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_7_R1_val_1_val_1_val_1.fq.gz ../../data/Haws-10/zr3644_7_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_7_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_7_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_7_R1_val_1_val_1_val_1.fq.gz to zr3644_7_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_7_R1_val_1_val_1_val_1.fq.gz to zr3644_7_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_7_R1_val_1_val_1_val_1.fq.gz (81572749 sequences in total) Writing a C -> T converted version of the input file zr3644_7_R2_val_2_val_2_val_2.fq.gz to zr3644_7_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file zr3644_7_R2_val_2_val_2_val_2.fq.gz to zr3644_7_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_7_R2_val_2_val_2_val_2.fq.gz (81572749 sequences in total) Input files are zr3644_7_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_7_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_7_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_7_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_7_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_7_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:2:1101:2700:1000_1:N:0:NCGGTCAG+AACGAGGC/1 99 NC_088853.1_CT_converted 4679845 12 115M = 4679871 140 TTGTTGAAATTTAATTTTTTGAAAAAAATTTGATATTTTTTTAATTGGTTTGGATGGTGAAAATATGATTTTAGTTTGAAATATATGAAATTTATGTTTAAATATTTTAGTAATT FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFF:FFFFFFF:FFFFF AS:i:0 XS:i:-24 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YS:i:-6 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2700:1000_2:N:0:NCGGTCAG+AACGAGGC/2 147 NC_088853.1_CT_converted 4679871 12 114M = 4679845 -140 AATTTGATATTTTTTTAATTGGTTTGGATGGTGAAAATATGATTTTAGTTTGAAATATATGAAATTTATGTTTAAATATTTTAGTAATTGTTATAGGATTGTTTTTTAGTATTT FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-6 XS:i:-12 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:108A5 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from zr3644_7_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_7_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:2:1101:2700:1000_1:N:0:NCGGTCAG+AACGAGGC/1 77 * 0 0 * * 0 0 TTATTAAAATTTAATTTTTTAAAAAAAATTTAATATTTTTTTAATTAATTTAAATAATAAAAATATAATTTTAATTTAAAATATATAAAATTTATATTTAAATATTTTAATAATT FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFF:FFFFFFF:FFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2700:1000_2:N:0:NCGGTCAG+AACGAGGC/2 141 * 0 0 * * 0 0 AAATATTAAAAAATAATTTTATAATAATTATTAAAATATTTAAATATAAATTTTATATATTTTAAATTAAAATTATATTTTTATTATTTAAATTAATTAAAAAAATATTAAATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from zr3644_7_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq and zr3644_7_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:2:1101:2700:1000_1:N:0:NCGGTCAG+AACGAGGC/1 77 * 0 0 * * 0 0 TTATTAAAATTTAATTTTTTAAAAAAAATTTAATATTTTTTTAATTAATTTAAATAATAAAAATATAATTTTAATTTAAAATATATAAAATTTATATTTAAATATTTTAATAATT FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF:FFFFF:FFFFFFF:FFFFFFF:FFFFF YT:Z:UP A00742:173:HMGVGDSXY:2:1101:2700:1000_2:N:0:NCGGTCAG+AACGAGGC/2 141 * 0 0 * * 0 0 AAATATTAAAAAATAATTTTATAATAATTATTAAAATATTTAAATATAAATTTTATATATTTTAAATTAAAATTATATTTTTATTATTTAAATTAATTAAAAAAATATTAAATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from zr3644_7_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq and zr3644_7_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:2:1101:2700:1000_1:N:0:NCGGTCAG+AACGAGGC/1 83 NC_088853.1_GA_converted 29358294 1 115M = 29358269 -140 AATTACTAAAATATTTAAACATAAATTTCATATATTTCAAACTAAAATCATATTTTCACCATCCAAACCAATTAAAAAAATATCAAATTTTTTTCAAAAAATTAAATTTCAACAA FFFFF:FFFFFFF:FFFFFFF:FFFFF:FFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:115 YS:i:-6 YT:Z:CP A00742:173:HMGVGDSXY:2:1101:2700:1000_2:N:0:NCGGTCAG+AACGAGGC/2 163 NC_088853.1_GA_converted 29358269 1 114M = 29358294 140 AAATACTAAAAAACAATCCTATAACAATTACTAAAATATTTAAACATAAATTTCATATATTTCAAACTAAAATCATATTTTCACCATCCAAACCAATTAAAAAAATATCAAATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF AS:i:-6 XS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:5T108 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to zr3644_7_pe.bam <<< Reading in the sequence files ../../data/Haws-10/zr3644_7_R1_val_1_val_1_val_1.fq.gz and ../../data/Haws-10/zr3644_7_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:2:1114:19886:8531_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1220:1208:8437_1:N:0:ACGGTCAG+AACGAGGC 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:2:1249:1949:25316_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3895 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1250:23357:23156_1:N:0:ACGGTCAG+AACGAGGC NW_027062577.1 1 Processed 12000000 sequence pairs so far 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:2:1319:32723:18490_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3891 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1356:23990:11068_1:N:0:ACGGTCAG+AACGAGGC NW_027062569.1 16385 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1411:20094:18568_1:N:0:ACGGTCAG+AACGAGGC NW_027062573.1 1900 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1420:20437:10394_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1512:5529:27946_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1516:20347:10802_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1530:24469:23891_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3891 Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1577:2519:4633_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3885 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1577:5439:5181_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3885 Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1616:18792:34413_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:1621:1470:4476_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3894 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:2:1657:14145:26584_1:N:0:ACGGTCAG+AACGAGGC NW_027062569.1 16385 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 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 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2246:2410:21167_1:N:0:ACGGTCAG+AACGAGGC NW_027062577.1 1 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 Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far Processed 59000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2358:7771:26318_1:N:0:ACGGTCAG+AACGAGGC NW_027062568.1 15482 Processed 60000000 sequence pairs so far Processed 61000000 sequence pairs so far Processed 62000000 sequence pairs so far Processed 63000000 sequence pairs so far Processed 64000000 sequence pairs so far Processed 65000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2445:30373:2143_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2451:15573:19382_1:N:0:ACGGTCAG+AACGAGGC NC_088853.1 76070884 Processed 66000000 sequence pairs so far Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2502:12988:33630_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Processed 69000000 sequence pairs so far Processed 70000000 sequence pairs so far Processed 71000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2536:11758:5603_1:N:0:ACGGTCAG+AACGAGGC NW_027062577.1 1 Processed 72000000 sequence pairs so far Processed 73000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2559:24451:9392_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3905 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2559:25870:12414_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 3905 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2562:23502:19742_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Processed 74000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2602:22354:3944_1:N:0:ACGGTCAG+AACGAGGC NW_027062575.1 49318 Processed 75000000 sequence pairs so far Processed 76000000 sequence pairs so far Processed 77000000 sequence pairs so far Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2628:7437:18192_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2628:6922:18208_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Chromosomal sequence could not be extracted for A00742:173:HMGVGDSXY:2:2628:7446:18208_1:N:0:ACGGTCAG+AACGAGGC NW_027062570.1 1 Processed 78000000 sequence pairs so far Processed 79000000 sequence pairs so far Processed 80000000 sequence pairs so far Processed 81000000 sequence pairs so far 81572749 reads; of these: 81572749 (100.00%) were paired; of these: 58705557 (71.97%) aligned concordantly 0 times 9934807 (12.18%) aligned concordantly exactly 1 time 12932385 (15.85%) aligned concordantly >1 times 28.03% overall alignment rate 81572749 reads; of these: 81572749 (100.00%) were paired; of these: 58660911 (71.91%) aligned concordantly 0 times 9936670 (12.18%) aligned concordantly exactly 1 time 12975168 (15.91%) aligned concordantly >1 times 28.09% overall alignment rate 81572749 reads; of these: 81572749 (100.00%) were paired; of these: 58703181 (71.96%) aligned concordantly 0 times 9908585 (12.15%) aligned concordantly exactly 1 time 12960983 (15.89%) aligned concordantly >1 times 28.04% overall alignment rate 81572749 reads; of these: 81572749 (100.00%) were paired; of these: 58744329 (72.01%) aligned concordantly 0 times 9908956 (12.15%) aligned concordantly exactly 1 time 12919464 (15.84%) aligned concordantly >1 times 27.99% overall alignment rate Processed 81572749 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 326290996. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 326290996. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 326290996. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 326290996. Successfully deleted the temporary files zr3644_7_R1_val_1_val_1_val_1.fq.gz_C_to_T.fastq, zr3644_7_R1_val_1_val_1_val_1.fq.gz_G_to_A.fastq, zr3644_7_R2_val_2_val_2_val_2.fq.gz_C_to_T.fastq and zr3644_7_R2_val_2_val_2_val_2.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 81572749 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1802698980 Total methylated C's in CpG context: 24128276 Total methylated C's in CHG context: 1542890 Total methylated C's in CHH context: 6881292 Total methylated C's in Unknown context: 101023 Total unmethylated C's in CpG context: 202773934 Total unmethylated C's in CHG context: 307930814 Total unmethylated C's in CHH context: 1259441774 Total unmethylated C's in Unknown context: 6045670 C methylated in CpG context: 10.6% 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 7h 9m 33s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe