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/1B2_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/1B2_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/1B2_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/1B2_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1B2_R1_001.fastp-trim.fq.gz to 1B2_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1B2_R1_001.fastp-trim.fq.gz (50777554 sequences in total) Writing a G -> A converted version of the input file 1B2_R2_001.fastp-trim.fq.gz to 1B2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1B2_R2_001.fastp-trim.fq.gz (50777554 sequences in total) Input files are 1B2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B2_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 1B2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B2_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: LH00526:197:22KFKVLT4:3:1101:49019:1056_1:N:0:GAACGAAG+GGTGATTC/1 99 ptg000007l_CT_converted 10654359 1 125M = 10654539 305 GAAAATTGTAAATTTTTTTGATGTTATTTTTAATTTGTTTGATGGGAAGTATATGGTTTATATTAAATTTGGAAATATTTTATTTTATGTAAATAGGAAATTGAATTATTTATTTTGTATTATTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:49019:1056_2:N:0:GAACGAAG+GGTGATTC/2 147 ptg000007l_CT_converted 10654539 1 125M = 10654359 -305 TTTGTTTAATTATTTAGTATTTATTTATTAGAAAGTTTTAGATGATAGTGGATATTATTATTGTTTTATATTTATATTAAGTTTTATGTAGTTTTTAAATTTTATAAGAATGAATTGTTGTTGTA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1B2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B2_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: LH00526:197:22KFKVLT4:3:1101:49019:1056_1:N:0:GAACGAAG+GGTGATTC/1 83 ptg000015l_GA_converted 1111257 1 125M = 1111077 -305 CAATAATACAAAATAAATAATTCAATTTCCTATTTACATAAAATAAAATATTTCCAAATTTAATATAAACCATATACTTCCCATCAAACAAATTAAAAATAACATCAAAAAAATTTACAATTTTC IIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:49019:1056_2:N:0:GAACGAAG+GGTGATTC/2 163 ptg000015l_GA_converted 1111077 1 125M = 1111257 305 TACAACAACAATTCATTCTTATAAAATTTAAAAACTACATAAAACTTAATATAAATATAAAACAATAATAATATCCACTATCATCTAAAACTTTCTAATAAATAAATACTAAATAATTAAACAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to 1B2_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/1B2_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/1B2_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1154:16804:5272_1:N:0:GAACGAAG+GGTGATTC ptg000174l 27225 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1166:27955:6253_1:N:0:GAACGAAG+GGTGATTC ptg000117l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1166:27971:6253_1:N:0:GAACGAAG+GGTGATTC ptg000117l 1 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1190:49570:16212_1:N:0:GAACGAAG+GGTGATTC ptg000057l 39952 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1210:19159:14532_1:N:0:GAACGAAG+GGTGATTC ptg000048l 1 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1225:48688:23174_1:N:0:GAACGAAG+GGTGATTC ptg000065l 49685 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1225:48696:23188_1:N:0:GAACGAAG+GGTGATTC ptg000065l 49685 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1241:23343:7766_1:N:0:GAACGAAG+GGTGATTC ptg000048l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1263:8510:10161_1:N:0:GAACGAAG+GGTGATTC ptg000066l 66350 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 LH00526:197:22KFKVLT4:3:1326:49651:19322_1:N:0:GAACGAAG+GGTGATTC ptg000115l 42068 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1342:43937:7864_1:N:0:GAACGAAG+GGTGATTC ptg000090l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1342:43929:7878_1:N:0:GAACGAAG+GGTGATTC ptg000090l 1 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1360:50144:4320_1:N:0:GAACGAAG+GGTGATTC ptg000020l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1360:50160:4320_1:N:0:GAACGAAG+GGTGATTC ptg000020l 1 Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1395:17905:12220_1:N:0:GAACGAAG+GGTGATTC ptg000024l 2 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1407:37221:27979_1:N:0:GAACGAAG+GGTGATTC ptg000159l 21858 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1417:32786:7864_1:N:0:GAACGAAG+GGTGATTC ptg000039l 1 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1467:42165:24379_1:N:0:GAACGAAG+GGTGATTC ptg000006l 3 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 LH00526:197:22KFKVLT4:3:2154:36533:13929_1:N:0:GAACGAAG+GGTGATTC ptg000066l 66413 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2194:25115:18986_1:N:0:GAACGAAG+GGTGATTC ptg000015l 14997091 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2321:26175:3816_1:N:0:GAACGAAG+GGTGATTC ptg000130l 2 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2365:23496:9237_1:N:0:GAACGAAG+GGTGATTC ptg000057l 39952 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2394:5564:5398_1:N:0:GAACGAAG+GGTGATTC ptg000033l 2625597 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2395:38872:20163_1:N:0:GAACGAAG+GGTGATTC ptg000060c 2 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2424:11091:9643_1:N:0:GAACGAAG+GGTGATTC ptg000056l 59113 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2476:34122:18664_1:N:0:GAACGAAG+GGTGATTC ptg000117l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2476:34113:18678_1:N:0:GAACGAAG+GGTGATTC ptg000117l 1 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1147:19936:22376_1:N:0:GAACGAAG+GGTGATTC ntLink_0 96482 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1147:35036:28371_1:N:0:GAACGAAG+GGTGATTC ptg000117l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1163:3590:21816_1:N:0:GAACGAAG+GGTGATTC ptg000160l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1167:31815:21844_1:N:0:GAACGAAG+GGTGATTC ntLink_2 1 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1189:38572:26648_1:N:0:GAACGAAG+GGTGATTC ptg000127l 27810 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1206:50686:4222_1:N:0:GAACGAAG+GGTGATTC ptg000048l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1212:44504:18538_1:N:0:GAACGAAG+GGTGATTC ptg000055l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1213:45960:29604_1:N:0:GAACGAAG+GGTGATTC ptg000055l 50534 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1216:8760:3423_1:N:0:GAACGAAG+GGTGATTC ptg000096l 1 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 LH00526:197:22KFKVLT4:4:1330:7409:10021_1:N:0:GAACGAAG+GGTGATTC ntLink_2 399379 Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1383:10249:25864_1:N:0:GAACGAAG+GGTGATTC ptg000133l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1384:27583:19476_1:N:0:GAACGAAG+GGTGATTC ptg000025l 1 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1420:2254:3059_1:N:0:GAACGAAG+GGTGATTC ptg000002l 1 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1445:22016:22558_1:N:0:GAACGAAG+GGTGATTC ptg000089l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1445:22007:22572_1:N:0:GAACGAAG+GGTGATTC ptg000089l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1446:15550:25878_1:N:0:GAACGAAG+GGTGATTC ptg000048l 1 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1462:41663:29226_1:N:0:GAACGAAG+GGTGATTC ptg000025l 1 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1482:29339:6603_1:N:0:GAACGAAG+GGTGATTC ptg000095l 1 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2123:15218:14826_1:N:0:GAACGAAG+GGTGATTC ptg000039l 2 Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2181:42877:28974_1:N:0:GAACGAAG+GGTGATTC ptg000133l 37099 Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2218:32746:20849_1:N:0:GAACGAAG+GGTGATTC ptg000066l 66347 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2227:48299:14181_1:N:0:GAACGAAG+GGTGATTC ptg000066l 66345 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2233:21473:26802_1:N:0:GAACGAAG+GGTGATTC ptg000098l 55079 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2234:19458:21465_1:N:0:GAACGAAG+GGTGATTC ptg000060c 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2239:13745:24407_1:N:0:GAACGAAG+GGTGATTC ptg000048l 1 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2283:30156:7794_1:N:0:GAACGAAG+GGTGATTC ptg000066l 66350 Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2357:8243:10035_1:N:0:GAACGAAG+GGTGATTC ptg000095l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2377:11949:12164_1:N:0:GAACGAAG+GGTGATTC ptg000101l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2381:11973:23412_1:N:0:GAACGAAG+GGTGATTC ptg000115l 42066 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2390:29978:2583_1:N:0:GAACGAAG+GGTGATTC ptg000083l 1 Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far 50777554 reads; of these: 50777554 (100.00%) were paired; of these: 20027697 (39.44%) aligned concordantly 0 times 10181731 (20.05%) aligned concordantly exactly 1 time 20568126 (40.51%) aligned concordantly >1 times 60.56% overall alignment rate 50777554 reads; of these: 50777554 (100.00%) were paired; of these: 20042106 (39.47%) aligned concordantly 0 times 10176867 (20.04%) aligned concordantly exactly 1 time 20558581 (40.49%) aligned concordantly >1 times 60.53% overall alignment rate Processed 50777554 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 203110216. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 203110216. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 203110216. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 203110216. Successfully deleted the temporary files 1B2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 50777554 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1480544688 Total methylated C's in CpG context: 22368224 Total methylated C's in CHG context: 1446104 Total methylated C's in CHH context: 5924437 Total methylated C's in Unknown context: 74522 Total unmethylated C's in CpG context: 206179220 Total unmethylated C's in CHG context: 247430910 Total unmethylated C's in CHH context: 997195793 Total unmethylated C's in Unknown context: 3986120 C methylated in CpG context: 9.8% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 1.8% Bismark completed in 0d 3h 26m 54s ==================== Bismark run complete ====================