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/1D7_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/1D7_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/1D7_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/1D7_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1D7_R1_001.fastp-trim.fq.gz to 1D7_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1D7_R1_001.fastp-trim.fq.gz (36725215 sequences in total) Writing a G -> A converted version of the input file 1D7_R2_001.fastp-trim.fq.gz to 1D7_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1D7_R2_001.fastp-trim.fq.gz (36725215 sequences in total) Input files are 1D7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D7_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 1D7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D7_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: LH00526:197:22KFKVLT4:3:1101:51075:1056_1:N:0:AGGAACAC+GACATTCC/1 99 ptg000031l_CT_converted 14283047 6 125M = 14283144 222 AGTAATGTTTTAAAAAATTAAAAATTAATTAATTAAAGTTTGAAATTTGATTGGAAAGTATGTAATTTATTTAAAATTGATTGAAATTAATAATAATAATTAAATTGTAAATATAGGAATAGTAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIII9IIII9II9IIIIIIIIIII9III99IIIIIIIIIIIIII-IIII9IIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:-6 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:-6 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:51075:1056_2:N:0:AGGAACAC+GACATTCC/2 147 ptg000031l_CT_converted 14283144 6 125M = 14283047 -222 AATTAAATTGTGAATATAGGAATAGTAAATTAAAGAAATATATTTTTGTTGTATTAAGGTATAAGAATGGTAGTAGTTGAGGAATTTTGTTGGTTGTTATGAATTTTTTTAATATTTGAATATTA IIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIII-9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-24 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:11A113 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1D7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D7_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: LH00526:197:22KFKVLT4:3:1101:51075:1056_1:N:0:AGGAACAC+GACATTCC/1 77 * 0 0 * * 0 0 AGTAATGTTTTAAAAAATTAAAAATTAATTAATTAAAGTTTGAAATTTGATTGGAAAGTATGTAATTTATTTAAAATTGATTGAAATTAATAATAATAATTAAATTGTAAATATAGGAATAGTAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIII9IIII9II9IIIIIIIIIII9III99IIIIIIIIIIIIII-IIII9IIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:51075:1056_2:N:0:AGGAACAC+GACATTCC/2 141 * 0 0 * * 0 0 TAATATTCAAATATTAAAAAAATTCATAACAACCAACAAAATTCCTCAACTACTACCATTCTTATACCTTAATACAACAAAAATATATTTCTTTAATTTACTATTCCTATATTCACAATTTAATT IIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9-IIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIII YT:Z:UP >>> Writing bisulfite mapping results to 1D7_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/1D7_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/1D7_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1139:19102:9026_1:N:0:AGGAACAC+GACATTCC ptg000127l 27820 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1151:6648:8116_1:N:0:AGGAACAC+GACATTCC ptg000096l 56399 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1169:16440:3185_1:N:0:AGGAACAC+GACATTCC ptg000102l 64514 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1216:27130:14798_1:N:0:AGGAACAC+GACATTCC ptg000046l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1230:50185:8340_1:N:0:AGGAACAC+GACATTCC ptg000007l 12295855 Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1291:25519:21255_1:N:0:AGGAACAC+GACATTCC ptg000066l 66354 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1305:26482:9643_1:N:0:AGGAACAC+GACATTCC ptg000065l 49673 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1324:37375:27517_1:N:0:AGGAACAC+GACATTCC ptg000017l 2 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1374:48153:24715_1:N:0:AGGAACAC+GACATTCC ptg000098l 55097 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 LH00526:197:22KFKVLT4:3:1480:32649:21661_1:N:0:AGGAACAC+GACATTCC ptg000118l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1498:28384:21788_1:N:0:AGGAACAC+GACATTCC ptg000115l 42062 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2116:43274:5763_1:N:0:AGGAACAC+GACATTCC ptg000015l 14997092 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2125:47360:1854_1:N:0:AGGAACAC+GACATTCC ptg000047l 1 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2144:22072:25990_1:N:0:AGGAACAC+GACATTCC ptg000015l 14997092 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2174:8712:13453_1:N:0:AGGAACAC+GACATTCC ptg000048l 1 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2206:42877:25360_1:N:0:AGGAACAC+GACATTCC ptg000125l 18884 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2232:8801:22124_1:N:0:AGGAACAC+GACATTCC ptg000066l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2250:36905:15386_1:N:0:AGGAACAC+GACATTCC ptg000024l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2269:30828:25458_1:N:0:AGGAACAC+GACATTCC ptg000094l 36831 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2307:13697:5328_1:N:0:AGGAACAC+GACATTCC ntLink_0 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2323:17581:27181_1:N:0:AGGAACAC+GACATTCC ptg000075l 54138 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2353:23804:19490_1:N:0:AGGAACAC+GACATTCC ptg000129l 33365 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2358:28344:28833_1:N:0:AGGAACAC+GACATTCC ptg000133l 37098 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2411:41283:3241_1:N:0:AGGAACAC+GACATTCC ptg000098l 55082 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2425:42392:11240_1:N:0:AGGAACAC+GACATTCC ptg000133l 37126 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2452:39398:25304_1:N:0:AGGAACAC+GACATTCC ptg000048l 1 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1123:44302:3675_1:N:0:AGGAACAC+GACATTCC ptg000093l 36779 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1157:43047:6351_1:N:0:AGGAACAC+GACATTCC ptg000048l 1 Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1198:41906:17011_1:N:0:AGGAACAC+GACATTCC ptg000033l 2625625 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1219:49497:19532_1:N:0:AGGAACAC+GACATTCC ptg000065l 49688 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1257:31055:22740_1:N:0:AGGAACAC+GACATTCC ptg000092l 1 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1309:30812:24029_1:N:0:AGGAACAC+GACATTCC ptg000128l 1 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1332:18439:24743_1:N:0:AGGAACAC+GACATTCC ptg000075l 2 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1377:38192:24729_1:N:0:AGGAACAC+GACATTCC ptg000118l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1384:38038:9699_1:N:0:AGGAACAC+GACATTCC ptg000092l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1387:18819:23889_1:N:0:AGGAACAC+GACATTCC ptg000083l 1 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 LH00526:197:22KFKVLT4:4:1467:16116:27447_1:N:0:AGGAACAC+GACATTCC ptg000093l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1471:30245:1196_1:N:0:AGGAACAC+GACATTCC ptg000089l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1486:37132:25752_1:N:0:AGGAACAC+GACATTCC ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1492:6333:14041_1:N:0:AGGAACAC+GACATTCC ptg000121l 50751 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2125:23998:10469_1:N:0:AGGAACAC+GACATTCC ptg000096l 2 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2177:17427:18986_1:N:0:AGGAACAC+GACATTCC ptg000066l 66361 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 LH00526:197:22KFKVLT4:4:2366:40522:23132_1:N:0:AGGAACAC+GACATTCC ptg000065l 49687 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2367:28506:15162_1:N:0:AGGAACAC+GACATTCC ptg000006l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2393:33960:26592_1:N:0:AGGAACAC+GACATTCC ptg000015l 14997105 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2415:38418:10833_1:N:0:AGGAACAC+GACATTCC ptg000127l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2459:40652:29016_1:N:0:AGGAACAC+GACATTCC ptg000056l 1 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2490:47951:3045_1:N:0:AGGAACAC+GACATTCC ptg000096l 1 36725215 reads; of these: 36725215 (100.00%) were paired; of these: 13446148 (36.61%) aligned concordantly 0 times 7711148 (21.00%) aligned concordantly exactly 1 time 15567919 (42.39%) aligned concordantly >1 times 63.39% overall alignment rate 36725215 reads; of these: 36725215 (100.00%) were paired; of these: 13452705 (36.63%) aligned concordantly 0 times 7719293 (21.02%) aligned concordantly exactly 1 time 15553217 (42.35%) aligned concordantly >1 times 63.37% overall alignment rate Processed 36725215 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 146900860. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 146900860. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 146900860. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 146900860. Successfully deleted the temporary files 1D7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D7_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 36725215 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1113933528 Total methylated C's in CpG context: 17398195 Total methylated C's in CHG context: 1180166 Total methylated C's in CHH context: 4745927 Total methylated C's in Unknown context: 54876 Total unmethylated C's in CpG context: 157041470 Total unmethylated C's in CHG context: 188458540 Total unmethylated C's in CHH context: 745109230 Total unmethylated C's in Unknown context: 2708454 C methylated in CpG context: 10.0% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 2.0% Bismark completed in 0d 2h 35m 5s ==================== Bismark run complete ====================