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/1A6_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/1A6_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/1A6_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/1A6_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1A6_R1_001.fastp-trim.fq.gz to 1A6_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1A6_R1_001.fastp-trim.fq.gz (36668236 sequences in total) Writing a G -> A converted version of the input file 1A6_R2_001.fastp-trim.fq.gz to 1A6_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1A6_R2_001.fastp-trim.fq.gz (36668236 sequences in total) Input files are 1A6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A6_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 1A6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A6_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: LH00526:197:22KFKVLT4:3:1101:40061:1070_1:N:0:CAGGTTCA+GGCGTTAT/1 99 ptg000025l_CT_converted 18456402 2 125M = 18456449 170 GAGGTTATTGATTTTTGATGAATGAATTTTGAGTATTGTTATGGGTTGTTAATTAGAGTTTGGTGAATTATATTGTTAAATTAAAGTATTTGGAGTTATTAATTTTTTAGTTAAAGAAGTATAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-48 XS:i:-48 XN:i:0 XM:i:8 XO:i:0 XG:i:0 NM:i:8 MD:Z:19A24T16T0A8T2T16A26A6 YS:i:-65 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:40061:1070_2:N:0:CAGGTTCA+GGCGTTAT/2 147 ptg000025l_CT_converted 18456449 2 119M2I4M = 18456402 -170 GTTAATTAGAGTTTGGTGAATTATATTGTTAAATTAAAGTATTTGGAGTTATTAATTTTTTAGTTAAAGAAGTATAAATTGTTAGTGTTGAAGTTTAGAAATTTTTAGATAAGAGAGTAATATTA 9IIIIIIIIIIIIIIII9IIIIIIIIII9I9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-65 XS:i:-74 XN:i:0 XM:i:9 XO:i:1 XG:i:2 NM:i:11 MD:Z:14T0A8T2T16A26A11G29G0A8 YS:i:-48 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1A6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A6_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: LH00526:197:22KFKVLT4:3:1101:40061:1070_1:N:0:CAGGTTCA+GGCGTTAT/1 83 ptg000008l_GA_converted 18479915 35 125M = 18479868 -172 TTTATACTTCTTTAACTAAAAAATTAATAACTCCAAATACTTTAATTTAACAATATAATTCACCAAACTCTAATTAACAACCCATAACAATACTCAAAATTCATTCATCAAAAATCAATAACCTC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:-54 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:40061:1070_2:N:0:CAGGTTCA+GGCGTTAT/2 163 ptg000008l_GA_converted 18479868 35 125M = 18479915 172 TAATATTACTCTCTTATCTAAAAATTTCTAAACTTCAACACTAACAATTTATACTTCTTTAACTAAAAAATTAATAACTCCAAATACTTTAATTTAACAATATAATTCACCAAACTCTAATTAAC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9I9IIIIIIIIII9IIIIIIIIIIIIIIII9 AS:i:0 XS:i:-80 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 1A6_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/1A6_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/1A6_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1117:26871:26844_1:N:0:CAGGTTCA+GGCGTTAT ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1117:26879:26858_1:N:0:CAGGTTCA+GGCGTTAT ptg000039l 1 Processed 1000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:3:1260:13907:23034_1:N:0:CAGGTTCA+GGCGTTAT ptg000066l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1265:48251:2163_1:N:0:CAGGTTCA+GGCGTTAT ptg000113l 59543 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1290:22784:2821_1:N:0:CAGGTTCA+GGCGTTAT ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1314:44714:6015_1:N:0:CAGGTTCA+GGCGTTAT ptg000096l 56415 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1363:24370:20555_1:N:0:CAGGTTCA+GGCGTTAT ptg000045l 1 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1404:13066:27405_1:N:0:CAGGTTCA+GGCGTTAT ptg000115l 42068 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1411:34939:2205_1:N:0:CAGGTTCA+GGCGTTAT ptg000057l 39951 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1424:40846:20079_1:N:0:CAGGTTCA+GGCGTTAT ptg000018l 2 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1474:5880:19448_1:N:0:CAGGTTCA+GGCGTTAT ptg000055l 50545 Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2111:22833:26494_1:N:0:CAGGTTCA+GGCGTTAT ptg000046l 1 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2133:8251:1168_1:N:0:CAGGTTCA+GGCGTTAT ptg000012l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2148:30804:6673_1:N:0:CAGGTTCA+GGCGTTAT ptg000121l 50724 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2155:48704:26340_1:N:0:CAGGTTCA+GGCGTTAT ptg000048l 1 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2220:49731:5202_1:N:0:CAGGTTCA+GGCGTTAT ptg000146l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2229:34712:19490_1:N:0:CAGGTTCA+GGCGTTAT ptg000015l 14997092 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2263:19766:17347_1:N:0:CAGGTTCA+GGCGTTAT ptg000093l 1 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2458:26175:1574_1:N:0:CAGGTTCA+GGCGTTAT ptg000048l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2459:23432:8536_1:N:0:CAGGTTCA+GGCGTTAT ntLink_0 96474 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2459:22436:25388_1:N:0:CAGGTTCA+GGCGTTAT ptg000083l 120284 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1115:30488:6099_1:N:0:CAGGTTCA+GGCGTTAT ptg000066l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1121:46308:28581_1:N:0:CAGGTTCA+GGCGTTAT ptg000116l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1121:46317:28595_1:N:0:CAGGTTCA+GGCGTTAT ptg000116l 2 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1130:45378:1140_1:N:0:CAGGTTCA+GGCGTTAT ntLink_0 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1150:36565:10539_1:N:0:CAGGTTCA+GGCGTTAT ptg000082l 1 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1184:25730:1532_1:N:0:CAGGTTCA+GGCGTTAT ptg000046l 2 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1230:16756:20121_1:N:0:CAGGTTCA+GGCGTTAT ptg000093l 1 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1347:39252:13397_1:N:0:CAGGTTCA+GGCGTTAT ptg000065l 49673 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1347:39268:13397_1:N:0:CAGGTTCA+GGCGTTAT ptg000065l 49673 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1387:1218:5216_1:N:0:CAGGTTCA+GGCGTTAT ptg000020l 17861297 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 LH00526:197:22KFKVLT4:4:1473:47538:16002_1:N:0:CAGGTTCA+GGCGTTAT ptg000160l 32601 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1493:27777:6897_1:N:0:CAGGTTCA+GGCGTTAT ptg000065l 49690 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 LH00526:197:22KFKVLT4:4:2240:30237:21633_1:N:0:CAGGTTCA+GGCGTTAT ptg000065l 49673 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 LH00526:197:22KFKVLT4:4:2361:7458:10357_1:N:0:CAGGTTCA+GGCGTTAT ptg000137l 40575 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2487:39778:5623_1:N:0:CAGGTTCA+GGCGTTAT ptg000064l 127032 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2487:39786:5637_1:N:0:CAGGTTCA+GGCGTTAT ptg000064l 127032 36668236 reads; of these: 36668236 (100.00%) were paired; of these: 13430324 (36.63%) aligned concordantly 0 times 7579519 (20.67%) aligned concordantly exactly 1 time 15658393 (42.70%) aligned concordantly >1 times 63.37% overall alignment rate 36668236 reads; of these: 36668236 (100.00%) were paired; of these: 13419074 (36.60%) aligned concordantly 0 times 7576719 (20.66%) aligned concordantly exactly 1 time 15672443 (42.74%) aligned concordantly >1 times 63.40% overall alignment rate Processed 36668236 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 146672944. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 146672944. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 146672944. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 146672944. Successfully deleted the temporary files 1A6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A6_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 36668236 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1110406250 Total methylated C's in CpG context: 16642042 Total methylated C's in CHG context: 1088809 Total methylated C's in CHH context: 4354648 Total methylated C's in Unknown context: 54124 Total unmethylated C's in CpG context: 159061488 Total unmethylated C's in CHG context: 187637249 Total unmethylated C's in CHH context: 741622014 Total unmethylated C's in Unknown context: 2846703 C methylated in CpG context: 9.5% 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 34m 24s ==================== Bismark run complete ====================