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/79M_R1.fastp-trim.fq.gz ../data/79M_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/79M_R1.fastp-trim.fq.gz and ../data/79M_R2.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 79M_R1.fastp-trim.fq.gz to 79M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 79M_R1.fastp-trim.fq.gz (88168089 sequences in total) Writing a G -> A converted version of the input file 79M_R2.fastp-trim.fq.gz to 79M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 79M_R2.fastp-trim.fq.gz (88168089 sequences in total) Input files are 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_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 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_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: LH00469:254:22HGFVLT4:2:1101:1906:1028_1:N:0:GTCATCGA+GATCCACT/1 99 NC_086380.1_CT_converted 73522031 17 131M = 73522189 289 TTGAGTAATGAATTGTGAAAATAAGGTTATGGTTAAATAAAATTTGTGTGATTATTGTATAGATTATAAAATATTTTTATATATTTAATATAGTTTATTTATGGTGTATAGTATTTGATAAAATGATTAAA III9IIIIIIIIIIIIIIIIIII9IIIII-IIIIII9IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIII9IIIIIIIIIIIII9IIIIII9IIIIIIIIIIIIIIIIIIIIIIII-I9IIIIIIII AS:i:-6 XS:i:-36 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:29A101 YS:i:0 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:1906:1028_2:N:0:GTCATCGA+GATCCACT/2 147 NC_086380.1_CT_converted 73522189 17 131M = 73522031 -289 ATTATGAAAATAAGGTTAAGGTTAGATAATATTTGTTTATTAGATATGTTTATTTTTTAATAATTTTATATAATAAATATAGTAGATTTATTGTTTAAAGTATGAGAAAAATAGATTAAATATAAAAATTT IIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII9I9IIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIII9IIIII9IIIII9IIIIIIIIII9IIIII9IIIII9IIIIIII AS:i:0 XS:i:-32 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:131 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_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: LH00469:254:22HGFVLT4:2:1101:1906:1028_1:N:0:GTCATCGA+GATCCACT/1 83 NC_086376.1_GA_converted 19370512 22 131M = 19370354 -289 TTTAATCATTTTATCAAATACTATACACCATAAATAAACTATATTAAATATATAAAAATATTTTATAATCTATACAATAATCACACAAATTTTATTTAACCATAACCTTATTTTCACAATTCATTACTCAA IIIIIIII9I-IIIIIIIIIIIIIIIIIIIIIIII9IIIIII9IIIIIIIIIIIII9IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII9IIIIII-IIIII9IIIIIIIIIIIIIIIIIII9III AS:i:-6 XS:i:-120 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:101T29 YS:i:0 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:1906:1028_2:N:0:GTCATCGA+GATCCACT/2 163 NC_086376.1_GA_converted 19370354 22 131M = 19370512 289 AAATTTTTATATTTAATCTATTTTTCTCATACTTTAAACAATAAATCTACTATATTTATTATATAAAATTATTAAAAAATAAACATATCTAATAAACAAATATTATCTAACCTTAACCTTATTTTCATAAT IIIIIII9IIIII9IIIII9IIIIIIIIII9IIIII9IIIII9IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIII9I9IIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIII AS:i:0 XS:i:-50 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:131 YS:i:-6 YT:Z:CP >>> Writing bisulfite mapping results to 79M_pe.bam <<< Reading in the sequence files ../data/79M_R1.fastp-trim.fq.gz and ../data/79M_R2.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1105:50444:23917_1:N:0:GTCATCGA+GATCCACT NW_026963298.1 2 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1119:13543:28175_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 1 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1137:19636:18664_1:N:0:GTCATCGA+GATCCACT NW_026963317.1 4467291 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1137:19645:18678_1:N:0:GTCATCGA+GATCCACT NW_026963317.1 4467291 Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1158:37577:18258_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 1 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 LH00469:254:22HGFVLT4:2:1190:17945:7360_1:N:0:GTCATCGA+GATCCACT NW_026963814.1 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 LH00469:254:22HGFVLT4:2:1227:38014:13999_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 3 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1251:23844:19925_1:N:0:GTCATCGA+GATCCACT NW_026963743.1 1 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1253:14158:15512_1:N:0:GTCATCGA+GATCCACT NW_026963374.1 73858 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1255:45507:19854_1:N:0:GTCATCGA+GATCCACT NW_026963406.1 16968 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1261:48121:26256_1:N:0:GTCATCGA+GATCCACT NW_026963487.1 25724 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1262:2821:21774_1:N:0:GTCATCGA+GATCCACT NW_026963450.1 23509 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1271:23415:11814_1:N:0:GTCCTCGA+GATCCACT NW_026963377.1 2 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1295:44051:7444_1:N:0:GTCATCGA+GATCCACT NW_026963377.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1295:41882:19882_1:N:0:GTCATCGA+GATCCACT NW_026963350.1 1 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1303:32997:9629_1:N:0:GTCATCGA+GATCCACT NW_026963337.1 658963 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1361:31216:23609_1:N:0:GTCATCGA+GATCCACT NW_026963699.1 2 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 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1454:48356:16016_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1454:48348:16030_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 1 Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1459:24257:10553_1:N:0:GTCATCGA+GATCCACT NW_026963295.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1466:49060:5020_1:N:0:GTCATCGA+GATCCACT NW_026963699.1 2 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1482:29590:11800_1:N:0:GTCATCGA+GATCCACT NC_086378.1 1 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2147:38669:10147_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 1 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2151:6211:12178_1:N:0:GTCATCGA+GATCCACT NW_026963402.1 95204 Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2172:6341:5370_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2177:35627:1238_1:N:0:GTCATCGA+GATCCACT NW_026963290.1 8139873 Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2185:1081:14307_1:N:0:GTCATCGA+GATCCACT NW_026963293.1 1 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2221:44310:19070_1:N:0:GTCATCGA+GATCCACT NW_026963402.1 95213 Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2252:29307:1532_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 2 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2318:51900:3493_1:N:0:GTCATCGA+GATCCAAT NW_026963699.1 3 Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2344:15817:28918_1:N:0:GTCATCGA+GATCCACT NW_026963443.1 56335 Processed 57000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2364:41696:12444_1:N:0:GTCATCGA+GATCCACT NW_026963401.1 21036 Processed 58000000 sequence pairs so far Processed 59000000 sequence pairs so far Processed 60000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2404:30529:27909_1:N:0:GTCATCGA+GATCCACT NW_026963684.1 18469 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2407:49052:12851_1:N:0:GTCATCGA+GATCCACT NW_026963307.1 3 Processed 61000000 sequence pairs so far Processed 62000000 sequence pairs so far Processed 63000000 sequence pairs so far Processed 64000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2450:3816:4978_1:N:0:GTCATCGA+GATCCACT NW_026963317.1 4467312 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2450:3824:4992_1:N:0:GTCATCGA+GATCCACT NW_026963317.1 4467312 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2450:3816:5006_1:N:0:GTCATCGA+GATCCACT NW_026963317.1 4467312 Processed 65000000 sequence pairs so far Processed 66000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2474:29331:18300_1:N:0:GTCATCGA+GATCCACT NW_026963487.1 2 Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1109:15234:26424_1:N:0:GTCATCGA+GATCCACT NW_026963677.1 25109 Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1114:4601:21297_1:N:0:GTCATCGA+GATCCACT NW_026963684.1 18480 Processed 69000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1160:15404:29380_1:N:0:GTCATCGA+GATCCACT NW_026963293.1 1 Processed 70000000 sequence pairs so far Processed 71000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1220:45151:2317_1:N:0:GTCATCGA+GATCCACT NW_026963299.1 1 Processed 72000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1256:50282:3157_1:N:0:GTCATCGA+GATCCACT NW_026963684.1 18461 Processed 73000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1310:4310:12949_1:N:0:GTCATCGA+GATCCACT NW_026963517.1 8510 Processed 74000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1338:8736:23160_1:N:0:GTCATCGA+GATCCACT NW_026963494.1 1 Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:1340:45062:20149_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 3 Processed 75000000 sequence pairs so far Processed 76000000 sequence pairs so far Processed 77000000 sequence pairs so far Processed 78000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2107:28376:20093_1:N:0:GTCATCGA+GATCCACT NW_026963415.1 2 Processed 79000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2133:2748:22236_1:N:0:GTCATCGA+GATCCACT NW_026963749.1 2 Processed 80000000 sequence pairs so far Processed 81000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2213:33782:14658_1:N:0:GTCATCGA+GATCCACT NW_026963598.1 2 Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2217:47142:14532_1:N:0:GTCATCGA+GATCCACT NW_026963699.1 1 Processed 82000000 sequence pairs so far Processed 83000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2308:45224:9727_1:N:0:GTCATCGA+GATCCACT NW_026963453.1 73102 Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2314:51374:1098_1:N:0:GTCATCGA+GATCCACT NW_026963308.1 1 Processed 84000000 sequence pairs so far Processed 85000000 sequence pairs so far Processed 86000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2427:18690:5034_1:N:0:GTCATCGA+GATCCACT NW_026963418.1 81291 Processed 87000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:122:22HTFTLT4:7:2456:9125:4754_1:N:0:GTCATCGA+GATCCACT NW_026963725.1 1 Processed 88000000 sequence pairs so far 88168089 reads; of these: 88168089 (100.00%) were paired; of these: 38092969 (43.20%) aligned concordantly 0 times 17408571 (19.74%) aligned concordantly exactly 1 time 32666549 (37.05%) aligned concordantly >1 times 56.80% overall alignment rate 88168089 reads; of these: 88168089 (100.00%) were paired; of these: 38096864 (43.21%) aligned concordantly 0 times 17400791 (19.74%) aligned concordantly exactly 1 time 32670434 (37.05%) aligned concordantly >1 times 56.79% overall alignment rate Processed 88168089 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 352672356. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 352672356. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 352672356. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 352672356. Successfully deleted the temporary files 79M_R1.fastp-trim.fq.gz_C_to_T.fastq and 79M_R2.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 88168089 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2147649385 Total methylated C's in CpG context: 28299299 Total methylated C's in CHG context: 3207786 Total methylated C's in CHH context: 14994187 Total methylated C's in Unknown context: 372662 Total unmethylated C's in CpG context: 219549241 Total unmethylated C's in CHG context: 340654143 Total unmethylated C's in CHH context: 1540944729 Total unmethylated C's in Unknown context: 11939840 C methylated in CpG context: 11.4% C methylated in CHG context: 0.9% C methylated in CHH context: 1.0% C methylated in unknown context (CN or CHN): 3.0% Bismark completed in 0d 6h 5m 11s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe