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/1H9_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/1H9_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/1H9_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/1H9_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1H9_R1_001.fastp-trim.fq.gz to 1H9_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1H9_R1_001.fastp-trim.fq.gz (41293598 sequences in total) Writing a G -> A converted version of the input file 1H9_R2_001.fastp-trim.fq.gz to 1H9_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1H9_R2_001.fastp-trim.fq.gz (41293598 sequences in total) Input files are 1H9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H9_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 1H9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H9_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: LH00526:197:22KFKVLT4:3:1101:40304:1070_1:N:0:GGACATCA+TGCAGGTA/1 99 ptg000004l_CT_converted 3787886 4 99M9I17M = 3788034 273 TTTTGTTGAATTTTATTATGGTTTGAGTGTAGTAGTAAAAGATTTGTTTTTGTAGTTATGTTGTATGTGTAAAATTAATGATAAAATGTAATAAAGAGTAGTAAAAAGTTTTTTTTTGTGGTTAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-68 XS:i:-116 XN:i:0 XM:i:6 XO:i:1 XG:i:9 NM:i:15 MD:Z:20A17G20A1A22T9G21 YS:i:-18 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:40304:1070_2:N:0:GGACATCA+TGCAGGTA/2 147 ptg000004l_CT_converted 3788034 4 125M = 3787886 -273 ATTTAAGTATAGTATTTTTTGTTGTAGGTAGGTTTTTTGTAAGTTTTAGATTAAATGTAAGAAAAGTAAGTAAAATTTTTTATTTTTTTTATTTATTTTATAGTTATTTTTGATTTGAATAATGT II9IIIIIIIIII99IIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIII AS:i:-18 XS:i:-84 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:1A25T69A27 YS:i:-68 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1H9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H9_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: LH00526:197:22KFKVLT4:3:1101:40304:1070_1:N:0:GGACATCA+TGCAGGTA/1 83 ptg000004l_GA_converted 2507005 7 17M9I99M = 2506848 -273 ATAACCACAAAAAAAAACTTTTTACTACTCTTTATTACATTTTATCATTAATTTTACACATACAACATAACTACAAAAACAAATCTTTTACTACTACACTCAAACCATAATAAAATTCAACAAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-56 XS:i:-66 XN:i:0 XM:i:4 XO:i:1 XG:i:9 NM:i:13 MD:Z:30A0A29A23A30 YS:i:-26 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:40304:1070_2:N:0:GGACATCA+TGCAGGTA/2 163 ptg000004l_GA_converted 2506848 7 119M1I5M = 2507005 273 ACATTATTCAAATCAAAAATAACTATAAAATAAATAAAAAAAATAAAAAATTTTACTTACTTTTCTTACATTTAATCTAAAACTTACAAAAAACCTACCTACAACAAAAAATACTATACTTAAAT IIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII99IIIIIIIIII9II AS:i:-26 XS:i:-48 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:27T8T60A26 YS:i:-56 YT:Z:CP >>> Writing bisulfite mapping results to 1H9_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/1H9_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/1H9_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1114:19127:16829_1:N:0:GGACATCA+TGCAGGTA ntLink_0 1 Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1188:50800:14447_1:N:0:GGACATCA+TGCAGGTA ptg000055l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1197:40717:6967_1:N:0:GGACATCA+TGCAGGTA ptg000066l 1 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1242:14150:11324_1:N:0:GGACATCA+TGCAGGTA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1242:14166:11324_1:N:0:GGACATCA+TGCAGGTA ptg000039l 1 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1340:10468:9237_1:N:0:GGACATCA+TGCAGGTA ptg000066l 66366 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 LH00526:197:22KFKVLT4:3:1441:47190:6379_1:N:0:GGACATCA+TGCAGGTA ptg000097l 1 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1488:48453:5511_1:N:0:GGACATCA+TGCAGGTA ptg000024l 1 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2141:7951:27517_1:N:0:GGACATCA+TGCAGGTA ptg000144l 2 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2170:38961:23202_1:N:0:GGACATCA+TGCAGGTA ptg000015l 14997091 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2237:17395:22376_1:N:0:GGACATCA+TGCAGGTA ptg000106l 40370 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2271:47458:23006_1:N:0:GGACATCA+TGCAGGTA ptg000075l 1 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2324:44358:3213_1:N:0:GGACATCA+TGCAGGTA ptg000039l 1 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2393:30520:3353_1:N:0:GGACATCA+TGCAGGTA ptg000129l 33365 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2404:19199:16367_1:N:0:GGACATCA+TGCAGGTA ptg000066l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2413:11827:10049_1:N:0:GGACATCA+TGCAGGTA ptg000063l 30351 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2425:7150:20359_1:N:0:GGACATCA+TGCAGGTA ntLink_1 163091 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1105:37528:25850_1:N:0:GGACATCA+TGCAGGTA ptg000107l 1 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1173:35060:4292_1:N:0:GGACATCA+TGCAGGTA ptg000007l 12295855 Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:1337:48979:10960_1:N:0:GGACATCA+TGCAGGTA ptg000129l 33365 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1337:48987:10974_1:N:0:GGACATCA+TGCAGGTA ptg000129l 33365 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1403:45451:27237_1:N:0:GGACATCA+TGCAGGTA ptg000088l 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 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2191:13179:9503_1:N:0:GGACATCA+TGCAGGTA ptg000117l 30008 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2286:46397:28287_1:N:0:GGACATCA+TGCAGGTA ptg000077l 42461 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2302:30739:7654_1:N:0:GGACATCA+TGCAGGTA ptg000066l 66348 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2309:38386:17725_1:N:0:GGACATCA+TGCAGGTA ntLink_3 131404 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2336:16440:23272_1:N:0:GGACATCA+TGCAGGTA ntLink_2 399363 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2380:34316:21269_1:N:0:GGACATCA+TGCAGGTA ptg000077l 42461 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2421:13648:3900_1:N:0:GGACATCA+TGCAGGTA ptg000077l 42461 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2464:33256:12458_1:N:0:GGACATCA+TGCAGGTA ptg000106l 1 Processed 41000000 sequence pairs so far 41293598 reads; of these: 41293598 (100.00%) were paired; of these: 15621887 (37.83%) aligned concordantly 0 times 8302823 (20.11%) aligned concordantly exactly 1 time 17368888 (42.06%) aligned concordantly >1 times 62.17% overall alignment rate 41293598 reads; of these: 41293598 (100.00%) were paired; of these: 15640632 (37.88%) aligned concordantly 0 times 8293178 (20.08%) aligned concordantly exactly 1 time 17359788 (42.04%) aligned concordantly >1 times 62.12% overall alignment rate Processed 41293598 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 165174392. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 165174392. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 165174392. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 165174392. Successfully deleted the temporary files 1H9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H9_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 41293598 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1207558736 Total methylated C's in CpG context: 17946276 Total methylated C's in CHG context: 1257538 Total methylated C's in CHH context: 4986356 Total methylated C's in Unknown context: 60943 Total unmethylated C's in CpG context: 174400262 Total unmethylated C's in CHG context: 204267546 Total unmethylated C's in CHH context: 804700758 Total unmethylated C's in Unknown context: 3082560 C methylated in CpG context: 9.3% 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 50m 42s ==================== Bismark run complete ====================