# reads processed: 1440285 # reads with at least one alignment: 922756 (64.07%) # reads that failed to align: 517529 (35.93%) Reported 4071812 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/F-Ptuh/data/Pocillopora_meandrina_HIv1.assembly.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/F-Ptuh/output/05-Ptuh-sRNA-ShortStack_4.1.0/ShortStack_out', 'pad': 200, 'readfile': [ '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/data/sRNA-trimmed-reads/sRNA-POC-47-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/data/sRNA-trimmed-reads/sRNA-POC-48-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/data/sRNA-trimmed-reads/sRNA-POC-50-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/data/sRNA-trimmed-reads/sRNA-POC-53-S1-TP2-fastp-adapters-polyG-31bp-merged.fq.gz', '/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/data/sRNA-trimmed-reads/sRNA-POC-57-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 22 Oct 2024 17:16:23 -0700 PDT Condensing reads Tue 22 Oct 2024 17:20:15 -0700 PDT Required bowtie indices not found. Building them ... Completed Beginning alignment phase Tue 22 Oct 2024 17:23:55 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/output/05-Ptuh-sRNA-ShortStack_4.1.0/ShortStack_out/sRNA-POC-47-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 2 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 1311407 # reads with at least one alignment: 866692 (66.09%) # reads that failed to align: 444715 (33.91%) Reported 2987681 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 344182/1440285 (23.9%) reads: 2142481/12068141 (17.8%) Multi-mapped placed with guidance (P): sequences: 297739/1440285 (20.7%) reads: 3265526/12068141 (27.1%) Multi-mapped randomly placed (R): sequences: 237492/1440285 (16.5%) reads: 1858434/12068141 (15.4%) Very highly multi-mapped (>=20 hits)(H): sequences: 43343/1440285 (3.0%) reads: 269989/12068141 (2.2%) Not mapped (no hits)(N): sequences: 517529/1440285 (35.9%) reads: 4531711/12068141 (37.6%) Tue 22 Oct 2024 17:24:44 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/output/05-Ptuh-sRNA-ShortStack_4.1.0/ShortStack_out/sRNA-POC-48-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 2 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 1328681 # reads with at least one alignment: 857874 (64.57%) # reads that failed to align: 470807 (35.43%) Reported 3564032 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 451678/1311407 (34.4%) reads: 1810393/14046678 (12.9%) Multi-mapped placed with guidance (P): sequences: 208370/1311407 (15.9%) reads: 4560871/14046678 (32.5%) Multi-mapped randomly placed (R): sequences: 174120/1311407 (13.3%) reads: 2218939/14046678 (15.8%) Very highly multi-mapped (>=20 hits)(H): sequences: 32524/1311407 (2.5%) reads: 174344/14046678 (1.2%) Not mapped (no hits)(N): sequences: 444715/1311407 (33.9%) reads: 5282131/14046678 (37.6%) Tue 22 Oct 2024 17:25:22 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/output/05-Ptuh-sRNA-ShortStack_4.1.0/ShortStack_out/sRNA-POC-50-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 2 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 1830547 # reads with at least one alignment: 1207821 (65.98%) # reads that failed to align: 622726 (34.02%) Reported 4465148 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 344123/1328681 (25.9%) reads: 1968136/12142097 (16.2%) Multi-mapped placed with guidance (P): sequences: 265175/1328681 (20.0%) reads: 3124840/12142097 (25.7%) Multi-mapped randomly placed (R): sequences: 212266/1328681 (16.0%) reads: 1450932/12142097 (11.9%) Very highly multi-mapped (>=20 hits)(H): sequences: 36310/1328681 (2.7%) reads: 242594/12142097 (2.0%) Not mapped (no hits)(N): sequences: 470807/1328681 (35.4%) reads: 5355595/12142097 (44.1%) Tue 22 Oct 2024 17:26:02 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/output/05-Ptuh-sRNA-ShortStack_4.1.0/ShortStack_out/sRNA-POC-53-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 2 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... # reads processed: 1978327 # reads with at least one alignment: 1364508 (68.97%) # reads that failed to align: 613819 (31.03%) Reported 3741206 alignments Converting to sorted bam format Uniquely mapped (U): sequences: 613604/1830547 (33.5%) reads: 2885541/15732765 (18.3%) Multi-mapped placed with guidance (P): sequences: 304523/1830547 (16.6%) reads: 4649182/15732765 (29.6%) Multi-mapped randomly placed (R): sequences: 241663/1830547 (13.2%) reads: 4110262/15732765 (26.1%) Very highly multi-mapped (>=20 hits)(H): sequences: 48031/1830547 (2.6%) reads: 281547/15732765 (1.8%) Not mapped (no hits)(N): sequences: 622726/1830547 (34.0%) reads: 3806233/15732765 (24.2%) Tue 22 Oct 2024 17:26:54 -0700 PDT Aligning /home/shared/8TB_HDD_02/shedurkin/deep-dive-expression/F-Ptuh/output/05-Ptuh-sRNA-ShortStack_4.1.0/ShortStack_out/sRNA-POC-57-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 2 chunks [bam_sort_core] merging from 0 files and 40 in-memory blocks... Converting to sorted bam format Uniquely mapped (U): sequences: 880580/1978327 (44.5%) reads: 2380882/14137264 (16.8%) Multi-mapped placed with guidance (P): sequences: 253815/1978327 (12.8%) reads: 5550229/14137264 (39.3%) Multi-mapped randomly placed (R): sequences: 187794/1978327 (9.5%) reads: 3643939/14137264 (25.8%) Very highly multi-mapped (>=20 hits)(H): sequences: 42319/1978327 (2.1%) reads: 190750/14137264 (1.3%) Not mapped (no hits)(N): sequences: 613819/1978327 (31.0%) reads: 2371464/14137264 (16.8%) Tue 22 Oct 2024 17:27:39 -0700 PDT Merging and indexing alignments Tue 22 Oct 2024 17:28:08 -0700 PDT Defining small RNA clusters de novo With 68126945 total reads and mincov of 1 reads per million, the min read depth is 68 Tue 22 Oct 2024 17:28:15 -0700 PDT Analyzing cluster properties using 40 threads # reads processed: 49415 # reads with at least one alignment: 171 (0.35%) # reads that failed to align: 49244 (99.65%) Reported 1856 alignments [bam_sort_core] merging from 0 files and 40 in-memory blocks... Tue 22 Oct 2024 17:28:25 -0700 PDT Completed Tue 22 Oct 2024 17:28:25 -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 22 Oct 2024 17:28:31 -0700 PDT Analyzing cluster properties using 40 threads Tue 22 Oct 2024 17:28:31 -0700 PDT Completed Writing final files Found a total of 37 MIRNA loci Non-MIRNA loci by DicerCall: N 7053 23 35 22 34 24 15 21 15 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 22 Oct 2024 17:28:40 -0700 PDT Run Completed! real 12m18.158s user 200m22.234s sys 25m26.799s