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/2D2_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/2D2_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/2D2_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/2D2_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 2D2_R1_001.fastp-trim.fq.gz to 2D2_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 2D2_R1_001.fastp-trim.fq.gz (30040067 sequences in total) Writing a G -> A converted version of the input file 2D2_R2_001.fastp-trim.fq.gz to 2D2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 2D2_R2_001.fastp-trim.fq.gz (30040067 sequences in total) Input files are 2D2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D2_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 2D2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D2_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: LH00652:104:22KG7JLT4:7:1101:5676:1084_1:N:0:GTAACCGA+GGTGTCTT/1 77 * 0 0 * * 0 0 ATAAAATTTTGTGTTGGTAAGTTTTGTTTTATTGTTAATTTAAAAGTTATTGTTGGTTATATTAAAGTATGGAATATGATATAAAATAGGATTATTTTGAAATTTTTGTGTTTGGAAAAATTGAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III9IIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00652:104:22KG7JLT4:7:1101:5676:1084_2:N:0:GTAACCGA+GGTGTCTT/2 141 * 0 0 * * 0 0 CTATTTTAAATACAATAAAAATACCAAAAAATCTAAATAAAACTTAAAAATAACAATAACATATTAATAAAATAACAATTTACCACTAATTAAATTAATATTTATAATTAAATAAATTCTTATAT IIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIII-IIIII-II9IIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 2D2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D2_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: LH00652:104:22KG7JLT4:7:1101:5676:1084_1:N:0:GTAACCGA+GGTGTCTT/1 83 ptg000024l_GA_converted 7591048 42 125M = 7590874 -299 ATCAATTTTTCCAAACACAAAAATTTCAAAATAATCCTATTTTATATCATATTCCATACTTTAATATAACCAACAATAACTTTTAAATTAACAATAAAACAAAACTTACCAACACAAAATTTTAT IIIIIIIIIIIIIIIIIIIIIII9III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:47T1C75 YS:i:-6 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:5676:1084_2:N:0:GTAACCGA+GGTGTCTT/2 163 ptg000024l_GA_converted 7590874 42 125M = 7591048 299 CTATTTTAAATACAATAAAAATACCAAAAAATCTAAATAAAACTTAAAAATAACAATAACATATTAATAAAATAACAATTTACCACTAATTAAATTAATATTTATAATTAAATAAATTCTTATAT IIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIII-IIIII-II9IIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:78A46 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to 2D2_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/2D2_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/2D2_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1105:49758:18501_1:N:0:GTAACCGA+GGTGTCTT ptg000066l 66368 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1148:10239:8286_1:N:0:GTAACCGA+GGTGTCTT ptg000075l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1148:10255:8286_1:N:0:GTAACCGA+GGTGTCTT ptg000075l 1 Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1236:9171:11369_1:N:0:GTAACCGA+GGTGTCTT ptg000077l 42477 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1248:12795:26306_1:N:0:GTAACCGA+GGTGTCTT ptg000066l 1 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1289:41976:25620_1:N:0:GTAACCGA+GGTGTCTT ptg000124l 54842 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1348:26953:14816_1:N:0:GTAACCGA+GGTGTCTT ptg000109l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1350:49289:14578_1:N:0:GTAACCGA+GGTGTCTT ptg000127l 27807 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1383:11080:3550_1:N:0:GTAACCGA+GGTGTCTT ptg000128l 44528 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1383:11096:3550_1:N:0:GTAACCGA+GGTGTCTT ptg000128l 44528 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1383:11088:3564_1:N:0:GTAACCGA+GGTGTCTT ptg000128l 44528 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1383:11096:3578_1:N:0:GTAACCGA+GGTGTCTT ptg000128l 44528 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2101:34129:8160_1:N:0:GTAACCGA+GGTGTCTT ptg000066l 66350 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2109:28660:26236_1:N:0:GTAACCGA+GGTGTCTT ptg000050l 26313 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2110:9130:4573_1:N:0:GTAACCGA+GGTGTCTT ptg000035l 10246571 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2123:37106:22677_1:N:0:GTAACCGA+GGTGTCTT ptg000056l 59099 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2142:25286:13779_1:N:0:GTAACCGA+GGTGTCTT ptg000048l 76434 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2146:6356:15937_1:N:0:GTAACCGA+GGTGTCTT ptg000125l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2162:11509:21892_1:N:0:GTAACCGA+GGTGTCTT ptg000075l 1 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2191:32341:3354_1:N:0:GTAACCGA+GGTGTCTT ptg000090l 49493 Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2252:38068:18599_1:N:0:GTAACCGA+GGTGTCTT ptg000106l 40385 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2280:12561:29263_1:N:0:GTAACCGA+GGTGTCTT ptg000129l 33337 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2337:30375:25507_1:N:0:GTAACCGA+GGTGTCTT ptg000130l 35796 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2355:15853:26054_1:N:0:GTAACCGA+GGTGTCTT ptg000116l 48489 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2425:22819:14242_1:N:0:GTAACCGA+GGTGTCTT ptg000098l 55091 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2430:11768:10711_1:N:0:GTAACCGA+GGTGTCTT ptg000106l 40377 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2466:45220:4139_1:N:0:GTAACCGA+GGTGTCTT ptg000048l 2 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1115:19097:23994_1:N:0:GTAACCGA+GGTGTCTT ptg000048l 76435 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1119:36806:23952_1:N:0:GTAACCGA+GGTGTCTT ptg000128l 44526 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1148:1607:12981_1:N:0:GTAACCGA+GGTGTCTT ptg000024l 3 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1152:47372:24401_1:N:0:GTAACCGA+GGTGTCTT ptg000116l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1170:48585:24569_1:N:0:GTAACCGA+GGTGTCTT ptg000117l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1184:37235:18389_1:N:0:GTAACCGA+GGTGTCTT ptg000096l 1 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1265:12836:22677_1:N:0:GTAACCGA+GGTGTCTT ptg000082l 2 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1365:45511:27567_1:N:0:GTAACCGA+GGTGTCTT ptg000098l 55086 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1367:34290:4405_1:N:0:GTAACCGA+GGTGTCTT ntLink_0 1 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1438:14632:19202_1:N:0:GTAACCGA+GGTGTCTT ptg000127l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1450:25618:12056_1:N:0:GTAACCGA+GGTGTCTT ptg000127l 1 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1487:40414:4055_1:N:0:GTAACCGA+GGTGTCTT ptg000048l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1488:45576:23532_1:N:0:GTAACCGA+GGTGTCTT ptg000045l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2113:5183:5414_1:N:0:GTAACCGA+GGTGTCTT ptg000048l 2 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2135:49847:1645_1:N:0:GTAACCGA+GGTGTCTT ptg000066l 66407 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2169:46814:7263_1:N:0:GTAACCGA+GGTGTCTT ptg000039l 1139416 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2218:14632:29291_1:N:0:GTAACCGA+GGTGTCTT ptg000096l 1 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2238:11727:7446_1:N:0:GTAACCGA+GGTGTCTT ptg000048l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2266:49750:1869_1:N:0:GTAACCGA+GGTGTCTT ptg000015l 14997133 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2271:16072:22565_1:N:0:GTAACCGA+GGTGTCTT ptg000129l 33353 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2273:15619:28814_1:N:0:GTAACCGA+GGTGTCTT ptg000006l 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2281:19874:16568_1:N:0:GTAACCGA+GGTGTCTT ptg000057l 39950 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2329:28506:3410_1:N:0:GTAACCGA+GGTGTCTT ntLink_1 163091 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2352:21265:16624_1:N:0:GTAACCGA+GGTGTCTT ptg000121l 50749 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2362:25132:7964_1:N:0:GTAACCGA+GGTGTCTT ptg000075l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2371:34137:16385_1:N:0:GTAACCGA+GGTGTCTT ptg000055l 50538 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2383:47962:16428_1:N:0:GTAACCGA+GGTGTCTT ptg000075l 54150 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2383:47979:16456_1:N:0:GTAACCGA+GGTGTCTT ptg000075l 54150 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2397:12876:20757_1:N:0:GTAACCGA+GGTGTCTT ptg000098l 55094 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2411:37615:15825_1:N:0:GTAACCGA+GGTGTCTT ptg000127l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2411:37591:15839_1:N:0:GTAACCGA+GGTGTCTT ptg000127l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2411:37607:15839_1:N:0:GTAACCGA+GGTGTCTT ptg000127l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2411:37599:15853_1:N:0:GTAACCGA+GGTGTCTT ptg000127l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2418:38740:27525_1:N:0:GTAACCGA+GGTGTCTT ptg000046l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2422:36645:22159_1:N:0:GTAACCGA+GGTGTCTT ptg000115l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2422:36636:22173_1:N:0:GTAACCGA+GGTGTCTT ptg000115l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2422:36636:22201_1:N:0:GTAACCGA+GGTGTCTT ptg000115l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2439:15303:9996_1:N:0:GTAACCGA+GGTGTCTT ptg000113l 59560 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2471:26532:26194_1:N:0:GTAACCGA+GGTGTCTT ptg000130l 35807 Processed 30000000 sequence pairs so far 30040067 reads; of these: 30040067 (100.00%) were paired; of these: 11840983 (39.42%) aligned concordantly 0 times 6224127 (20.72%) aligned concordantly exactly 1 time 11974957 (39.86%) aligned concordantly >1 times 60.58% overall alignment rate 30040067 reads; of these: 30040067 (100.00%) were paired; of these: 11846274 (39.43%) aligned concordantly 0 times 6226454 (20.73%) aligned concordantly exactly 1 time 11967339 (39.84%) aligned concordantly >1 times 60.57% overall alignment rate Processed 30040067 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 120160268. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 120160268. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 120160268. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 120160268. Successfully deleted the temporary files 2D2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 30040067 Final Cytosine Methylation Report ================================= Total number of C's analysed: 895699250 Total methylated C's in CpG context: 15014074 Total methylated C's in CHG context: 832284 Total methylated C's in CHH context: 3401739 Total methylated C's in Unknown context: 46290 Total unmethylated C's in CpG context: 124131965 Total unmethylated C's in CHG context: 150446463 Total unmethylated C's in CHH context: 601872725 Total unmethylated C's in Unknown context: 2348995 C methylated in CpG context: 10.8% 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 2m 41s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe