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 /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/ (absolute path is '/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/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/8TB_HDD_03/sr320/github/timeseries_molecular/D-Apul/code'): /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/01.20-D-Apul-WGBS-trimming-fastp-FastQC-MultiQC/1D8_R1_001.fastp-trim.fq.gz /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/01.20-D-Apul-WGBS-trimming-fastp-FastQC-MultiQC/1D8_R2_001.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/8TB_HDD_03/sr320/github/timeseries_molecular/D-Apul/output/14-Apul-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/8TB_HDD_03/sr320/github/timeseries_molecular/D-Apul/code Now reading in and storing sequence information of the genome specified in: /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/01.20-D-Apul-WGBS-trimming-fastp-FastQC-MultiQC/1D8_R1_001.fastp-trim.fq.gz and /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/01.20-D-Apul-WGBS-trimming-fastp-FastQC-MultiQC/1D8_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1D8_R1_001.fastp-trim.fq.gz to 1D8_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1D8_R1_001.fastp-trim.fq.gz (40093521 sequences in total) Writing a G -> A converted version of the input file 1D8_R2_001.fastp-trim.fq.gz to 1D8_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1D8_R2_001.fastp-trim.fq.gz (40093521 sequences in total) Input files are 1D8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D8_R2_001.fastp-trim.fq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/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 1D8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D8_R2_001.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: LH00526:197:22KFKVLT4:3:1101:44997:1070_1:N:0:CTTACAGC+CTTCACCA/1 77 * 0 0 * * 0 0 TGTTTTTAATTGTGTATTTATGAATTTTTAAGTATATTTATATGTTGTTTTGTATTATTGTAATAGTATATTTTTTGTGGATTTTGTGTGAATATGGTAAAATTTGATAGTTGTTGAATGTATTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:44997:1070_2:N:0:CTTACAGC+CTTCACCA/2 141 * 0 0 * * 0 0 TTCTAAAAATAACCAAAAATAACAATAAACATACAAATTACAATACTACTAACAAACATATCAAAAAATAAATATATATAATCCACCCCACCCCAAAAAATATAAAAACACATTATCATAAAATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIII9III9I-I-I9I-IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1D8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D8_R2_001.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: LH00526:197:22KFKVLT4:3:1101:44997:1070_1:N:0:CTTACAGC+CTTCACCA/1 83 ptg000019l_GA_converted 2931611 42 125M = 2931316 -420 AAATACATTCAACAACTATCAAATTTTACCATATTCACACAAAATCCACAAAAAATATACTATTACAATAATACAAAACAACATATAAATATACTTAAAAATTCATAAATACACAATTAAAAACA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:33C69T21 YS:i:-30 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:44997:1070_2:N:0:CTTACAGC+CTTCACCA/2 163 ptg000019l_GA_converted 2931316 42 125M = 2931611 420 TTCTAAAAATAACCAAAAATAACAATAAACATACAAATTACAATACTACTAACAAACATATCAAAAAATAAATATATATAATCCACCCCACCCCAAAAAATATAAAAACACATTATCATAAAATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIII9III9I-I-I9I-IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII AS:i:-30 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:43A4A25C6C30A12 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to 1D8_pe.bam <<< Reading in the sequence files /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/01.20-D-Apul-WGBS-trimming-fastp-FastQC-MultiQC/1D8_R1_001.fastp-trim.fq.gz and /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/01.20-D-Apul-WGBS-trimming-fastp-FastQC-MultiQC/1D8_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1137:18180:1798_1:N:0:CTTACAGC+CTTCACCA ptg000105l 83244 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1157:33159:16128_1:N:0:CTTACAGC+CTTCACCA ptg000089l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1167:22194:10763_1:N:0:CTTACAGC+CTTCACCA ptg000065l 49681 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1177:17184:20639_1:N:0:CTTACAGC+CTTCACCA ptg000094l 1 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1208:24241:25962_1:N:0:CTTACAGC+CTTCACCA ptg000066l 1 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1237:18932:25430_1:N:0:CTTACAGC+CTTCACCA ptg000092l 28991 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1262:11811:23328_1:N:0:CTTACAGC+CTTCACCA ptg000006l 3 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1269:46357:13201_1:N:0:CTTACAGC+CTTCACCA ptg000092l 2 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1283:37245:22334_1:N:0:CTTACAGC+CTTCACCA ptg000095l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1284:27089:5146_1:N:0:CTTACAGC+CTTCACCA ptg000088l 96913 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1322:24540:28889_1:N:0:CTTACAGC+CTTCACCA ptg000057l 39950 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1356:29388:9237_1:N:0:CTTACAGC+CTTCACCA ptg000075l 1 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1387:6745:18454_1:N:0:CTTACAGC+CTTCACCA ptg000095l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1387:6762:18454_1:N:0:CTTACAGC+CTTCACCA ptg000095l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1409:11690:12584_1:N:0:CTTACAGC+CTTCACCA ptg000066l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1411:44552:20303_1:N:0:CTTACAGC+CTTCACCA ptg000117l 30013 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1421:42529:25205_1:N:0:CTTACAGC+CTTCACCA ptg000066l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1421:42537:25219_1:N:0:CTTACAGC+CTTCACCA ptg000066l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1434:44480:22614_1:N:0:CTTACAGC+CTTCACCA ptg000080l 40111 Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1458:40571:22180_1:N:0:CTTACAGC+CTTCACCA ptg000096l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1459:36784:8536_1:N:0:CTTACAGC+CTTCACCA ptg000089l 46433 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1467:50581:10343_1:N:0:CTTACAGC+CTTCACCA ptg000029c 1802557 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1484:21554:22600_1:N:0:CTTACAGC+CTTCACCA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2112:18811:1434_1:N:0:CTTACAGC+CTTCACCA ptg000101l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2113:25657:5048_1:N:0:CTTACAGC+CTTCACCA ntLink_0 96484 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2215:22800:25962_1:N:0:CTTACAGC+CTTCACCA ptg000174l 27225 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2271:3832:7640_1:N:0:CTTACAGC+CTTCACCA ptg000057l 39958 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2272:37326:6869_1:N:0:CTTACAGC+CTTCACCA ptg000109l 39630 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2350:7830:14504_1:N:0:CTTACAGC+CTTCACCA ptg000124l 54835 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2431:33191:25065_1:N:0:CTTACAGC+CTTCACCA ptg000011l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2437:42238:2569_1:N:0:CTTACAGC+CTTCACCA ptg000185l 34505 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2438:33555:8018_1:N:0:CTTACAGC+CTTCACCA ptg000011l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2442:31775:14574_1:N:0:CTTACAGC+CTTCACCA ptg000092l 1 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2467:6033:13019_1:N:0:CTTACAGC+CTTCACCA ptg000096l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2470:41016:9839_1:N:0:CTTACAGC+CTTCACCA ptg000047l 1 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2493:49060:12753_1:N:0:CTTACAGC+CTTCACCA ptg000092l 1 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1150:20777:17753_1:N:0:CTTACAGC+CTTCACCA ptg000066l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1164:26984:4796_1:N:0:CTTACAGC+CTTCACCA ptg000098l 55067 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1164:4366:21143_1:N:0:CTTACAGC+CTTCACCA ptg000125l 2 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1174:1486:24421_1:N:0:CTTACAGC+CTTCACCA ptg000012l 3 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1183:25965:28441_1:N:0:CTTACAGC+CTTCACCA ptg000160l 32573 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1190:3128:27601_1:N:0:CTTACAGC+CTTCACCA ptg000096l 1 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1204:23100:17291_1:N:0:CTTACAGC+CTTCACCA ptg000146l 38996 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1214:40239:14574_1:N:0:CTTACAGC+CTTCACCA ptg000066l 2 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1236:37415:15372_1:N:0:CTTACAGC+CTTCACCA ptg000021l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1236:37423:15386_1:N:0:CTTACAGC+CTTCACCA ptg000021l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1236:37415:15400_1:N:0:CTTACAGC+CTTCACCA ptg000021l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1236:37431:15400_1:N:0:CTTACAGC+CTTCACCA ptg000021l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1251:49691:9699_1:N:0:CTTACAGC+CTTCACCA ptg000057l 39950 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1254:28837:22516_1:N:0:CTTACAGC+CTTCACCA ptg000092l 2 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1287:41040:7640_1:N:0:CTTACAGC+CTTCACCA ptg000129l 33365 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1302:20761:24673_1:N:0:CTTACAGC+CTTCACCA ptg000116l 48493 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1307:28894:27629_1:N:0:CTTACAGC+CTTCACCA ptg000099l 50587 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1364:51665:21297_1:N:0:CTTACAGC+CTTCACCA ptg000120l 1 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1384:8801:7135_1:N:0:CTTACAGC+CTTCACCA ptg000027l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1387:11423:4418_1:N:0:CTTACAGC+CTTCACCA ptg000113l 1 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1413:4213:17039_1:N:0:CTTACAGC+CTTCACCA ptg000093l 36783 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1413:4188:17053_1:N:0:CTTACAGC+CTTCACCA ptg000093l 36783 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1414:3363:28960_1:N:0:CTTACAGC+CTTCACCA ptg000077l 42471 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1440:40636:25626_1:N:0:CTTACAGC+CTTCACCA ptg000089l 46440 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1450:26045:20009_1:N:0:CTTACAGC+CTTCACCA ptg000096l 1 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2166:46972:9531_1:N:0:CTTACAGC+CTTCACCA ptg000048l 1 Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2183:2780:13747_1:N:0:CTTACAGC+CTTCACCA ptg000113l 2 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2248:47085:29590_1:N:0:CTTACAGC+CTTCACCA ptg000136l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2248:47077:29604_1:N:0:CTTACAGC+CTTCACCA ptg000136l 1 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2274:18058:17865_1:N:0:CTTACAGC+CTTCACCA ptg000047l 2 Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2370:41243:12472_1:N:0:CTTACAGC+CTTCACCA ptg000039l 2 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2418:38232:3675_1:N:0:CTTACAGC+CTTCACCA ptg000093l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2442:50929:9377_1:N:0:CTTACAGC+CTTCACCA ptg000045l 1 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2457:20551:23300_1:N:0:CTTACAGC+CTTCACCA ptg000096l 2 Processed 40000000 sequence pairs so far 40093521 reads; of these: 40093521 (100.00%) were paired; of these: 15760416 (39.31%) aligned concordantly 0 times 8084935 (20.17%) aligned concordantly exactly 1 time 16248170 (40.53%) aligned concordantly >1 times 60.69% overall alignment rate 40093521 reads; of these: 40093521 (100.00%) were paired; of these: 15771543 (39.34%) aligned concordantly 0 times 8086395 (20.17%) aligned concordantly exactly 1 time 16235583 (40.49%) aligned concordantly >1 times 60.66% overall alignment rate Processed 40093521 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 160374084. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 160374084. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 160374084. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 160374084. Successfully deleted the temporary files 1D8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D8_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 40093521 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1165119537 Total methylated C's in CpG context: 19528501 Total methylated C's in CHG context: 1166830 Total methylated C's in CHH context: 4748206 Total methylated C's in Unknown context: 60753 Total unmethylated C's in CpG context: 160274036 Total unmethylated C's in CHG context: 195860184 Total unmethylated C's in CHH context: 783541780 Total unmethylated C's in Unknown context: 3080662 C methylated in CpG context: 10.9% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 1.9% Bismark completed in 0d 2h 47m 13s ==================== Bismark run complete ====================