Bowtie 2 seems to be working fine (tested command '/home/shared/bowtie2-2.4.4-linux-x86_64/bowtie2 --version' [2.4.4]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools found here: '/usr/bin/samtools' Reference genome folder provided is ../../data/Haws-10/ (absolute path is '/home/shared/16TB_HDD_01/sr320/github/project-oyster-oa/data/Haws-10/)' FastQ format assumed (by default) Attention: using more than 4 cores per alignment thread has been reported to have diminishing returns. If possible try to limit -p to a value of 4 Each Bowtie 2 instance is going to be run with 8 threads. Please monitor performance closely and tune down if necessary! Input files to be analysed (in current folder '/home/shared/16TB_HDD_01/sr320/github/project-oyster-oa/code/Haws'): ../../data/Haws-10/zr3644_20_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_20_R2_val_2_val_2_val_2.fq.gz Library was specified to be not strand-specific (non-directional), therefore alignments to all four possible bisulfite strands (OT, CTOT, OB and CTOB) will be reported Output will be written into the directory: /home/shared/16TB_HDD_01/sr320/github/project-oyster-oa/analyses/Haws-10/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /home/shared/16TB_HDD_01/sr320/github/project-oyster-oa/code/Haws Now reading in and storing sequence information of the genome specified in: /home/shared/16TB_HDD_01/sr320/github/project-oyster-oa/data/Haws-10/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../../data/Haws-10/zr3644_20_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_20_R2_val_2_val_2_val_2.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file zr3644_20_R1_val_1_val_1_val_1.fq.gz to zr3644_20_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_20_R1_val_1_val_1_val_1.fq.gz to zr3644_20_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file zr3644_20_R1_val_1_val_1_val_1.fq.gz (53390071 sequences in total) Writing a C -> T converted version of the input file zr3644_20_R2_val_2_val_2_val_2.fq.gz to zr3644_20_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_20_R2_val_2_val_2_val_2.fq.gz to zr3644_20_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file zr3644_20_R2_val_2_val_2_val_2.fq.gz (53390071 sequences in total) Input files are zr3644_20_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_20_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_20_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_20_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq (FastQ) Now running 4 individual instances of Bowtie 2 against the bisulfite genome of /home/shared/16TB_HDD_01/sr320/github/project-oyster-oa/data/Haws-10/ with the specified options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Now starting a Bowtie 2 paired-end alignment for CTread1GAread2CTgenome (reading in sequences from zr3644_20_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_20_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: A00742:173:HMGVGDSXY:2:1101:2790:1000_1:N:0:NCGTTGGT+GGAAGTAT/1 99 NC_088855.1_CT_converted 39293629 24 42M1D73M = 39293780 265 AAATAATGTTGGTATATATTAATTTGTATTTTTTTTTAATTATTTATAATTTAATTTAAATTATATGAGTGTTTTTTAAATTAGTAAAGTGATTGTATTTGTGTTTAAGGTGATT FFFFFFFF,FF,F:F:FFF:F::FFFF:F,F:FFFF:FFFFFFFFFFFFFF::FFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFF,FF:FFF:FFF,FF:FF:,FFFF:FFFFF AS:i:-32 XN:i:0 XM:i:4 XO:i:1 XG:i:1 NM:i:5 MD:Z:27T14^T22T6G37A5 YS:i:-30 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2790:1000_2:N:0:NCGTTGGT+GGAAGTAT/2 147 NC_088855.1_CT_converted 39293780 24 114M = 39293629 -265 GTATGTTTTTAATGATAGAATGTGTATAAAGAGAGGTTATTTTTTTTAATAGAAGTTTTTGTTTTTGAGTTGATGGAATAAATTTTAGATTGTTAAGTTTTGTTTTATTTTTTA :FFFF:FFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-30 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:28T3A46G22G10G0 YS:i:-32 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_20_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_20_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: A00742:173:HMGVGDSXY:2:1101:2790:1000_1:N:0:NCGTTGGT+GGAAGTAT/1 77 * 0 0 * * 0 0 AAATAATATTAATATATATTAATTTATATTTTTTTTTAATTATTTATAATTTAATTTAAATTATATAAATATTCTTTAAATTAATAAAATAATTATATTTATATTTAAAATAATT FFFFFFFF,FF,F:F:FFF:F::FFFF:F,F:FFFF:FFFFFFFFFFFFFF::FFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFF,FF:FFF:FFF,FF:FF:,FFFF:FFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2790:1000_2:N:0:NCGTTGGT+GGAAGTAT/2 141 * 0 0 * * 0 0 TAAAAAATAAAATAAAATTTAATAATTTAAAATTTATTTTATTAATTTAAAAATAAAAATTTTTATTAAAAAAAATAATTTTTTTTTATATATATTTTATTATTAAAAATATAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF:FFFF: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_20_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_20_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: A00742:173:HMGVGDSXY:2:1101:2790:1000_1:N:0:NCGTTGGT+GGAAGTAT/1 77 * 0 0 * * 0 0 AAATAATATTAATATATATTAATTTATATTTTTTTTTAATTATTTATAATTTAATTTAAATTATATAAATATTCTTTAAATTAATAAAATAATTATATTTATATTTAAAATAATT FFFFFFFF,FF,F:F:FFF:F::FFFF:F,F:FFFF:FFFFFFFFFFFFFF::FFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFF,FF:FFF:FFF,FF:FF:,FFFF:FFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2790:1000_2:N:0:NCGTTGGT+GGAAGTAT/2 141 * 0 0 * * 0 0 TAAAAAATAAAATAAAATTTAATAATTTAAAATTTATTTTATTAATTTAAAAATAAAAATTTTTATTAAAAAAAATAATTTTTTTTTATATATATTTTATTATTAAAAATATAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF:FFFF: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_20_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_20_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: A00742:173:HMGVGDSXY:2:1101:2790:1000_1:N:0:NCGTTGGT+GGAAGTAT/1 83 NC_088857.1_GA_converted 2912681 6 115M = 2912532 -264 AATCACCTTAAACACAAATACAATCACTTTACTAATTTAAAAAACACTCATATAATTTAAATTAAATTATAAATAATTAAAAAAAAATACAAATTAATATATACCAACATTATTT FFFFF:FFFF,:FF:FF,FFF:FFF:FF,FFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFF::FFFFFFFFFFFFFF:FFFF:F,F:FFFF::F:FFF:F:F,FF,FFFFFFFF AS:i:-24 XS:i:-32 XN:i:0 XM:i:4 XO:i:0 XG:i:0 NM:i:4 MD:Z:5T37C6A36A27 YS:i:-30 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2790:1000_2:N:0:NCGTTGGT+GGAAGTAT/2 163 NC_088857.1_GA_converted 2912532 6 114M = 2912681 264 TAAAAAATAAAACAAAACTTAACAATCTAAAATTTATTCCATCAACTCAAAAACAAAAACTTCTATTAAAAAAAATAACCTCTCTTTATACACATTCTATCATTAAAAACATAC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF:FFFF: AS:i:-30 XS:i:-30 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0C10C22C46T3A28 YS:i:-24 YT:Z:CP >>> Writing bisulfite mapping results to zr3644_20_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_20_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_20_R2_val_2_val_2_val_2.fq.gz Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1116:14398:32534_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1130:26793:28040_1:N:0:GCGTTGGT+GGAAGTAT NW_027062573.1 1898 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1131:28275:28197_1:N:0:GCGTTGGT+GGAAGTAT NW_027062573.1 1899 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1138:31331:6151_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1141:20735:4335_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 3908 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1141:20292:28244_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 3909 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1203:23529:15060_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1227:5647:27117_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1303:24876:20682_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1353:6334:24048_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 3895 Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1454:20952:10191_1:N:0:GCGTTGGT+GGAAGTAT NW_027062577.1 1 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1554:26350:27085_1:N:0:GCGTTGGT+GGAAGTAT NW_027062568.1 15472 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1554:25681:33317_1:N:0:GCGTTGGT+GGAAGTAT NW_027062568.1 15471 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1559:18683:27211_1:N:0:GCGTTGGT+GGAAGTAT NW_027062577.1 3 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1571:10484:28103_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1640:13250:30639_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1670:19135:33442_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2133:28375:27618_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2276:24071:33599_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 2 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2276:24297:33802_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 2 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2302:25409:34945_1:N:0:GCGTTGGT+GGAAGTAT NW_027062580.1 24118 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2323:28809:6480_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2539:14769:33270_1:N:0:GCGTTGGT+GGAAGTAT NW_027062570.1 1 Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far 53390071 reads; of these: 53390071 (100.00%) were paired; of these: 39056646 (73.15%) aligned concordantly 0 times 6316851 (11.83%) aligned concordantly exactly 1 time 8016574 (15.02%) aligned concordantly >1 times 26.85% overall alignment rate 53390071 reads; of these: 53390071 (100.00%) were paired; of these: 38020865 (71.21%) aligned concordantly 0 times 6746787 (12.64%) aligned concordantly exactly 1 time 8622419 (16.15%) aligned concordantly >1 times 28.79% overall alignment rate 53390071 reads; of these: 53390071 (100.00%) were paired; of these: 37985119 (71.15%) aligned concordantly 0 times 6748179 (12.64%) aligned concordantly exactly 1 time 8656773 (16.21%) aligned concordantly >1 times 28.85% overall alignment rate 53390071 reads; of these: 53390071 (100.00%) were paired; of these: 39081853 (73.20%) aligned concordantly 0 times 6320304 (11.84%) aligned concordantly exactly 1 time 7987914 (14.96%) aligned concordantly >1 times 26.80% overall alignment rate Processed 53390071 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 213560284. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 213560284. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 213560284. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 213560284. Successfully deleted the temporary files zr3644_20_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_20_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_20_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_20_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 53390071 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1179732756 Total methylated C's in CpG context: 15669638 Total methylated C's in CHG context: 1036445 Total methylated C's in CHH context: 4543913 Total methylated C's in Unknown context: 72189 Total unmethylated C's in CpG context: 133751818 Total unmethylated C's in CHG context: 202852620 Total unmethylated C's in CHH context: 821878322 Total unmethylated C's in Unknown context: 3843645 C methylated in CpG context: 10.5% C methylated in CHG context: 0.5% C methylated in CHH context: 0.5% C methylated in unknown context (CN or CHN): 1.8% Bismark completed in 0d 4h 42m 1s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe