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/1H5_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/1H5_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/1H5_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/1H5_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1H5_R1_001.fastp-trim.fq.gz to 1H5_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1H5_R1_001.fastp-trim.fq.gz (40897003 sequences in total) Writing a G -> A converted version of the input file 1H5_R2_001.fastp-trim.fq.gz to 1H5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1H5_R2_001.fastp-trim.fq.gz (40897003 sequences in total) Input files are 1H5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H5_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 1H5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H5_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: LH00526:197:22KFKVLT4:3:1101:39786:1070_1:N:0:CGGCATTA+TGACTGAC/1 99 ptg000088l_CT_converted 27944 1 125M = 28010 191 TTTTATTGTTTGTGAAATTATAATGTATGTTTAAAGTAAGGATTGTTTTGATTTAGTAAATGGAAAATATTGGTGAAAGTAATTGTTTTTAAGGATAGTGGTAGAAGATATTTTTATTATTATGT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 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 LH00526:197:22KFKVLT4:3:1101:39786:1070_2:N:0:CGGCATTA+TGACTGAC/2 147 ptg000088l_CT_converted 28010 1 125M = 27944 -191 ATATTGGTGAAAGTAATTGTTTTTAAGGATAGTGGTAGAAGATATTTTTATTATTATGTATTTATTAATAGGATTGTATTAGTTATGTTAAGGTGTTTTATTTTTTTGAATAATGTATTATATAG IIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 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 1H5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H5_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: LH00526:197:22KFKVLT4:3:1101:39786:1070_1:N:0:CGGCATTA+TGACTGAC/1 83 ptg000047l_GA_converted 245594 1 125M = 245528 -191 ACATAATAATAAAAATATCTTCTACCACTATCCTTAAAAACAATTACTTTCACCAATATTTTCCATTTACTAAATCAAAACAATCCTTACTTTAAACATACATTATAATTTCACAAACAATAAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 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 LH00526:197:22KFKVLT4:3:1101:39786:1070_2:N:0:CGGCATTA+TGACTGAC/2 163 ptg000047l_GA_converted 245528 1 125M = 245594 191 CTATATAATACATTATTCAAAAAAATAAAACACCTTAACATAACTAATACAATCCTATTAATAAATACATAATAATAAAAATATCTTCTACCACTATCCTTAAAAACAATTACTTTCACCAATAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII 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 1H5_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/1H5_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/1H5_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1176:14166:11240_1:N:0:CGGCATTA+TGACTGAC ptg000066l 66348 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1295:20616:28371_1:N:0:CGGCATTA+TGACTGAC ptg000025l 21443089 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1306:10678:24673_1:N:0:CGGCATTA+TGACTGAC ptg000002l 1 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1345:25091:1770_1:N:0:CGGCATTA+TGACTGAC ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1345:25099:1784_1:N:0:CGGCATTA+TGACTGAC ptg000039l 1 Processed 7000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:3:1465:47903:6995_1:N:0:CGGCATTA+TGACTGAC ptg000024l 3 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2134:46066:28974_1:N:0:CGGCATTA+TGACTGAC ptg000015l 14997093 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2139:18479:3746_1:N:0:CGGCATTA+TGACTGAC ptg000033l 2625597 Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2223:25584:7416_1:N:0:CGGCATTA+TGACTGAC ptg000066l 66382 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2243:42546:2709_1:N:0:CGGCATTA+TGACTGAC ptg000066l 66382 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 LH00526:197:22KFKVLT4:3:2395:22695:21549_1:N:0:CGGCATTA+TGACTGAC ptg000025l 21443081 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2426:8971:3283_1:N:0:CGGCATTA+TGACTGAC ptg000025l 21443081 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1148:19653:8326_1:N:0:CGGCATTA+TGACTGAC ptg000159l 2 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1159:15606:19252_1:N:0:CGGCATTA+TGACTGAC ptg000066l 66349 Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1298:9651:14041_1:N:0:CGGCATTA+TGACTGAC ptg000113l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1298:9643:14055_1:N:0:CGGCATTA+TGACTGAC ptg000113l 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 LH00526:197:22KFKVLT4:4:1371:2351:24827_1:N:0:CGGCATTA+TGACTGAC ptg000092l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1403:3824:4068_1:N:0:CGGCATTA+TGACTGAC ptg000066l 66350 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1433:29266:15414_1:N:0:CGGCATTA+TGACTGAC ptg000118l 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 LH00526:197:22KFKVLT4:4:2152:20543:14630_1:N:0:CGGCATTA+TGACTGAC ptg000036l 6407224 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2177:22428:9181_1:N:0:CGGCATTA+TGACTGAC ptg000002l 2 Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2249:27235:2093_1:N:0:CGGCATTA+TGACTGAC ptg000024l 2 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2305:16529:11268_1:N:0:CGGCATTA+TGACTGAC ntLink_4 304046 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2305:16545:11268_1:N:0:CGGCATTA+TGACTGAC ntLink_4 304046 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2317:18536:8382_1:N:0:CGGCATTA+TGACTGAC ptg000066l 66382 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2340:23173:25261_1:N:0:CGGCATTA+TGACTGAC ptg000066l 66348 Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far 40897003 reads; of these: 40897003 (100.00%) were paired; of these: 15337119 (37.50%) aligned concordantly 0 times 7337781 (17.94%) aligned concordantly exactly 1 time 18222103 (44.56%) aligned concordantly >1 times 62.50% overall alignment rate 40897003 reads; of these: 40897003 (100.00%) were paired; of these: 15328711 (37.48%) aligned concordantly 0 times 7339153 (17.95%) aligned concordantly exactly 1 time 18229139 (44.57%) aligned concordantly >1 times 62.52% overall alignment rate Processed 40897003 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 163588012. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 163588012. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 163588012. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 163588012. Successfully deleted the temporary files 1H5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 40897003 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1063935910 Total methylated C's in CpG context: 15496649 Total methylated C's in CHG context: 1152540 Total methylated C's in CHH context: 4428306 Total methylated C's in Unknown context: 59125 Total unmethylated C's in CpG context: 161277940 Total unmethylated C's in CHG context: 181964213 Total unmethylated C's in CHH context: 699616262 Total unmethylated C's in Unknown context: 2648646 C methylated in CpG context: 8.8% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 2.2% Bismark completed in 0d 2h 33m 39s ==================== Bismark run complete ====================