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/ (absolute path is '/home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/)' 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-mytilus-methylation/code'): ../data/240M_R1.fastp-trim.fq.gz ../data/240M_R2.fastp-trim.fq.gz Library is assumed to be strand-specific (directional), alignments to strands complementary to the original top or bottom strands will be ignored (i.e. not performed!) Output will be written into the directory: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/output/01.2-bismark/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/code Now reading in and storing sequence information of the genome specified in: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../data/240M_R1.fastp-trim.fq.gz and ../data/240M_R2.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 240M_R1.fastp-trim.fq.gz to 240M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 240M_R1.fastp-trim.fq.gz (87578771 sequences in total) Writing a G -> A converted version of the input file 240M_R2.fastp-trim.fq.gz to 240M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 240M_R2.fastp-trim.fq.gz (87578771 sequences in total) Input files are 240M_R1.fastp-trim.fq.gz_C_to_T.fastq and 240M_R2.fastp-trim.fq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/ with the specified options: -q --score-min L,0,-1.0 -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 240M_R1.fastp-trim.fq.gz_C_to_T.fastq and 240M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1348:1014_1:N:0:AGTTCGTC+ACTCGATC/1 77 * 0 0 * * 0 0 ATGTTGTTATGGTTATTTGATTGAATTATGTTTATGGGTTGATAATAAAATAATGTTAGTTAATTAGAAAATGTGTTATATTTAAAATTAAATTATATTAATATAATAAGATATTATTAAATATATTTAGT IIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIII9I9IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIII9IIIIII YT:Z:UP LH00469:254:22HGFVLT4:2:1101:1348:1014_2:N:0:AGTTCGTC+ACTCGATC/2 141 * 0 0 * * 0 0 AAAAATCCAAACTCATCAAAAATAAAAATTTAAAAAAAAATAAATTTATTATTCTTCCCTATTCTAAACAACACTATAATACTAAATATATTTAATAATATCTTATTATATTAATATAATTTAATTTTAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIII9IIIIIIIIIIIII-IIIIII9I-IIIIIIIIIIIII9-IIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIII9I YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 240M_R1.fastp-trim.fq.gz_C_to_T.fastq and 240M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:1348:1014_1:N:0:AGTTCGTC+ACTCGATC/1 83 NC_086380.1_GA_converted 28180887 24 131M = 28180813 -205 ACTAAATATATTTAATAATATCTTATTATATTAATATAATTTAATTTTAAATATAACACATTTTCTAATTAACTAACATTATTTTATTATCAACCCATAAACATAATTCAATCAAATAACCATAACAACAT IIIIII9IIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII9I9IIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-18 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:13T65C18T32 YS:i:-82 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:1348:1014_2:N:0:AGTTCGTC+ACTCGATC/2 163 NC_086380.1_GA_converted 28180813 24 25M5I1M1I99M = 28180887 205 AAAAATCCAAACTCATCAAAAATAAAAATTTAAAAAAAAATAAATTTATTATTCTTCCCTATTCTAAACAACACTATAATACTAAATATATTTAATAATATCTTATTATATTAATATAATTTAATTTTAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIII9IIIIIIIIIIIII-IIIIII9I-IIIIIIIIIIIII9-IIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIII9I AS:i:-82 XN:i:0 XM:i:9 XO:i:2 XG:i:6 NM:i:15 MD:Z:2T1T2T5A0T3T7C46C13T37 YS:i:-18 YT:Z:CP >>> Writing bisulfite mapping results to 240M_pe.bam <<< Reading in the sequence files ../data/240M_R1.fastp-trim.fq.gz and ../data/240M_R2.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1115:48429:8242_1:N:0:AGTTCGTC+ACTCGATC NW_026963639.1 24139 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 LH00469:254:22HGFVLT4:2:1147:4342:5356_1:N:0:AGTTCGTC+ACTCGATC NW_026963564.1 19023 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1149:50055:18286_1:N:0:AGTTCGTC+ACTCGATC NW_026963650.1 20974 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1207:15186:21577_1:N:0:AGTTCGTC+ACTCGATC NW_026963389.1 32297 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1218:4642:20723_1:N:0:AGTTCGTC+ACTCGATC NW_026963307.1 124878 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1221:15647:9545_1:N:0:AGTTCGTC+ACTCGATC NW_026963362.1 2 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1241:29736:3788_1:N:0:AGTTCGTC+ACTCGATC NW_026963372.1 501151 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 LH00469:254:22HGFVLT4:2:1275:27025:16044_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 1 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1288:20405:2317_1:N:0:AGTTTGTC+ACTCGATC NW_026963364.1 1 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1306:6292:8900_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 2 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 LH00469:254:22HGFVLT4:2:1340:28004:2051_1:N:0:AGTTCGTC+ACTCGATC NW_026963307.1 124871 Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1361:36978:18538_1:N:0:AGTTCGTC+ACTCGATC NW_026963675.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1361:36970:18552_1:N:0:AGTTCGTC+ACTCGATC NW_026963675.1 1 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1373:20421:1252_1:N:0:AGTTCGTC+ACTCGATC NW_026963786.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1377:7919:19000_1:N:0:AGTTCGTC+ACTCGATC NW_026963316.1 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1382:8582:9082_1:N:0:AGTTCGTC+ACTCGATC NW_026963788.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1386:42109:24113_1:N:0:AGTTCGTC+ACTCGATC NW_026963307.1 124877 Processed 27000000 sequence pairs so far 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1466:32406:18524_1:N:0:AGTTCGTC+ACTCGATC NW_026963506.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1473:27081:26340_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 3 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1484:3662:15638_1:N:0:AGTTCGTC+ACTCGATC NW_026963307.1 124871 Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2133:25989:8564_1:N:0:AGTTCGTC+ACTCGATC NW_026963309.1 2 Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2166:10873:4614_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 3 Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2181:14652:20149_1:N:0:AGTTCGTC+ACTCGATC NW_026963319.1 249906 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2237:48857:21760_1:N:0:AGTTCGTC+ACTCGATC NW_026963550.1 1 Processed 49000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2242:41898:22656_1:N:0:AGTTCGTC+ACTCGATC NW_026963583.1 63477 Processed 50000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2254:36808:11324_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 2 Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2282:46923:22194_1:N:0:AGTTCGTC+ACTCGATC NW_026963684.1 18490 Processed 53000000 sequence pairs so far Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2316:28133:25780_1:N:0:AGTTCGTC+ACTCGATC NW_026963684.1 18488 Processed 56000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2333:23691:27111_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2333:23707:27111_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 1 Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2348:40814:20583_1:N:0:AGTTCGTC+ACTCGATC NW_026963772.1 1 Processed 59000000 sequence pairs so far Processed 60000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2378:5046:9293_1:N:0:AGTTCGTC+ACTCGATC NW_026963335.1 1 Processed 61000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2389:34987:18818_1:N:0:AGTTCGTC+ACTCGATC NW_026963616.1 36405 Processed 62000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2403:32009:6687_1:N:0:AGTTCGTC+ACTCGATC NW_026963636.1 18735 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 LH00469:254:22HGFVLT4:2:2435:37350:19518_1:N:0:AGTTCGTC+ACTCGATC NW_026963598.1 1 Processed 66000000 sequence pairs so far Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Processed 69000000 sequence pairs so far Processed 70000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1110:39551:14083_1:N:0:AGTTCGTC+ACTCGATC NW_026963482.1 3 Processed 71000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1154:22558:24309_1:N:0:AGTTCGTC+ACTCGATC NW_026963684.1 18470 Processed 72000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1186:34405:3774_1:N:0:AGTTCGTC+ACTCGATC NW_026963576.1 1 Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1191:43347:5553_1:N:0:AGTTCGTC+ACTCGATC NW_026963684.1 18467 Processed 73000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1257:22323:16983_1:N:0:AGTTCGTC+ACTCGATC NW_026963335.1 2 Processed 74000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1277:6600:11758_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 1 Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1290:15906:18930_1:N:0:AGTTCGTC+ACTCGATC NC_086380.1 80675424 Processed 75000000 sequence pairs so far Processed 76000000 sequence pairs so far Processed 77000000 sequence pairs so far Processed 78000000 sequence pairs so far Processed 79000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1496:47312:19140_1:N:0:AGTTCGTC+ACTCGATC NW_026963415.1 3 Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2103:12143:18272_1:N:0:AGTTCGTC+ACTCGATC NW_026963418.1 1 Processed 80000000 sequence pairs so far Processed 81000000 sequence pairs so far Processed 82000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2261:30262:18202_1:N:0:AGTTCGTC+ACTCGATC NW_026963307.1 124869 Processed 83000000 sequence pairs so far Processed 84000000 sequence pairs so far Processed 85000000 sequence pairs so far Processed 86000000 sequence pairs so far Processed 87000000 sequence pairs so far 87578771 reads; of these: 87578771 (87578771100.00 reads; of these:% ) were paired; of these: 87578771 (32669028 (37.30%) aligned concordantly 0 times 15008543 (100.0017.14%%) were paired; of these:) aligned concordantly exactly 1 time 3267905639901200 ( (37.3145.56%%) aligned concordantly 0 times) aligned concordantly >1 times 62.7015011776% ( overall alignment rate17.14 %) aligned concordantly exactly 1 time 39887939 (45.55%) aligned concordantly >1 times 62.69% overall alignment rate Processed 87578771 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 350315084. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 350315084. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 350315084. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 350315084. Successfully deleted the temporary files 240M_R1.fastp-trim.fq.gz_C_to_T.fastq and 240M_R2.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 87578771 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2169746117 Total methylated C's in CpG context: 34269450 Total methylated C's in CHG context: 3531041 Total methylated C's in CHH context: 15925445 Total methylated C's in Unknown context: 422447 Total unmethylated C's in CpG context: 238403186 Total unmethylated C's in CHG context: 354608507 Total unmethylated C's in CHH context: 1523008488 Total unmethylated C's in Unknown context: 12170352 C methylated in CpG context: 12.6% C methylated in CHG context: 1.0% C methylated in CHH context: 1.0% C methylated in unknown context (CN or CHN): 3.4% Bismark completed in 0d 6h 2m 59s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe