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/6--1E3_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/6--1E3_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/6--1E3_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/6--1E3_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 6--1E3_R1_001.fastp-trim.fq.gz to 6--1E3_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 6--1E3_R1_001.fastp-trim.fq.gz (48546692 sequences in total) Writing a G -> A converted version of the input file 6--1E3_R2_001.fastp-trim.fq.gz to 6--1E3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 6--1E3_R2_001.fastp-trim.fq.gz (48546692 sequences in total) Input files are 6--1E3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 6--1E3_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 6--1E3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 6--1E3_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: LH00526:197:22KFKVLT4:3:1101:41874:1070_1:N:0:CAGTGCTT+ACCTGGAA/1 77 * 0 0 * * 0 0 AATGTGGTTTTTTATGTTATGTTATGTATGTATTAATTATGTTTATTTTGTGTATTTGTGTTGTTAATATTGTTTTTTATTTTGTAAGTTTGGGATGAATTGTAGTTTAATAAATGATATAATTG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIII9III99IIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:41874:1070_2:N:0:CAGTGCTT+ACCTGGAA/2 141 * 0 0 * * 0 0 TTAACAACTAAAACAAAACAAAACCTCAAAAAAACAAAACAACCAAAATTTTTCAAAAATACTTATCATAATATCCATAAAACCTACTTTAACAAAAAAACTACTCAAAATAAATTCTACTTTAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII9IIIIIIIIII9IIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIII9II YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 6--1E3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 6--1E3_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: LH00526:197:22KFKVLT4:3:1101:41874:1070_1:N:0:CAGTGCTT+ACCTGGAA/1 83 ptg000002l_GA_converted 1955991 42 125M = 1955786 -330 CAATTATATCATTTATTAAACTACAATTCATCCCAAACTTACAAAATAAAAAACAATATTAACAACACAAATACACAAAATAAACATAATTAATACATACATAACATAACATAAAAAACCACATT IIIIIIIIIIIIIII99III9IIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS: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 LH00526:197:22KFKVLT4:3:1101:41874:1070_2:N:0:CAGTGCTT+ACCTGGAA/2 163 ptg000002l_GA_converted 1955786 42 125M = 1955991 330 TTAACAACTAAAACAAAACAAAACCTCAAAAAAACAAAACAACCAAAATTTTTCAAAAATACTTATCATAATATCCATAAAACCTACTTTAACAAAAAAACTACTCAAAATAAATTCTACTTTAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII9IIIIIIIIII9IIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIII9II AS: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 6--1E3_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/6--1E3_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/6--1E3_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1145:45451:4208_1:N:0:CAGTGCTT+ACCTGGAA ptg000018l 2 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:3:1357:17379:14111_1:N:0:CAGTGCTT+ACCTGGAA ptg000054l 36043 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2127:40676:24659_1:N:0:CAGTGCTT+ACCTGGAA ptg000101l 1 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2149:7579:4432_1:N:0:CAGTGCTT+ACCTGGAA ptg000004l 15492363 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2151:46648:25360_1:N:0:CAGTGCTT+ACCTGGAA ptg000066l 66400 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2194:38993:17403_1:N:0:CAGTGCTT+ACCTGGAA ptg000083l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2196:48259:3886_1:N:0:CAGTGCTT+ACCTGGAA ptg000179l 40681 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2220:17816:22964_1:N:0:CAGTGCTT+ACCTGGAA ptg000025l 21443080 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2231:20616:22432_1:N:0:CAGTGCTT+ACCTGGAA ptg000056l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2248:26102:11366_1:N:0:CAGTGCTT+ACCTGGAA ptg000082l 2 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2314:33612:6603_1:N:0:CAGTGCTT+ACCTGGAA ptg000121l 50746 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2360:28667:12360_1:N:0:CAGTGCTT+ACCTGGAA ptg000066l 66349 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2410:26814:14896_1:N:0:CAGTGCTT+ACCTGGAA ptg000114l 1 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2488:46810:16114_1:N:0:CAGTGCTT+ACCTGGAA ptg000117l 1 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1105:32608:20695_1:N:0:CAGTGCTT+ACCTGGAA ptg000077l 42465 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1111:19386:18426_1:N:0:CAGTGCTT+ACCTGGAA ptg000006l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1119:29072:21017_1:N:0:CAGTGCTT+ACCTGGAA ntLink_0 96467 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1119:29088:21017_1:N:0:CAGTGCTT+ACCTGGAA ptg000025l 1 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1149:36177:26788_1:N:0:CAGTGCTT+ACCTGGAA ptg000011l 1 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1237:11374:28175_1:N:0:CAGTGCTT+ACCTGGAA ptg000056l 2 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1267:27259:9643_1:N:0:CAGTGCTT+ACCTGGAA ptg000182l 35550 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1285:7255:29282_1:N:0:CAGTGCTT+ACCTGGAA ptg000184l 44395 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1310:29574:6673_1:N:0:CAGTGCTT+ACCTGGAA ptg000066l 66417 Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1371:43031:1644_1:N:0:CAGTGCTT+ACCTGGAA ptg000113l 2 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1387:7450:17011_1:N:0:CAGTGCTT+ACCTGGAA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1387:7441:17025_1:N:0:CAGTGCTT+ACCTGGAA ptg000039l 1 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2102:45855:17459_1:N:0:CAGTGCTT+ACCTGGAA ptg000097l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2102:45847:17473_1:N:0:CAGTGCTT+ACCTGGAA ptg000097l 1 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2169:12637:28932_1:N:0:CAGTGCTT+ACCTGGAA ptg000144l 3 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2170:37561:7079_1:N:0:CAGTGCTT+ACCTGGAA ptg000066l 66347 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2194:34704:14770_1:N:0:CAGTGCTT+ACCTGGAA ptg000015l 14997092 Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2319:44957:15288_1:N:0:CAGTGCTT+ACCTGGAA ptg000066l 66365 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2368:25746:20891_1:N:0:CAGTGCTT+ACCTGGAA ptg000057l 39951 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2380:42756:22516_1:N:0:CAGTGCTT+ACCTGGAA ntLink_0 2 Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2410:6454:27867_1:N:0:CAGTGCTT+ACCTGGAA ptg000048l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2419:25115:25598_1:N:0:CAGTGCTT+ACCTGGAA ptg000066l 66352 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2431:45329:25458_1:N:0:CAGTGCTT+ACCTGGAA ptg000096l 2 Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far 48546692 reads; of these: 48546692 (100.00%) were paired; of these: 18831198 (38.79%) aligned concordantly 0 times 9923533 (20.44%) aligned concordantly exactly 1 time 19791961 (40.77%) aligned concordantly >1 times 61.21% overall alignment rate 48546692 reads; of these: 48546692 (100.00%) were paired; of these: 18823181 (38.77%) aligned concordantly 0 times 9919050 (20.43%) aligned concordantly exactly 1 time 19804461 (40.79%) aligned concordantly >1 times 61.23% overall alignment rate Processed 48546692 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 194186768. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 194186768. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 194186768. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 194186768. Successfully deleted the temporary files 6--1E3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 6--1E3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 48546692 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1436479493 Total methylated C's in CpG context: 24070974 Total methylated C's in CHG context: 1638645 Total methylated C's in CHH context: 6647993 Total methylated C's in Unknown context: 80407 Total unmethylated C's in CpG context: 199216544 Total unmethylated C's in CHG context: 241507702 Total unmethylated C's in CHH context: 963397635 Total unmethylated C's in Unknown context: 4003728 C methylated in CpG context: 10.8% 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 3h 17m 32s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe