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/1C6_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/1C6_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/1C6_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/1C6_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1C6_R1_001.fastp-trim.fq.gz to 1C6_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1C6_R1_001.fastp-trim.fq.gz (36941218 sequences in total) Writing a G -> A converted version of the input file 1C6_R2_001.fastp-trim.fq.gz to 1C6_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1C6_R2_001.fastp-trim.fq.gz (36941218 sequences in total) Input files are 1C6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C6_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 1C6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C6_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: LH00526:197:22KFKVLT4:3:1101:39203:1070_1:N:0:CATGAGCA+GCAAGATC/1 99 ptg000120l_CT_converted 17037 1 125M = 17300 388 TTTTAAAGTTTATGTTAGGGGTGGGTTTTGATATGATGTTGGGTGGTTGTGTTGGTAATTGGATGTTTTATAAGTTTAAAAGGAAAGAAAATTAATTTTGGTTTATTTTTGTTTGTTTAATGTTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9-IIIIIIIIIIIIIIIII-IIIIIIII-IIIIIIIIIIIIIIIIIIIIIII9IIII 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 LH00526:197:22KFKVLT4:3:1101:39203:1070_2:N:0:CATGAGCA+GCAAGATC/2 147 ptg000120l_CT_converted 17300 1 125M = 17037 -388 TAATTGATTGATTATGGGAAAGAAGTATTTTAGTTTTTTTAGTAAGGTATTTTGAAGATATATTTTTTGGAAGAGAAATTTTGTTTTGTAGTTGTAGTAAATATTATTATTATGGTGGATTATAT IIII9IIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 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 1C6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C6_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: LH00526:197:22KFKVLT4:3:1101:39203:1070_1:N:0:CATGAGCA+GCAAGATC/1 83 ptg000088l_GA_converted 49939 1 125M = 49676 -388 AAACATTAAACAAACAAAAATAAACCAAAATTAATTTTCTTTCCTTTTAAACTTATAAAACATCCAATTACCAACACAACCACCCAACATCATATCAAAACCCACCCCTAACATAAACTTTAAAA IIII9IIIIIIIIIIIIIIIIIIIIIII-IIIIIIII-IIIIIIIIIIIIIIIII-9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII 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 LH00526:197:22KFKVLT4:3:1101:39203:1070_2:N:0:CATGAGCA+GCAAGATC/2 163 ptg000088l_GA_converted 49676 1 125M = 49939 388 ATATAATCCACCATAATAATAATATTTACTACAACTACAAAACAAAATTTCTCTTCCAAAAAATATATCTTCAAAATACCTTACTAAAAAAACTAAAATACTTCTTTCCCATAATCAATCAATTA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIII9IIII 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 1C6_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/1C6_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/1C6_R2_001.fastp-trim.fq.gz 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 LH00526:197:22KFKVLT4:3:1267:27300:15680_1:N:0:CATGAGCA+GCAAGATC ptg000024l 1 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1304:38329:18972_1:N:0:CATGAGCA+GCAAGATC ptg000174l 27262 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1332:23618:16563_1:N:0:CATGAGCA+GCAAGATC ptg000093l 36787 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1364:8056:7444_1:N:0:CATGAGCA+GCAAGATC ptg000015l 14997092 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1394:40223:27068_1:N:0:CATGAGCA+GCAAGATC ptg000096l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1405:13146:20261_1:N:0:CATGAGCA+GCAAGATC ptg000059l 1573515 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1412:41526:26746_1:N:0:CATGAGCA+GCAAGATC ptg000093l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1424:29930:26200_1:N:0:CATGAGCA+GCAAGATC ptg000025l 21443081 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2109:49327:5903_1:N:0:CATGAGCA+GCAAGATC ptg000096l 2 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2168:13964:25542_1:N:0:CATGAGCA+GCAAGATC ptg000140l 2 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2343:25220:10960_1:N:0:CATGAGCA+GCAAGATC ptg000095l 40248 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2435:28789:1308_1:N:0:CATGAGCA+GCAAGATC ptg000025l 21443080 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1102:47223:21619_1:N:0:CATGAGCA+GCAAGATC ptg000160l 1 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1156:23545:8088_1:N:0:CATGAGCA+GCAAGATC ptg000077l 42461 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1223:8769:13523_1:N:0:CATGAGCA+GCAAGATC ptg000039l 1 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1267:3395:26998_1:N:0:CATGAGCA+GCAAGATC ptg000097l 1 Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1338:5111:8340_1:N:0:CATGAGCA+GCAAGATC ptg000066l 66343 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1392:20438:20471_1:N:0:CATGAGCA+GCAAGATC ptg000025l 21443081 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1435:14894:11744_1:N:0:CATGAGCA+GCAAGATC ptg000066l 66350 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1455:45742:23230_1:N:0:CATGAGCA+GCAAGATC ptg000066l 66364 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2127:27462:26634_1:N:0:CATGAGCA+GCAAGATC ptg000095l 1 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2189:46260:8298_1:N:0:CATGAGCA+GCAAGATC ptg000059l 1573514 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2197:36161:14994_1:N:0:CATGAGCA+GCAAGATC ptg000055l 1 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2220:40765:5146_1:N:0:CATGAGCA+GCAAGATC ptg000033l 2625597 Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2265:25309:12206_1:N:0:CATGAGCA+GCAAGATC ptg000017l 1 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2355:42165:15162_1:N:0:CATGAGCA+GCAAGATC ptg000092l 2 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2440:28684:17403_1:N:0:CATGAGCA+GCAAGATC ptg000092l 2 Processed 36000000 sequence pairs so far 36941218 reads; of these: 36941218 (100.00%) were paired; of these: 13049105 (35.32%) aligned concordantly 0 times 7157443 (19.38%) aligned concordantly exactly 1 time 16734670 (45.30%) aligned concordantly >1 times 64.68% overall alignment rate 36941218 reads; of these: 36941218 (100.00%) were paired; of these: 13041322 (35.30%) aligned concordantly 0 times 7155918 (19.37%) aligned concordantly exactly 1 time 16743978 (45.33%) aligned concordantly >1 times 64.70% overall alignment rate Processed 36941218 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 147764872. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 147764872. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 147764872. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 147764872. Successfully deleted the temporary files 1C6_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C6_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 36941218 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1073918442 Total methylated C's in CpG context: 16537207 Total methylated C's in CHG context: 1273633 Total methylated C's in CHH context: 4890760 Total methylated C's in Unknown context: 54723 Total unmethylated C's in CpG context: 157884399 Total unmethylated C's in CHG context: 183711970 Total unmethylated C's in CHH context: 709620473 Total unmethylated C's in Unknown context: 2624150 C methylated in CpG context: 9.5% C methylated in CHG context: 0.7% C methylated in CHH context: 0.7% C methylated in unknown context (CN or CHN): 2.0% Bismark completed in 0d 2h 30m 25s ==================== Bismark run complete ====================