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_8_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_8_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_8_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_8_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_8_R1_val_1_val_1_val_1.fq.gz to zr3644_8_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_8_R1_val_1_val_1_val_1.fq.gz to zr3644_8_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_8_R1_val_1_val_1_val_1.fq.gz (42336845 sequences in total) Writing a C -> T converted version of the input file zr3644_8_R2_val_2_val_2_val_2.fq.gz to zr3644_8_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_8_R2_val_2_val_2_val_2.fq.gz to zr3644_8_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_8_R2_val_2_val_2_val_2.fq.gz (42336845 sequences in total) Input files are zr3644_8_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_8_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_8_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_8_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_8_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_8_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:2:1101:2899:1000_1:N:0:NGCAAGCC+CGGATGCT/1 77 * 0 0 * * 0 0 TTAATTAAATATATTAAAATAAATTTTAAATTTAAAAATAATATTAATTATATATTATATATATTAATATTATTTTTATTATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF: YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2899:1000_2:N:0:NGCAAGCC+CGGATGCT/2 141 * 0 0 * * 0 0 ATTAATATATATAATATATAATTAATATTATTTTTAAATTTAAAATTTATTTTAATATATTTAATTAAAAATAATTAAATAAT ,FFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFF:FFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_8_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_8_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:2:1101:2899:1000_1:N:0:NGCAAGCC+CGGATGCT/1 99 NC_088854.1_GA_converted 59297255 2 29M3I1M3I47M = 59297240 -92 TTAATCAAATATACTAAAATAAACTTTAAATCTAAAAATAACATTAACTATATATCATATACATCAATATTATTTTTATTATC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF: AS:i:-46 XS:i:-46 XN:i:0 XM:i:3 XO:i:2 XG:i:6 NM:i:9 MD:Z:41T18T15T0 YS:i:-40 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2899:1000_2:N:0:NGCAAGCC+CGGATGCT/2 147 NC_088854.1_GA_converted 59297240 2 44M3I1M3I32M = 59297255 92 ATCACTCAATTATTTTTAATCAAATATACTAAAATAAACTTTAAATCTAAAAATAACATTAACTATATATCATATACATCAAT FFFFFF:FFFFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF, AS:i:-40 XS:i:-46 XN:i:0 XM:i:2 XO:i:2 XG:i:6 NM:i:8 MD:Z:56T18T1 YS:i:-46 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_8_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_8_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:2:1101:2899:1000_1:N:0:NGCAAGCC+CGGATGCT/1 83 NC_088856.1_CT_converted 10368273 2 3M1I43M6I30M = 10368287 91 GATAATAAAAATAATATTGATGTATATGATATATAGTTAATGTTATTTTTAGATTTAAAGTTTATTTTAGTATATTTGATTAA :FFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-55 XS:i:-51 XN:i:0 XM:i:4 XO:i:2 XG:i:7 NM:i:11 MD:Z:0A14A18A35A5 YS:i:-41 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2899:1000_2:N:0:NGCAAGCC+CGGATGCT/2 163 NC_088856.1_CT_converted 10368287 2 32M6I45M = 10368273 -91 ATTGATGTATATGATATATAGTTAATGTTATTTTTAGATTTAAAGTTTATTTTAGTATATTTGATTAAAAATAATTGAGTGAT ,FFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFF:FFFFFF AS:i:-41 XS:i:-46 XN:i:0 XM:i:3 XO:i:1 XG:i:6 NM:i:9 MD:Z:1A18A35A20 YS:i:-55 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_8_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_8_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:2:1101:2899:1000_1:N:0:NGCAAGCC+CGGATGCT/1 77 * 0 0 * * 0 0 TTAATTAAATATATTAAAATAAATTTTAAATTTAAAAATAATATTAATTATATATTATATATATTAATATTATTTTTATTATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFF: YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2899:1000_2:N:0:NGCAAGCC+CGGATGCT/2 141 * 0 0 * * 0 0 ATTAATATATATAATATATAATTAATATTATTTTTAAATTTAAAATTTATTTTAATATATTTAATTAAAAATAATTAAATAAT ,FFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFFFF:FFFFFF YT:Z:UP >>> Writing bisulfite mapping results to zr3644_8_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_8_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_8_R2_val_2_val_2_val_2.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1139:19217:31673_1:N:0:GGCAAGCC+CGGATGCT NW_027062582.1 18705 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1165:30644:7373_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1221:7328:8735_1:N:0:GGCAAGCC+CGGATGCT NW_027062568.1 15483 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1221:17336:20212_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 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:2:1341:14968:13542_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 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:2:1443:32199:20306_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 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:2:1528:11749:20682_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 3893 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1546:31810:28745_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1627:6985:2879_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1634:12572:29277_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 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:2:2125:27082:36652_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2162:2799:3208_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2276:5683:32252_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 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:2:2466:18249:18912_1:N:0:GGCAAGCC+CGGATGCT NW_027062570.1 1 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far 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 42336845 reads; of these: 42336845 (100.00%) were paired; of these: 30270715 (71.50%) aligned concordantly 0 times 5149859 (12.16%) aligned concordantly exactly 1 time 6916271 (16.34%) aligned concordantly >1 times 28.50% overall alignment rate 42336845 reads; of these: 42336845 (100.00%) were paired; of these: 30270885 (71.50%) aligned concordantly 0 times 5135254 (12.13%) aligned concordantly exactly 1 time 6930706 (16.37%) aligned concordantly >1 times 28.50% overall alignment rate 42336845 reads; of these: 42336845 (100.00%) were paired; of these: 30246446 (71.44%) aligned concordantly 0 times 5147471 (12.16%) aligned concordantly exactly 1 time 6942928 (16.40%) aligned concordantly >1 times 28.56% overall alignment rate 42336845 reads; of these: 42336845 (100.00%) were paired; of these: 30296489 (71.56%) aligned concordantly 0 times 5137337 (12.13%) aligned concordantly exactly 1 time 6903019 (16.30%) aligned concordantly >1 times 28.44% overall alignment rate Processed 42336845 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 169347380. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 169347380. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 169347380. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 169347380. Successfully deleted the temporary files zr3644_8_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_8_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_8_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_8_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 42336845 Final Cytosine Methylation Report ================================= Total number of C's analysed: 932206335 Total methylated C's in CpG context: 12691048 Total methylated C's in CHG context: 826844 Total methylated C's in CHH context: 3689564 Total methylated C's in Unknown context: 56202 Total unmethylated C's in CpG context: 105885679 Total unmethylated C's in CHG context: 160821684 Total unmethylated C's in CHH context: 648291516 Total unmethylated C's in Unknown context: 3066767 C methylated in CpG context: 10.7% C methylated in CHG context: 0.5% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 1.8% Bismark completed in 0d 3h 41m 51s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe