_out/sRNA-ACR-178-S1-TP2-fastp-adapters-polyG-31bp-merged_condensed.fa First pass alignment with bowtie using 40 threads Second pass - placing multimappers using 40 threads to process 3 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... Converting to sorted bam format Uniquely mapped (U): sequences: 753475/2451637 (30.7%) reads: 4555160/14295278 (31.9%) Multi-mapped placed with guidance (P): sequences: 523983/2451637 (21.4%) reads: 3662233/14295278 (25.6%) Multi-mapped randomly placed (R): sequences: 242308/2451637 (9.9%) reads: 843497/14295278 (5.9%) Very highly multi-mapped (>=20 hits)(H): sequences: 193516/2451637 (7.9%) reads: 2385986/14295278 (16.7%) Not mapped (no hits)(N): sequences: 738355/2451637 (30.1%) reads: 2848402/14295278 (19.9%) Tue 08 Oct 2024 15:52:31 -0700 PDT Merging and indexing alignments Tue 08 Oct 2024 15:53:21 -0700 PDT Defining small RNA clusters de novo With 81364317 total reads and mincov of 1 reads per million, the min read depth is 81 Tue 08 Oct 2024 15:53:32 -0700 PDT Analyzing cluster properties using 40 threads # reads processed: 49415 # reads with at least one alignment: 143 (0.29%) # reads that failed to align: 49272 (99.71%) Reported 1613 alignments [bam_sort_core] merging from 0 files and 40 in-memory blocks... Tue 08 Oct 2024 15:53:49 -0700 PDT Completed Tue 08 Oct 2024 15:53:49 -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 Tue 08 Oct 2024 15:53:54 -0700 PDT Analyzing cluster properties using 40 threads Tue 08 Oct 2024 15:53:55 -0700 PDT Completed Writing final files Found a total of 39 MIRNA loci Non-MIRNA loci by DicerCall: N 20608 22 78 23 27 21 23 24 14 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 Tue 08 Oct 2024 15:54:04 -0700 PDT Run Completed! real 13m43.312s user 170m20.326s sys 55m0.571s