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_12_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_12_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_12_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_12_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_12_R1_val_1_val_1_val_1.fq.gz to zr3644_12_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_12_R1_val_1_val_1_val_1.fq.gz to zr3644_12_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_12_R1_val_1_val_1_val_1.fq.gz (46849571 sequences in total) Writing a C -> T converted version of the input file zr3644_12_R2_val_2_val_2_val_2.fq.gz to zr3644_12_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_12_R2_val_2_val_2_val_2.fq.gz to zr3644_12_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_12_R2_val_2_val_2_val_2.fq.gz (46849571 sequences in total) Input files are zr3644_12_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_12_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_12_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_12_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_12_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_12_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:2:1101:3134:1000_1:N:0:NCAAGTGG+AACATCGC/1 77 * 0 0 * * 0 0 GTTTAAGAGAAGTTTTAGGGAATAAAGAAAGGATAATTTATTAATTTTAATGTTATATATATTTATTTAAGAAATATTATGT FFFFF:F:F:FFFFFFFFFFFFF:F:FF,FFF,F,,FFFFFFF:FFFF:,FFFF:F,FFFFFFF,FFFF,:,:FF:FF:F:F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:3134:1000_2:N:0:NCAAGTGG+AACATCGC/2 141 * 0 0 * * 0 0 AAATAAATATATATAACATTAAAATTAATAAATTATCCTTTCTTTATTCCCTAAAACTTCTCTTAAACATTTTTATATCTTTT FF:FFFFFFFFFFF:FFFFFFFFFFFF:FF:,FF,F,,FFF:FFF,FFFF:FF::,,FF,F:FF::,,:FFFFFFF:F,FFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_12_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_12_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:2:1101:3134:1000_1:N:0:NCAAGTGG+AACATCGC/1 77 * 0 0 * * 0 0 ATTTAAAAAAAATTTTAAAAAATAAAAAAAAAATAATTTATTAATTTTAATATTATATATATTTATTTAAAAAATATTATAT FFFFF:F:F:FFFFFFFFFFFFF:F:FF,FFF,F,,FFFFFFF:FFFF:,FFFF:F,FFFFFFF,FFFF,:,:FF:FF:F:F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:3134:1000_2:N:0:NCAAGTGG+AACATCGC/2 141 * 0 0 * * 0 0 AAATAAATATATATAATATTAAAATTAATAAATTATTTTTTTTTTATTTTTTAAAATTTTTTTTAAATATTTTTATATTTTTT FF:FFFFFFFFFFF:FFFFFFFFFFFF:FF:,FF,F,,FFF:FFF,FFFF:FF::,,FF,F:FF::,,:FFFFFFF:F,FFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_12_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_12_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:2:1101:3134:1000_1:N:0:NCAAGTGG+AACATCGC/1 77 * 0 0 * * 0 0 ATTTAAAAAAAATTTTAAAAAATAAAAAAAAAATAATTTATTAATTTTAATATTATATATATTTATTTAAAAAATATTATAT FFFFF:F:F:FFFFFFFFFFFFF:F:FF,FFF,F,,FFFFFFF:FFFF:,FFFF:F,FFFFFFF,FFFF,:,:FF:FF:F:F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:3134:1000_2:N:0:NCAAGTGG+AACATCGC/2 141 * 0 0 * * 0 0 AAATAAATATATATAATATTAAAATTAATAAATTATTTTTTTTTTATTTTTTAAAATTTTTTTTAAATATTTTTATATTTTTT FF:FFFFFFFFFFF:FFFFFFFFFFFF:FF:,FF,F,,FFF:FFF,FFFF:FF::,,FF,F:FF::,,:FFFFFFF:F,FFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_12_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_12_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:2:1101:3134:1000_1:N:0:NCAAGTGG+AACATCGC/1 83 NC_088855.1_GA_converted 59376926 42 82M = 59376940 97 ACATAATATTTCTTAAATAAATATATATAACATTAAAATTAATAAATTATCCTTTCTTTATTCCCTAAAACTTCTCTTAAAC F:F:FF:FF:,:,FFFF,FFFFFFF,F:FFFF,:FFFF:FFFFFFF,,F,FFF,FF:F:FFFFFFFFFFFFF:F:F:FFFFF AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:63T9A8 YS:i:-12 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:3134:1000_2:N:0:NCAAGTGG+AACATCGC/2 163 NC_088855.1_GA_converted 59376940 42 83M = 59376926 -97 AAATAAATATATATAACATTAAAATTAATAAATTATCCTTTCTTTATTCCCTAAAACTTCTCTTAAACATTTTTATATCTTTT FF:FFFFFFFFFFF:FFFFFFFFFFFF:FF:,FF,F,,FFF:FFF,FFFF:FF::,,FF,F:FF::,,:FFFFFFF:F,FFFF AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:49T9A23 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to zr3644_12_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_12_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_12_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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1425:18539:7263_1:N:0:ACAAGTGG+AACCTCGC NW_027062570.1 3891 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1425:18530:7341_1:N:0:ACAAGTGG+AACATCGC NW_027062570.1 3891 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1434:24397:26334_1:N:0:ACAAGTGG+AACATCGC NW_027062568.1 15471 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1434:24505:28025_1:N:0:ACAAGTGG+AACATCGC NW_027062568.1 15471 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1506:26512:2754_1:N:0:ACAAGTGG+AACATCGC NW_027062582.1 18700 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1513:25030:36448_1:N:0:ACAAGTGG+AACATCGC NW_027062573.1 1898 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 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far 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 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2367:3893:9893_1:N:0:ACAAGTGG+AACATCGC NW_027062586.1 257924 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2429:13711:34945_1:N:0:ACAAGTGG+AACATCGC NW_027062580.1 2 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 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far 46849571 reads; of these: 46849571 (100.00%) were paired; of these: 33619852 (71.76%) aligned concordantly 0 times 5745306 (12.26%) aligned concordantly exactly 1 time 7484413 (15.98%) aligned concordantly >1 times 28.24% overall alignment rate 46849571 reads; of these: 46849571 (100.00%) were paired; of these: 33614264 (71.75%) aligned concordantly 0 times 5736636 (12.24%) aligned concordantly exactly 1 time 7498671 (16.01%) aligned concordantly >1 times 28.25% overall alignment rate 46849571 reads; of these: 46849571 (100.00%) were paired; of these: 33502623 (71.51%) aligned concordantly 0 times 5787662 (12.35%) aligned concordantly exactly 1 time 7559286 (16.14%) aligned concordantly >1 times 28.49% overall alignment rate 46849571 reads; of these: 46849571 (100.00%) were paired; of these: 33500513 (71.51%) aligned concordantly 0 times 5803181 (12.39%) aligned concordantly exactly 1 time 7545877 (16.11%) aligned concordantly >1 times 28.49% overall alignment rate Processed 46849571 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 187398284. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 187398284. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 187398284. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 187398284. Successfully deleted the temporary files zr3644_12_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_12_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_12_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_12_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 46849571 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1041306706 Total methylated C's in CpG context: 12700548 Total methylated C's in CHG context: 841033 Total methylated C's in CHH context: 3711172 Total methylated C's in Unknown context: 53055 Total unmethylated C's in CpG context: 117236954 Total unmethylated C's in CHG context: 174319861 Total unmethylated C's in CHH context: 732497138 Total unmethylated C's in Unknown context: 3530664 C methylated in CpG context: 9.8% 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 14m 3s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe