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_15_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_15_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_15_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_15_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_15_R1_val_1_val_1_val_1.fq.gz to zr3644_15_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_15_R1_val_1_val_1_val_1.fq.gz to zr3644_15_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_15_R1_val_1_val_1_val_1.fq.gz (58435477 sequences in total) Writing a C -> T converted version of the input file zr3644_15_R2_val_2_val_2_val_2.fq.gz to zr3644_15_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_15_R2_val_2_val_2_val_2.fq.gz to zr3644_15_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_15_R2_val_2_val_2_val_2.fq.gz (58435477 sequences in total) Input files are zr3644_15_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_15_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_15_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_15_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_15_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_15_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:2:1101:2157:1000_1:N:0:NCTCTACA+AGGAGGTA/1 99 NC_088853.1_CT_converted 6654978 0 114M = 6655043 193 ATAGAATAAAATATTATTTTATGGTTTTTTTAATTTTAAATATGAATTAAGGTATGTGTGTTTATAATATTTATGTGTTATTATTGTTTATTTTATTTGTTTTTATATTTTTAT FFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:,FFFFFFFFFFFFFFFFFFF,F,FFFFFF:FFFFF:FFFF::FFFFFFFFFFF:FFFFF AS:i:-24 XS:i:-24 XN:i:0 XM:i:4 XO:i:0 XG:i:0 NM:i:4 MD:Z:10T2G45T52T1 YS:i:-53 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2157:1000_2:N:0:NCTCTACA+AGGAGGTA/2 147 NC_088853.1_CT_converted 6655043 0 42M14D72M = 6654978 -193 AATATTTATGTGTTATTATTGTTTATTTTATTTGTTTTTATATTTTTATATGGATTTTGGAATATTTTGAATTTTTTTTATTTAATTGATGTTTATTTATAGTTTTGTTTTTGA FFF:FF:FFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF,FFFFFFFFF: AS:i:-53 XS:i:-53 XN:i:0 XM:i:1 XO:i:1 XG:i:14 NM:i:15 MD:Z:42^TTTTTTTGGAATAT65A6 YS:i:-24 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_15_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_15_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:2:1101:2157:1000_1:N:0:NCTCTACA+AGGAGGTA/1 77 * 0 0 * * 0 0 ATAAAATAAAATATTATTTTATAATTTTTTTAATTTTAAATATAAATTAAAATATATATATTTATAATATTTATATATTATTATTATTTATTTTATTTATTTTTATATTTTTAT FFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:,FFFFFFFFFFFFFFFFFFF,F,FFFFFF:FFFFF:FFFF::FFFFFFFFFFF:FFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2157:1000_2:N:0:NCTCTACA+AGGAGGTA/2 141 * 0 0 * * 0 0 TTAAAAATAAAATTATAAATAAATATTAATTAAATAAAAAAAATTTAAAATATTTTAAAATTTATATAAAAATATAAAAATAAATAAAATAAATAATAATAATATATAAATATT :FFFFFFFFF,FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:FF:FFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_15_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_15_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:2:1101:2157:1000_1:N:0:NCTCTACA+AGGAGGTA/1 77 * 0 0 * * 0 0 ATAAAATAAAATATTATTTTATAATTTTTTTAATTTTAAATATAAATTAAAATATATATATTTATAATATTTATATATTATTATTATTTATTTTATTTATTTTTATATTTTTAT FFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:,FFFFFFFFFFFFFFFFFFF,F,FFFFFF:FFFFF:FFFF::FFFFFFFFFFF:FFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2157:1000_2:N:0:NCTCTACA+AGGAGGTA/2 141 * 0 0 * * 0 0 TTAAAAATAAAATTATAAATAAATATTAATTAAATAAAAAAAATTTAAAATATTTTAAAATTTATATAAAAATATAAAAATAAATAAAATAAATAATAATAATATATAAATATT :FFFFFFFFF,FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:FF:FFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_15_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_15_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:2:1101:2157:1000_1:N:0:NCTCTACA+AGGAGGTA/1 83 NC_088854.1_GA_converted 15202773 0 83M1I30M = 15202694 -192 ATAAAAATATAAAAACAAATAAAATAAACAATAATAACACATAAATATTATAAACACACATACCTTAATTCATATTTAAAATTAAAAAAACCATAAAATAATATTTTATTCTAT FFFFF:FFFFFFFFFFF::FFFF:FFFFF:FFFFFF,F,FFFFFFFFFFFFFFFFFFF,:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFF AS:i:-32 XS:i:-26 XN:i:0 XM:i:4 XO:i:1 XG:i:1 NM:i:5 MD:Z:1A52A32C11C13 YS:i:-53 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2157:1000_2:N:0:NCTCTACA+AGGAGGTA/2 163 NC_088854.1_GA_converted 15202694 0 67M14D47M = 15202773 192 TCAAAAACAAAACTATAAATAAACATCAATTAAATAAAAAAAATTCAAAATATTCCAAAATCCATATAAAAATATAAAAACAAATAAAATAAACAATAATAACACATAAATATT :FFFFFFFFF,FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFF:FF:FFF AS:i:-53 XS:i:-59 XN:i:0 XM:i:1 XO:i:1 XG:i:14 NM:i:15 MD:Z:6T60^AAAAAATATTCCAA47 YS:i:-32 YT:Z:CP >>> Writing bisulfite mapping results to zr3644_15_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_15_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_15_R2_val_2_val_2_val_2.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1242:18087:20509_1:N:0:CCTCTACA+AGGAGGTA NW_027062573.1 1896 Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1317:6894:17691_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 3892 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1446:22625:4852_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 3891 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:2:1539:23339:11318_1:N:0:CCTCTACA+AGGAGGTA NW_027062586.1 257919 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1563:11460:26068_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 1 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1640:13783:3161_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 1 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1646:8368:13322_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 1 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far 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 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2234:2031:34287_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 3891 Processed 37000000 sequence pairs so far 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2413:30662:14011_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 3890 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2448:1434:9017_1:N:0:CCTCTACA+AGGAGGTA NW_027062582.1 18694 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 Processed 54000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2615:9796:7435_1:N:0:CGTCTACA+AGGAGGTA NW_027062578.1 34399 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2615:9869:7467_1:N:0:CCTCTACA+AGGAGGTA NW_027062578.1 34399 Processed 55000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2637:20311:33004_1:N:0:CCTCTACA+AGGAGGTA NW_027062570.1 1 Processed 56000000 sequence pairs so far Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far 58435477 reads; of these: 58435477 (100.00%) were paired; of these: 41911582 (71.72%) aligned concordantly 0 times 7183094 (12.29%) aligned concordantly exactly 1 time 9340801 (15.98%) aligned concordantly >1 times 28.28% overall alignment rate 58435477 reads; of these: 58435477 (100.00%) were paired; of these: 41913919 (71.73%) aligned concordantly 0 times 7177413 (12.28%) aligned concordantly exactly 1 time 9344145 (15.99%) aligned concordantly >1 times 28.27% overall alignment rate 58435477 reads; of these: 58435477 (100.00%) were paired; of these: 41945952 (71.78%) aligned concordantly 0 times 7172680 (12.27%) aligned concordantly exactly 1 time 9316845 (15.94%) aligned concordantly >1 times 28.22% overall alignment rate 58435477 reads; of these: 58435477 (100.00%) were paired; of these: 41943941 (71.78%) aligned concordantly 0 times 7180062 (12.29%) aligned concordantly exactly 1 time 9311474 (15.93%) aligned concordantly >1 times 28.22% overall alignment rate Processed 58435477 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 233741908. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 233741908. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 233741908. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 233741908. Successfully deleted the temporary files zr3644_15_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_15_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_15_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_15_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 58435477 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1298084628 Total methylated C's in CpG context: 18579183 Total methylated C's in CHG context: 1140569 Total methylated C's in CHH context: 5061560 Total methylated C's in Unknown context: 75308 Total unmethylated C's in CpG context: 145901050 Total unmethylated C's in CHG context: 222764259 Total unmethylated C's in CHH context: 904638007 Total unmethylated C's in Unknown context: 4303411 C methylated in CpG context: 11.3% C methylated in CHG context: 0.5% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 1.7% Bismark completed in 0d 5h 18m 56s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe