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_23_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_23_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_23_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_23_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_23_R1_val_1_val_1_val_1.fq.gz to zr3644_23_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_23_R1_val_1_val_1_val_1.fq.gz to zr3644_23_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_23_R1_val_1_val_1_val_1.fq.gz (39902509 sequences in total) Writing a C -> T converted version of the input file zr3644_23_R2_val_2_val_2_val_2.fq.gz to zr3644_23_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_23_R2_val_2_val_2_val_2.fq.gz to zr3644_23_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_23_R2_val_2_val_2_val_2.fq.gz (39902509 sequences in total) Input files are zr3644_23_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_23_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_23_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_23_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_23_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_23_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:2:1101:3206:1000_1:N:0:NTATTCCA+TTGTCTAC/1 99 NC_088855.1_CT_converted 52746955 42 115M = 52746971 128 AGTAGAAATAATTTATGGGGGAGTTAATGATGGAAAAAATGTTTATTGGGTATTTTAGAGATAGTATAGAAATTAATTTAATTAGTTTTAATATATATTGTTTATTAAAAATAAT FFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF,:FFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:F:FFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YS:i:-18 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:3206:1000_2:N:0:NTATTCCA+TTGTCTAC/2 147 NC_088855.1_CT_converted 52746971 42 112M = 52746955 -128 GGGGTAGTTAAAGATGGAAAAAATGTTTATTGGGTATATTAGAGATAGTATAGAAATTAATTTAATTAGTTTTAATATATATTGTTTATTAAAAATAATAGAGATAGTTATT :F,,,F,,,FF,:F,,,FFFFFF,,,:,F,,,,,,:,,:,F:F,F,F,,F:F,FFF::FFF,FFF:FF:,:::FF:FFFFF,F:,F,FF:FFFFFFFFFFFF:F:F:::F:, AS:i:-18 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:4G6T25T74 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_23_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_23_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:2:1101:3206:1000_1:N:0:NTATTCCA+TTGTCTAC/1 77 * 0 0 * * 0 0 AATAAAAATAATTTATAAAAAAATTAATAATAAAAAAAATATTTATTAAATATTTTAAAAATAATATAAAAATTAATTTAATTAATTTTAATATATATTATTTATTAAAAATAAT FFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF,:FFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:F:FFFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:3206:1000_2:N:0:NTATTCCA+TTGTCTAC/2 141 * 0 0 * * 0 0 AATAATTATTTTTATTATTTTTAATAAATAATATATATTAAAATTAATTAAATTAATTTTTATATTATTTTTAATATATTTAATAAATATTTTTTTTATTTTTAATTGTTTT ,:F:::F:F:FFFFFFFFFFFF:FF,F,:F,FFFFF:FF:::,:FF:FFF,FFF::FFF,F:F,,F,F,F:F,:,,:,,,,,,F,:,,,FFFFFF,,,F:,FF,,,F,,,F: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_23_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_23_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:2:1101:3206:1000_1:N:0:NTATTCCA+TTGTCTAC/1 77 * 0 0 * * 0 0 AATAAAAATAATTTATAAAAAAATTAATAATAAAAAAAATATTTATTAAATATTTTAAAAATAATATAAAAATTAATTTAATTAATTTTAATATATATTATTTATTAAAAATAAT FFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF,:FFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:F:FFFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:3206:1000_2:N:0:NTATTCCA+TTGTCTAC/2 141 * 0 0 * * 0 0 AATAATTATTTTTATTATTTTTAATAAATAATATATATTAAAATTAATTAAATTAATTTTTATATTATTTTTAATATATTTAATAAATATTTTTTTTATTTTTAATTGTTTT ,:F:::F:F:FFFFFFFFFFFF:FF,F,:F,FFFFF:FF:::,:FF:FFF,FFF::FFF,F:F,,F,F,F:F,:,,:,,,,,,F,:,,,FFFFFF,,,F:,FF,,,F,,,F: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_23_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_23_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:2:1101:3206:1000_1:N:0:NTATTCCA+TTGTCTAC/1 77 * 0 0 * * 0 0 AGTAGAAATAATTTATGGGGGAGTTAATGATGGAAAAAATGTTTATTGGGTATTTTAGAGATAGTATAGAAATTAATTTAATTAGTTTTAATATATATTGTTTATTAAAAATAAT FFFF:FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF,:FFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:F:FFFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:3206:1000_2:N:0:NTATTCCA+TTGTCTAC/2 141 * 0 0 * * 0 0 AATAACTATCTCTATTATTTTTAATAAACAATATATATTAAAACTAATTAAATTAATTTCTATACTATCTCTAATATACCCAATAAACATTTTTTCCATCTTTAACTACCCC ,:F:::F:F:FFFFFFFFFFFF:FF,F,:F,FFFFF:FF:::,:FF:FFF,FFF::FFF,F:F,,F,F,F:F,:,,:,,,,,,F,:,,,FFFFFF,,,F:,FF,,,F,,,F: YT:Z:UP >>> Writing bisulfite mapping results to zr3644_23_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_23_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_23_R2_val_2_val_2_val_2.fq.gz Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1102:20844:24690_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 3885 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1426:10167:4194_1:N:0:GTATTCCA+TTGTCTAC NW_027062577.1 1 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1502:25328:16078_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 1 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1662:20202:18035_1:N:0:GTATTCCA+TTGTCTAC NW_027062586.1 257915 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1667:2564:7341_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 1 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2105:32217:20870_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 3921 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2112:6361:6277_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2122:23619:25394_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 3921 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2239:7491:36479_1:N:0:GTATTCCA+TTGTCTAC NW_027062586.1 257932 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2265:18927:36714_1:N:0:GTATTCCA+TTGTCTAC NW_027062586.1 257915 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2332:17508:20948_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 3893 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:2:2566:8504:4507_1:N:0:GTATTCCA+TTGTCTAC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2576:5394:30311_1:N:0:GTATTCCA+TTGTCTAC NW_027062573.1 1893 Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far 39902509 reads; of these: 39902509 (100.00%) were paired; of these: 28463380 (71.33%) aligned concordantly 0 times 4947983 (12.40%) aligned concordantly exactly 1 time 6491146 (16.27%) aligned concordantly >1 times 28.67% overall alignment rate 39902509 reads; of these: 39902509 (100.00%) were paired; of these: 28505391 (71.44%) aligned concordantly 0 times 4919795 (12.33%) aligned concordantly exactly 1 time 6477323 (16.23%) aligned concordantly >1 times 28.56% overall alignment rate 39902509 reads; of these: 39902509 (100.00%) were paired; of these: 28435851 (71.26%) aligned concordantly 0 times 4945348 (12.39%) aligned concordantly exactly 1 time 6521310 (16.34%) aligned concordantly >1 times 28.74% overall alignment rate 39902509 reads; of these: 39902509 (100.00%) were paired; of these: 28531121 (71.50%) aligned concordantly 0 times 4923758 (12.34%) aligned concordantly exactly 1 time 6447630 (16.16%) aligned concordantly >1 times 28.50% overall alignment rate Processed 39902509 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 159610036. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 159610036. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 159610036. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 159610036. Successfully deleted the temporary files zr3644_23_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_23_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_23_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_23_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 39902509 Final Cytosine Methylation Report ================================= Total number of C's analysed: 886020469 Total methylated C's in CpG context: 11770515 Total methylated C's in CHG context: 774415 Total methylated C's in CHH context: 3436488 Total methylated C's in Unknown context: 49699 Total unmethylated C's in CpG context: 101065913 Total unmethylated C's in CHG context: 153146269 Total unmethylated C's in CHH context: 615826869 Total unmethylated C's in Unknown context: 2879731 C methylated in CpG context: 10.4% 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 3h 32m 30s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe