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_3_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_3_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_3_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_3_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_3_R1_val_1_val_1_val_1.fq.gz to zr3644_3_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_3_R1_val_1_val_1_val_1.fq.gz to zr3644_3_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_3_R1_val_1_val_1_val_1.fq.gz (38913609 sequences in total) Writing a C -> T converted version of the input file zr3644_3_R2_val_2_val_2_val_2.fq.gz to zr3644_3_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_3_R2_val_2_val_2_val_2.fq.gz to zr3644_3_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_3_R2_val_2_val_2_val_2.fq.gz (38913609 sequences in total) Input files are zr3644_3_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_3_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_3_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_3_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_3_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_3_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:2:1101:2121:1000_1:N:0:NTCTGCAG+TACATCCA/1 77 * 0 0 * * 0 0 TTAAAAATGAAAAGAATGTATGGATTTTGAATAGAATTAGTTTTAGGATTTATTTTTTTTTTTAGTTTTTAAATATTGAGGGGTTTTATTTATGTATGTGAATTAATTTTAAT FFFF,FFF:FFF,F:FFF,FFF::FFF,,,FFFFF:FF,FFFFFF:FFFFF:FFF:FFFFF,FF:::FFF,FFF,FFF,FFFFFFF,,,FF,,F,FF,FF,FFFFF:FFFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2121:1000_2:N:0:NTCTGCAG+TACATCCA/2 141 * 0 0 * * 0 0 TTTTTATTCATTAAAATTAATTCACATACCTAAAAAAAACCCCTCAATATTTAAAAACTAAAAAAAAAAATAAATCCTAAAACTAATTCTATTCAAAATCCATACATTCTTTT FFFFFFF,FFFFFFFFFFFFFFFFFFF,FFFFF,FFFFFFFFF,F:F,F:FFF:FFFFF:F,:FFFF:FF:F::F:,FFF,FF,F::FFFF:,:FF::F:FF,,,,,,,F,:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_3_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_3_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:2:1101:2121:1000_1:N:0:NTCTGCAG+TACATCCA/1 77 * 0 0 * * 0 0 TTAAAAATAAAAAAAATATATAAATTTTAAATAAAATTAATTTTAAAATTTATTTTTTTTTTTAATTTTTAAATATTAAAAAATTTTATTTATATATATAAATTAATTTTAAT FFFF,FFF:FFF,F:FFF,FFF::FFF,,,FFFFF:FF,FFFFFF:FFFFF:FFF:FFFFF,FF:::FFF,FFF,FFF,FFFFFFF,,,FF,,F,FF,FF,FFFFF:FFFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2121:1000_2:N:0:NTCTGCAG+TACATCCA/2 141 * 0 0 * * 0 0 TTTTTATTTATTAAAATTAATTTATATATTTAAAAAAAATTTTTTAATATTTAAAAATTAAAAAAAAAAATAAATTTTAAAATTAATTTTATTTAAAATTTATATATTTTTTT FFFFFFF,FFFFFFFFFFFFFFFFFFF,FFFFF,FFFFFFFFF,F:F,F:FFF:FFFFF:F,:FFFF:FF:F::F:,FFF,FF,F::FFFF:,:FF::F:FF,,,,,,,F,:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_3_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_3_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:2:1101:2121:1000_1:N:0:NTCTGCAG+TACATCCA/1 77 * 0 0 * * 0 0 TTAAAAATAAAAAAAATATATAAATTTTAAATAAAATTAATTTTAAAATTTATTTTTTTTTTTAATTTTTAAATATTAAAAAATTTTATTTATATATATAAATTAATTTTAAT FFFF,FFF:FFF,F:FFF,FFF::FFF,,,FFFFF:FF,FFFFFF:FFFFF:FFF:FFFFF,FF:::FFF,FFF,FFF,FFFFFFF,,,FF,,F,FF,FF,FFFFF:FFFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2121:1000_2:N:0:NTCTGCAG+TACATCCA/2 141 * 0 0 * * 0 0 TTTTTATTTATTAAAATTAATTTATATATTTAAAAAAAATTTTTTAATATTTAAAAATTAAAAAAAAAAATAAATTTTAAAATTAATTTTATTTAAAATTTATATATTTTTTT FFFFFFF,FFFFFFFFFFFFFFFFFFF,FFFFF,FFFFFFFFF,F:F,F:FFF:FFFFF:F,:FFFF:FF:F::F:,FFF,FF,F::FFFF:,:FF::F:FF,,,,,,,F,:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_3_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_3_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:2:1101:2121:1000_1:N:0:NTCTGCAG+TACATCCA/1 83 NC_088858.1_GA_converted 8555870 23 22M1D28M1D63M = 8555861 -124 ATTAAAATTAATTCACATACATAAATAAAACCCCTCAATATTTAAAAACTAAAAAAAAAAATAAATCCTAAAACTAATTCTATTCAAAATCCATACATTCTTTTCATTTTTAA F,FFFF:FFFFF,FF,FF,F,,FF,,,FFFFFFF,FFF,FFF,FFF:::FF,FFFFF:FFF:FFFFF:FFFFFF,FF:FFFFF,,,FFF::FFF,FFF:F,FFF:FFF,FFFF AS:i:-46 XN:i:0 XM:i:5 XO:i:2 XG:i:2 NM:i:7 MD:Z:20C1^A3A4A3A15^A54T8 YS:i:-28 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2121:1000_2:N:0:NTCTGCAG+TACATCCA/2 163 NC_088858.1_GA_converted 8555861 23 31M1D28M1D54M = 8555870 124 TTTTTATTCATTAAAATTAATTCACATACCTAAAAAAAACCCCTCAATATTTAAAAACTAAAAAAAAAAATAAATCCTAAAACTAATTCTATTCAAAATCCATACATTCTTTT FFFFFFF,FFFFFFFFFFFFFFFFFFF,FFFFF,FFFFFFFFF,F:F,F:FFF:FFFFF:F,:FFFF:FF:F::F:,FFF,FF,F::FFFF:,:FF::F:FF,,,,,,,F,:F AS:i:-28 XN:i:0 XM:i:2 XO:i:2 XG:i:2 NM:i:4 MD:Z:31^A8A3A15^A54 YS:i:-46 YT:Z:CP >>> Writing bisulfite mapping results to zr3644_3_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_3_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_3_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 Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1553:7012:23124_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 3892 Processed 16000000 sequence pairs so far 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:2:2159:7581:5916_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 1 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2176:4237:26584_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 3900 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:2:2271:29215:23406_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 3891 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2436:22200:33927_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 1 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2478:25771:34632_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 1 Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2523:19461:17973_1:N:0:CTCTGCAG+AACATCCA NW_027062570.1 1 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2553:22733:17159_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 3891 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2644:19904:18396_1:N:0:CTCTGCAG+TACATCCA NW_027062570.1 3936 Processed 38000000 sequence pairs so far 38913609 reads; of these: 38913609 (100.00%) were paired; of these: 27953259 (71.83%) aligned concordantly 0 times 4695846 (12.07%) aligned concordantly exactly 1 time 6264504 (16.10%) aligned concordantly >1 times 28.17% overall alignment rate 38913609 reads; of these: 38913609 (100.00%) were paired; of these: 27904803 (71.71%) aligned concordantly 0 times 4726343 (12.15%) aligned concordantly exactly 1 time 6282463 (16.14%) aligned concordantly >1 times 28.29% overall alignment rate 38913609 reads; of these: 38913609 (100.00%) were paired; of these: 27975660 (71.89%) aligned concordantly 0 times 4697127 (12.07%) aligned concordantly exactly 1 time 6240822 (16.04%) aligned concordantly >1 times 28.11% overall alignment rate 38913609 reads; of these: 38913609 (100.00%) were paired; of these: 27883370 (71.65%) aligned concordantly 0 times 4724352 (12.14%) aligned concordantly exactly 1 time 6305887 (16.20%) aligned concordantly >1 times 28.35% overall alignment rate Processed 38913609 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 155654436. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 155654436. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 155654436. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 155654436. Successfully deleted the temporary files zr3644_3_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_3_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_3_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_3_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 38913609 Final Cytosine Methylation Report ================================= Total number of C's analysed: 855764184 Total methylated C's in CpG context: 11302258 Total methylated C's in CHG context: 720284 Total methylated C's in CHH context: 3208919 Total methylated C's in Unknown context: 49154 Total unmethylated C's in CpG context: 96915341 Total unmethylated C's in CHG context: 146783931 Total unmethylated C's in CHH context: 596833451 Total unmethylated C's in Unknown context: 2844148 C methylated in CpG context: 10.4% C methylated in CHG context: 0.5% C methylated in CHH context: 0.5% C methylated in unknown context (CN or CHN): 1.7% Bismark completed in 0d 3h 24m 26s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe