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_11_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_11_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_11_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_11_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_11_R1_val_1_val_1_val_1.fq.gz to zr3644_11_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_11_R1_val_1_val_1_val_1.fq.gz to zr3644_11_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_11_R1_val_1_val_1_val_1.fq.gz (56063348 sequences in total) Writing a C -> T converted version of the input file zr3644_11_R2_val_2_val_2_val_2.fq.gz to zr3644_11_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_11_R2_val_2_val_2_val_2.fq.gz to zr3644_11_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_11_R2_val_2_val_2_val_2.fq.gz (56063348 sequences in total) Input files are zr3644_11_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_11_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_11_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_11_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_11_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_11_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:2:1101:1542:1000_1:N:0:NATGCCTT+TAATGTGT/1 77 * 0 0 * * 0 0 ATGTAAAAATTAAAAAAATTATTAATTTATTTTAATTAAATTTAAATTTTAAAATTTTAATTTTTTTATTTTAAAATTTAAATTAAAAAATATATTTATGAATTATTATATTAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFF:FFF,F:,,,F:F:F:FFF,::FF:FFF:FFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:1542:1000_2:N:0:NATGCCTT+TAATGTGT/2 141 * 0 0 * * 0 0 TTAAAATTAAATAAAATATTTTTTAATTAATATAATAATTCATAAATATATTTTTTAATTTAAATTTTAAAATAAAAAAATTAAAATTTTAAAATTTAAATTTAATTAAAATAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF,FFFFFFFFFFFFFFFFF:FFF:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_11_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_11_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:2:1101:1542:1000_1:N:0:NATGCCTT+TAATGTGT/1 99 NC_088860.1_GA_converted 18800748 42 115M = 18800774 140 ACATAAAAATTAAAAAAACTATTAATTCATTCTAATTAAATTCAAATTCCAAAATTTTAACTTTTTTATTTTAAAATTTAAATTAAAAAACACATCCACAAACTACTATACCAAC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFF:FFF,F:,,,F:F:F:FFF,::FF:FFF:FFF,F AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YS:i:0 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:1542:1000_2:N:0:NATGCCTT+TAATGTGT/2 147 NC_088860.1_GA_converted 18800774 42 114M = 18800748 -140 TCATTCTAATTAAATTCAAATTCCAAAATTTTAACTTTTTTATTTTAAAATTTAAATTAAAAAACACATCCACAAACTACTATACCAACCAAAAAATATTCTACTTAACTTCAA F:FFF:FFFFFFFFFFFFFFFFF,FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:114 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_11_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_11_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:2:1101:1542:1000_1:N:0:NATGCCTT+TAATGTGT/1 77 * 0 0 * * 0 0 ACATAAAAATTAAAAAAACTATTAATTCATTCTAATTAAATTCAAATTCCAAAATTTTAACTTTTTTATTTTAAAATTTAAATTAAAAAACACATCCACAAACTACTATACCAAC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFF:FFF,F:,,,F:F:F:FFF,::FF:FFF:FFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:1542:1000_2:N:0:NATGCCTT+TAATGTGT/2 141 * 0 0 * * 0 0 TTGAAGTTAAGTAGAATATTTTTTGGTTGGTATAGTAGTTTGTGGATGTGTTTTTTAATTTAAATTTTAAAATAAAAAAGTTAAAATTTTGGAATTTGAATTTAATTAGAATGA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF,FFFFFFFFFFFFFFFFF:FFF:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_11_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_11_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:2:1101:1542:1000_1:N:0:NATGCCTT+TAATGTGT/1 77 * 0 0 * * 0 0 ATGTAAAAATTAAAAAAATTATTAATTTATTTTAATTAAATTTAAATTTTAAAATTTTAATTTTTTTATTTTAAAATTTAAATTAAAAAATATATTTATGAATTATTATATTAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFF:FFF,F:,,,F:F:F:FFF,::FF:FFF:FFF,F YT:Z:UP A00742:173:HMGVGDSXY:2:1101:1542:1000_2:N:0:NATGCCTT+TAATGTGT/2 141 * 0 0 * * 0 0 TTAAAATTAAATAAAATATTTTTTAATTAATATAATAATTCATAAATATATTTTTTAATTTAAATTTTAAAATAAAAAAATTAAAATTTTAAAATTTAAATTTAATTAAAATAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFF,FFFFFFFFFFFFFFFFF:FFF:F YT:Z:UP >>> Writing bisulfite mapping results to zr3644_11_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_11_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_11_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:2:1121:16504:8656_1:N:0:TATGCCTT+TAATGTGT NW_027062586.1 257919 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1170:18349:25003_1:N:0:TATGCCTT+TAATGTGT NW_027062570.1 3921 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 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1459:22146:19867_1:N:0:TATGCCTT+TAATGTGT NW_027062568.1 15488 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2142:16423:14559_1:N:0:TATGCCTT+TAATGTGT NW_027062575.1 2 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 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2429:24297:21997_1:N:0:TATGCCTT+TAATGTGT NW_027062568.1 15482 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far 56063348 reads; of these: 56063348 (100.00%) were paired; of these: 40137802 (71.59%) aligned concordantly 0 times 6847866 (12.21%) aligned concordantly exactly 1 time 9077680 (16.19%) aligned concordantly >1 times 28.41% overall alignment rate 56063348 reads; of these: 56063348 (100.00%) were paired; of these: 40203702 (71.71%) aligned concordantly 0 times 6811962 (12.15%) aligned concordantly exactly 1 time 9047684 (16.14%) aligned concordantly >1 times 28.29% overall alignment rate 56063348 reads; of these: 56063348 (100.00%) were paired; of these: 40233706 (71.76%) aligned concordantly 0 times 6823635 (12.17%) aligned concordantly exactly 1 time 9006007 (16.06%) aligned concordantly >1 times 28.24% overall alignment rate 56063348 reads; of these: 56063348 (100.00%) were paired; of these: 40176407 (71.66%) aligned concordantly 0 times 6852555 (12.22%) aligned concordantly exactly 1 time 9034386 (16.11%) aligned concordantly >1 times 28.34% overall alignment rate Processed 56063348 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 224253392. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 224253392. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 224253392. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 224253392. Successfully deleted the temporary files zr3644_11_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_11_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_11_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_11_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 56063348 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1230748721 Total methylated C's in CpG context: 16690108 Total methylated C's in CHG context: 1066444 Total methylated C's in CHH context: 4783259 Total methylated C's in Unknown context: 72842 Total unmethylated C's in CpG context: 139942032 Total unmethylated C's in CHG context: 212487210 Total unmethylated C's in CHH context: 855779668 Total unmethylated C's in Unknown context: 4071864 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 5h 3m 50s ==================== Bismark run complete ====================