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/2H3_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/2H3_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/2H3_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/2H3_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 2H3_R1_001.fastp-trim.fq.gz to 2H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 2H3_R1_001.fastp-trim.fq.gz (32103792 sequences in total) Writing a G -> A converted version of the input file 2H3_R2_001.fastp-trim.fq.gz to 2H3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 2H3_R2_001.fastp-trim.fq.gz (32103792 sequences in total) Input files are 2H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2H3_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 2H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2H3_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: LH00652:104:22KG7JLT4:7:1101:10352:1084_1:N:0:GTCAGTCA+TAATGCCG/1 99 ptg000027l_CT_converted 162861 1 125M = 162795 -191 GTTGTTTTTTGTAAAAAATTGTTTTAAAAAATATTAAATATTTGAGAAATGAGAGTATTTGGAAAATTGGTTATTATGTAAAGGTTATAGTTTATGTAAAATTTTTATTTTAGTTTAAAAATTAA IIIIIIIIIIIII9II9IIIIIIIIIIIII9IIIIIIIIIIIIIII9I9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII AS:i:-18 XS:i:-42 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:60T0T31G31 YS:i:-44 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:10352:1084_2:N:0:GTCAGTCA+TAATGCCG/2 147 ptg000027l_CT_converted 162795 1 91M1D34M = 162861 191 TTTATTTTGGTTTAAAAATTAAAATTTTAAAAAATAAGTTGAAATTTATTATATAAATTATTTATAGTTGTTTTTTGTAAAAGATTGTTTTAAAAATATTAAATATTTGAGAAATGAGAGTATTT III9II9II-IIIIII9I9IIII9I9IIIII99IIIIII99I9I9IIII-IIIIIIIIIIIIIII9II9IIII9IIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-44 XS:i:-44 XN:i:0 XM:i:6 XO:i:1 XG:i:1 NM:i:7 MD:Z:32T3T3A9T3T27A8^A34 YS:i:-18 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 2H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2H3_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: LH00652:104:22KG7JLT4:7:1101:10352:1084_1:N:0:GTCAGTCA+TAATGCCG/1 83 ptg000027l_GA_converted 7749644 1 125M = 7749709 191 TTAATTTTTAAACTAAAATAAAAATTTTACATAAACTATAACCTTTACATAATAACCAATTTTCCAAATACTCTCATTTCTCAAATATTTAATATTTTTTAAAACAATTTTTTACAAAAAACAAC IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9I9IIIIIIIIIIIIIII9IIIIIIIIIIIII9II9IIIIIIIIIIIII AS:i:-12 XS:i:-24 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:64A51C8 YS:i:-44 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:10352:1084_2:N:0:GTCAGTCA+TAATGCCG/2 163 ptg000027l_GA_converted 7749709 1 29M1D96M = 7749644 -191 AAATACTCTCATTTCTCAAATATTTAATATTTTTAAAACAATCTTTTACAAAAAACAACTATAAATAATTTATATAATAAATTTCAACTTATTTTTTAAAATTTTAATTTTTAAACCAAAATAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIII9IIII9II9IIIIIIIIIIIIIII-IIII9I9I99IIIIII99IIIII9I9IIII9I9IIIIII-II9II9III AS:i:-44 XS:i:-44 XN:i:0 XM:i:6 XO:i:1 XG:i:1 NM:i:7 MD:Z:29^T13T7C9C13A9T3A36 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to 2H3_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/2H3_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/2H3_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1110:20974:10682_1:N:0:GTCAGTCA+TAATGCCG ptg000095l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1131:4147:21164_1:N:0:GTCAGTCA+TAATGCCG ptg000177l 1 Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1210:6032:6689_1:N:0:GTCAGTCA+TAATGCCG ptg000092l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1212:44087:4923_1:N:0:GTCAGTCA+TAATGCCG ptg000183l 26189 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1257:46531:3718_1:N:0:GTCAGTCA+TAATGCCG ptg000171l 1 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1341:20594:11453_1:N:0:GTCAGTCA+TAATGCCG ntLink_0 96475 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 LH00652:104:22KG7JLT4:7:1497:50988:11495_1:N:0:GTCAGTCA+TAATGCCG ptg000165l 24782 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2136:34209:18669_1:N:0:GTCAGTCA+TAATGCCG ptg000094l 36833 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2136:34201:18684_1:N:0:GTCAGTCA+TAATGCCG ptg000094l 36833 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2136:34193:18698_1:N:0:GTCAGTCA+TAATGCCG ptg000094l 36833 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2136:34201:18712_1:N:0:GTCAGTCA+TAATGCCG ptg000094l 36833 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2213:24954:21724_1:N:0:GTCAGTCA+TAATGCCG ptg000004l 2 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2303:48715:21962_1:N:0:GTCAGTCA+TAATGCCG ptg000047l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2317:39840:15839_1:N:0:GTCAGTCA+TAATGCCG ptg000006l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2331:34824:21640_1:N:0:GTCAGTCA+TAATGCCG ptg000004l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2331:34840:21640_1:N:0:GTCAGTCA+TAATGCCG ptg000004l 1 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2354:40042:5372_1:N:0:GTCAGTCA+TAATGCCG ptg000120l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2381:11727:15825_1:N:0:GTCAGTCA+TAATGCCG ptg000094l 36823 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2381:11744:15825_1:N:0:GTCAGTCA+TAATGCCG ptg000094l 36823 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2381:11735:15839_1:N:0:GTCAGTCA+TAATGCCG ptg000094l 36823 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2393:32583:7221_1:N:0:GTCAGTCA+TAATGCCG ptg000137l 40569 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2393:32591:7235_1:N:0:GTCAGTCA+TAATGCCG ptg000137l 40569 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2393:19842:9057_1:N:0:GTCAGTCA+TAATGCCG ptg000066l 66347 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2460:50624:20757_1:N:0:GTCAGTCA+TAATGCCG ptg000066l 66406 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1233:6970:1308_1:N:0:GTCAGTCA+TAATGCCG ptg000113l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1233:6962:1322_1:N:0:GTCAGTCA+TAATGCCG ptg000113l 1 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1292:36111:6577_1:N:0:GTCAGTCA+TAATGCCG ptg000123l 44478 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1347:34080:7992_1:N:0:GTCAGTCA+TAATGCCG ptg000039l 1 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1391:8257:11860_1:N:0:GTCAGTCA+TAATGCCG ptg000115l 42071 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1454:10409:29515_1:N:0:GTCAGTCA+TAATGCCG ptg000177l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1454:10417:29529_1:N:0:GTCAGTCA+TAATGCCG ptg000177l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1473:5797:2808_1:N:0:GTCAGTCA+TAATGCCG ptg000105l 83226 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1494:13984:15363_1:N:0:GTCAGTCA+TAATGCCG ptg000121l 50726 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2115:22972:15741_1:N:0:GTCAGTCA+TAATGCCG ptg000121l 50734 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2231:35099:13485_1:N:0:GTCAGTCA+TAATGCCG ntLink_4 304020 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2249:44112:26853_1:N:0:GTCAGTCA+TAATGCCG ptg000075l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2252:1736:1995_1:N:0:GTCAGTCA+TAATGCCG ptg000066l 66351 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2286:20408:23994_1:N:0:GTCAGTCA+TAATGCCG ptg000066l 66414 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2316:26799:3844_1:N:0:GTCAGTCA+TAATGCCG ntLink_1 163148 Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2416:37033:1168_1:N:0:GTCAGTCA+TAATGCCG ptg000066l 66345 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far 32103792 reads; of these: 32103792 (100.00%) were paired; of these: 13582677 (42.31%) aligned concordantly 0 times 6716191 (20.92%) aligned concordantly exactly 1 time 11804924 (36.77%) aligned concordantly >1 times 57.69% overall alignment rate 32103792 reads; of these: 32103792 (100.00%) were paired; of these: 13592495 (42.34%) aligned concordantly 0 times 6716572 (20.92%) aligned concordantly exactly 1 time 11794725 (36.74%) aligned concordantly >1 times 57.66% overall alignment rate Processed 32103792 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 128415168. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 128415168. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 128415168. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 128415168. Successfully deleted the temporary files 2H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2H3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 32103792 Final Cytosine Methylation Report ================================= Total number of C's analysed: 958031882 Total methylated C's in CpG context: 18592313 Total methylated C's in CHG context: 1028522 Total methylated C's in CHH context: 4211040 Total methylated C's in Unknown context: 55394 Total unmethylated C's in CpG context: 128078418 Total unmethylated C's in CHG context: 160762920 Total unmethylated C's in CHH context: 645358669 Total unmethylated C's in Unknown context: 2574713 C methylated in CpG context: 12.7% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 2.1% Bismark completed in 0d 2h 11m 44s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe