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_4_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_4_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_4_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_4_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_4_R1_val_1_val_1_val_1.fq.gz to zr3644_4_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_4_R1_val_1_val_1_val_1.fq.gz to zr3644_4_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_4_R1_val_1_val_1_val_1.fq.gz (49306248 sequences in total) Writing a C -> T converted version of the input file zr3644_4_R2_val_2_val_2_val_2.fq.gz to zr3644_4_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_4_R2_val_2_val_2_val_2.fq.gz to zr3644_4_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_4_R2_val_2_val_2_val_2.fq.gz (49306248 sequences in total) Input files are zr3644_4_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_4_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_4_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_4_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_4_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_4_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:2:1101:1868:1000_1:N:0:NTGACTCT+TGACGGCC/1 99 NC_088862.1_CT_converted 31229480 1 88M = 31229465 -103 TTTGAGGTAAATAAAGAGGTGATATTAGTAGTAAATTGTATGAAGAATTTAATGGTATTAATTGTTTTTATAGAATTTGTGTATAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,F AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:88 YS:i:0 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:1868:1000_2:N:0:NTGACTCT+TGACGGCC/2 147 NC_088862.1_CT_converted 31229465 1 88M = 31229480 103 GTGTTTTGGAGAAAGTTTGAGGTAAATAAAGAGGTGATATTAGTAGTAAATTGTATGAAGAATTTAATGGTATTAATTGTTTTTATAG FFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:88 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_4_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_4_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:2:1101:1868:1000_1:N:0:NTGACTCT+TGACGGCC/1 77 * 0 0 * * 0 0 TTTAAAATAAATAAAAAAATAATATTAATAATAAATTATATAAAAAATTTAATAATATTAATTATTTTTATAAAATTTATATATAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:1868:1000_2:N:0:NTGACTCT+TGACGGCC/2 141 * 0 0 * * 0 0 TTATAAAAATAATTAATATTATTAAATTTTTTATATAATTTATTATTAATATTATTTTTTTATTTATTTTAAATTTTTTTTAAAATAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:::FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_4_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_4_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:2:1101:1868:1000_1:N:0:NTGACTCT+TGACGGCC/1 77 * 0 0 * * 0 0 TTTAAAATAAATAAAAAAATAATATTAATAATAAATTATATAAAAAATTTAATAATATTAATTATTTTTATAAAATTTATATATAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:1868:1000_2:N:0:NTGACTCT+TGACGGCC/2 141 * 0 0 * * 0 0 TTATAAAAATAATTAATATTATTAAATTTTTTATATAATTTATTATTAATATTATTTTTTTATTTATTTTAAATTTTTTTTAAAATAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:::FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_4_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_4_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:2:1101:1868:1000_1:N:0:NTGACTCT+TGACGGCC/1 83 NC_088853.1_GA_converted 56929289 1 88M = 56929304 103 ATTTATACACAAATTCTATAAAAACAATTAATACCATTAAATTCTTCATACAATTTACTACTAATATCACCTCTTTATTTACCTCAAA F,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:88 YS:i:0 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:1868:1000_2:N:0:NTGACTCT+TGACGGCC/2 163 NC_088853.1_GA_converted 56929304 1 88M = 56929289 -103 CTATAAAAACAATTAATACCATTAAATTCTTCATACAATTTACTACTAATATCACCTCTTTATTTACCTCAAACTTTCTCCAAAACAC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:::FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFF AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:88 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to zr3644_4_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_4_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_4_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:2:1151:13973:27226_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1266:20934:14418_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1274:28094:16203_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 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:2:1369:26675:5666_1:N:0:CTGACTCT+TGACGGCC 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:2:1425:26223:7921_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 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:2:1478:32389:11804_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 3895 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1501:1904:12962_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1504:6081:31313_1:N:0:CTGACTCT+TGACGGCC 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:2:1538:1344:19132_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1569:3667:26036_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 3895 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1606:26630:22936_1:N:0:CTGACTCT+TGACGGCC NW_027062573.1 1894 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:2:1665:19822:4413_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 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:2:2167:2935:34695_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 3921 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2205:18168:2738_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 3894 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2235:31358:8140_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2235:30526:25645_1:N:0:CTGACTCT+TGACGGCC 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:2:2269:19687:18082_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 3921 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2434:24469:20666_1:N:0:CTGACTCT+TGACGGCC NW_027062573.1 1 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2506:25111:20619_1:N:0:CTGACTCT+TGCCGGCC NW_027062570.1 3902 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2536:4227:9596_1:N:0:CTGACTCT+TGACGGCC NW_027062573.1 1907 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2643:16649:8500_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 3890 Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2659:12780:26537_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2671:13132:32784_1:N:0:CTGACTCT+TGACGGCC NW_027062570.1 1 Processed 49000000 sequence pairs so far 49306248 reads; of these: 49306248 (100.00%) were paired; of these: 35346726 (71.69%) aligned concordantly 0 times 6021991 (12.21%) aligned concordantly exactly 1 time 7937531 (16.10%) aligned concordantly >1 times 28.31% overall alignment rate 49306248 reads; of these: 49306248 (100.00%) were paired; of these: 35346905 (71.69%) aligned concordantly 0 times 6025125 (12.22%) aligned concordantly exactly 1 time 7934218 (16.09%) aligned concordantly >1 times 28.31% overall alignment rate 49306248 reads; of these: 49306248 (100.00%) were paired; of these: 35347877 (71.69%) aligned concordantly 0 times 6025989 (12.22%) aligned concordantly exactly 1 time 7932382 (16.09%) aligned concordantly >1 times 28.31% overall alignment rate 49306248 reads; of these: 49306248 (100.00%) were paired; of these: 35336566 (71.67%) aligned concordantly 0 times 6025420 (12.22%) aligned concordantly exactly 1 time 7944262 (16.11%) aligned concordantly >1 times 28.33% overall alignment rate Processed 49306248 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 197224992. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 197224992. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 197224992. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 197224992. Successfully deleted the temporary files zr3644_4_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_4_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_4_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_4_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 49306248 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1095445149 Total methylated C's in CpG context: 14041365 Total methylated C's in CHG context: 897413 Total methylated C's in CHH context: 3904845 Total methylated C's in Unknown context: 59802 Total unmethylated C's in CpG context: 124009766 Total unmethylated C's in CHG context: 186504782 Total unmethylated C's in CHH context: 766086978 Total unmethylated C's in Unknown context: 3665861 C methylated in CpG context: 10.2% C methylated in CHG context: 0.5% C methylated in CHH context: 0.5% C methylated in unknown context (CN or CHN): 1.6% Bismark completed in 0d 4h 20m 36s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe