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_2_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_2_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_2_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_2_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_2_R1_val_1_val_1_val_1.fq.gz to zr3644_2_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_2_R1_val_1_val_1_val_1.fq.gz to zr3644_2_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_2_R1_val_1_val_1_val_1.fq.gz (55025914 sequences in total) Writing a C -> T converted version of the input file zr3644_2_R2_val_2_val_2_val_2.fq.gz to zr3644_2_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_2_R2_val_2_val_2_val_2.fq.gz to zr3644_2_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_2_R2_val_2_val_2_val_2.fq.gz (55025914 sequences in total) Input files are zr3644_2_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_2_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_2_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_2_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_2_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_2_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:2:1101:2013:1000_1:N:0:NTCATAAG+CCTTCTAA/1 77 * 0 0 * * 0 0 AATATAAATTAATTTTTTAAAAAAAATTTTATTTTAATTATATTATATAAAATATTATATTTTTATTAAATTAATTTAATTTTTTATTAATTATATAAATTTAATTTTTTTTA FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFFF,FFFFFFFFF:FFFFFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2013:1000_2:N:0:NTCATAAG+CCTTCTAA/2 141 * 0 0 * * 0 0 ATATTTAAATTTTTATATTTAATAAAAATTAAATTTTAAAATTATTTAATTAATTTTTAAAAAAAATTAAATTTATATAATTAATAAAAAATTAAATTAATTTAATAAAAATAT FFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFF:F:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFF:FFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_2_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_2_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:2:1101:2013:1000_1:N:0:NTCATAAG+CCTTCTAA/1 99 NC_088856.1_GA_converted 17317306 2 16M1D97M = 17317363 170 AATATAAACCAACCCCTTAAAAAAAATCCCATTTTAACTATATTATACAAAATATCACATTTTCACCAAACTAACTTAACCTCTTACTAACCATATAAACCCAATTCCTTTTA FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFFF,FFFFFFFFF:FFFFFFFF AS:i:-26 XS:i:-26 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:5T10^A17A4A74 YS:i:-38 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2013:1000_2:N:0:NTCATAAG+CCTTCTAA/2 147 NC_088856.1_GA_converted 17317363 2 92M1I21M = 17317306 -170 ACATTTTCACCAAACTAACTTAACCTCTTACTAACCATATAAACCCAATTCCTTTTAAAAATCAACTAAACAATCCCAAAACCCAATTCTCACTAAATATAAAAATCCAAATAT FFFFFFFFF:FFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:F:FFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-38 XS:i:-36 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:71T9A2C2C8A16 YS:i:-26 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_2_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_2_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:2:1101:2013:1000_1:N:0:NTCATAAG+CCTTCTAA/1 83 NC_088856.1_CT_converted 35977921 2 113M = 35977866 -168 TAAAAGGAATTGGGTTTATATGGTTAGTAAGAGGTTAAGTTAGTTTGGTGAAAATGTGATATTTTGTATAATATAGTTAAAATGGGATTTTTTTTAAGGGGTTGGTTTATATT FFFFFFFF:FFFFFFFFF,FFFFF:FFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF AS:i:-18 XS:i:-48 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:79T5A18T8 YS:i:-47 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2013:1000_2:N:0:NTCATAAG+CCTTCTAA/2 163 NC_088856.1_CT_converted 35977866 2 5M2I107M = 35977921 168 ATATTTGGATTTTTATATTTAGTGAGAATTGGGTTTTGGGATTGTTTAGTTGATTTTTAAAAGGAATTGGGTTTATATGGTTAGTAAGAGGTTAAGTTAGTTTGGTGAAAATGT FFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFF:F:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFF:FFFFFFFFF AS:i:-47 XS:i:-24 XN:i:0 XM:i:6 XO:i:1 XG:i:2 NM:i:8 MD:Z:18G5G2G7A0A12A62 YS:i:-18 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_2_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_2_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:2:1101:2013:1000_1:N:0:NTCATAAG+CCTTCTAA/1 77 * 0 0 * * 0 0 AATATAAATTAATTTTTTAAAAAAAATTTTATTTTAATTATATTATATAAAATATTATATTTTTATTAAATTAATTTAATTTTTTATTAATTATATAAATTTAATTTTTTTTA FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFFF,FFFFFFFFF:FFFFFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2013:1000_2:N:0:NTCATAAG+CCTTCTAA/2 141 * 0 0 * * 0 0 ATATTTAAATTTTTATATTTAATAAAAATTAAATTTTAAAATTATTTAATTAATTTTTAAAAAAAATTAAATTTATATAATTAATAAAAAATTAAATTAATTTAATAAAAATAT FFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFF:F:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFF:FFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to zr3644_2_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_2_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_2_R2_val_2_val_2_val_2.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1147:25057:1642_1:N:0:TTCATAAG+CCTTCTAA NW_027062569.1 16385 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 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1274:23990:29543_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 3895 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1326:25654:11381_1:N:0:TTCATAAG+CCTTCTAA NW_027062568.1 15460 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1347:30680:14105_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1402:15971:27586_1:N:0:TTCATAAG+CCTTCTAA NW_027062577.1 1 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:2:1453:14425:20149_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 3896 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1461:8838:35712_1:N:0:TTCATAAG+CCTTCTAA NC_088862.1 2 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1507:17644:28197_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1564:28908:27946_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 3893 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1603:9109:27821_1:N:0:TTCATAAG+CCTTCTAA NW_027062568.1 15471 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1642:17535:9408_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 3891 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2123:2437:33520_1:N:0:TTCATAAG+CCTTCTAA NW_027062582.1 18712 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2130:27651:23954_1:N:0:TTCATAAG+CCTTCTAA NW_027062586.1 257925 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2157:24677:19961_1:N:0:TTCATAAG+CCTTCTAA NW_027062578.1 34437 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2205:3848:12696_1:N:0:TTCATAAG+CCTTCTAA NW_027062573.1 1898 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:2:2259:13340:10254_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2259:14940:21731_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2260:14850:18286_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2260:14597:18662_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2277:12816:7717_1:N:0:TTCATAAG+CCTTCTAA NW_027062585.1 1 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:2:2455:32859:31939_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2462:10257:5321_1:N:0:TTCATAAG+CCTTCTAA NC_088862.1 2 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2576:7021:14497_1:N:0:TTCATAAG+CCTTCTAA NW_027062578.1 34400 Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2649:18285:15280_1:N:0:TTCATAAG+CCTTCTAA NW_027062570.1 1 Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far 55025914 reads; of these: 55025914 (100.00%) were paired; of these: 39600895 (71.97%) aligned concordantly 0 times 6661902 (12.11%) aligned concordantly exactly 1 time 8763117 (15.93%) aligned concordantly >1 times 28.03% overall alignment rate 55025914 reads; of these: 55025914 (100.00%) were paired; of these: 39658254 (72.07%) aligned concordantly 0 times 6662573 (12.11%) aligned concordantly exactly 1 time 8705087 (15.82%) aligned concordantly >1 times 27.93% overall alignment rate 55025914 reads; of these: 55025914 (100.00%) were paired; of these: 39647643 (72.05%) aligned concordantly 0 times 6663112 (12.11%) aligned concordantly exactly 1 time 8715159 (15.84%) aligned concordantly >1 times 27.95% overall alignment rate 55025914 reads; of these: 55025914 (100.00%) were paired; of these: 39603661 (71.97%) aligned concordantly 0 times 6669520 (12.12%) aligned concordantly exactly 1 time 8752733 (15.91%) aligned concordantly >1 times 28.03% overall alignment rate Processed 55025914 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 220103656. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 220103656. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 220103656. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 220103656. Successfully deleted the temporary files zr3644_2_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_2_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_2_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_2_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 55025914 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1219646118 Total methylated C's in CpG context: 15827434 Total methylated C's in CHG context: 1026185 Total methylated C's in CHH context: 4563014 Total methylated C's in Unknown context: 62203 Total unmethylated C's in CpG context: 137507065 Total unmethylated C's in CHG context: 208064918 Total unmethylated C's in CHH context: 852657502 Total unmethylated C's in Unknown context: 4058384 C methylated in CpG context: 10.3% C methylated in CHG context: 0.5% C methylated in CHH context: 0.5% C methylated in unknown context (CN or CHN): 1.5% Bismark completed in 0d 4h 47m 45s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe