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/2D1_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/2D1_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/2D1_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/2D1_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 2D1_R1_001.fastp-trim.fq.gz to 2D1_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 2D1_R1_001.fastp-trim.fq.gz (38150783 sequences in total) Writing a G -> A converted version of the input file 2D1_R2_001.fastp-trim.fq.gz to 2D1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 2D1_R2_001.fastp-trim.fq.gz (38150783 sequences in total) Input files are 2D1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D1_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 2D1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D1_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: LH00652:104:22KG7JLT4:7:1101:19332:1084_1:N:0:GTCGTTAC+GCTTAGCT/1 99 ptg000023l_CT_converted 9322463 6 90M = 9322447 -106 TATTAGAGGATGTTGTATATTAGAATATGTGGTTTATAATGTTTTTTTTTTATTTTTGAAGTATAGTATAGTTTTTTTTTTTTTTAAGGG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-I-I9II AS:i:-6 XS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:83G6 YS:i:0 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:19332:1084_2:N:0:GTCGTTAC+GCTTAGCT/2 147 ptg000023l_CT_converted 9322447 6 92M = 9322463 106 AAATTATTGTTATTATTATTAGAGGATGTTGTATATTAGAATATGTGGTTTATAATGTTTTTTTTTTATTTTTGAAGTATAGTATAGTTTTT IIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:-6 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:92 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 2D1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D1_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: LH00652:104:22KG7JLT4:7:1101:19332:1084_1:N:0:GTCGTTAC+GCTTAGCT/1 83 ptg000020l_GA_converted 13718287 1 90M = 13718301 106 CCCTTAAAAAAAAAAAAAACTATACTATACTTCAAAAATAAAAAAAAAACATTATAAACCACATATTCTAATATACAACATCCTCTAATA II9I-I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:6C83 YS:i:0 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:19332:1084_2:N:0:GTCGTTAC+GCTTAGCT/2 163 ptg000020l_GA_converted 13718301 1 92M = 13718287 -106 AAAAACTATACTATACTTCAAAAATAAAAAAAAAACATTATAAACCACATATTCTAATATACAACATCCTCTAATAATAATAACAATAATTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:92 YS:i:-6 YT:Z:CP >>> Writing bisulfite mapping results to 2D1_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/2D1_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/2D1_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1154:51579:3354_1:N:0:GTCGTTAC+GCTTAGCT ptg000092l 2 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1211:16800:23434_1:N:0:GTCGTTAC+GCTTAGCT ptg000046l 39585 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 LH00652:104:22KG7JLT4:7:1430:11695:16666_1:N:0:GTCGTTAC+GCTTAGCT ptg000053l 71552 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1486:19696:10935_1:N:0:GTCGTTAC+GCTTAGCT ptg000120l 31354 Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2113:24154:9183_1:N:0:GTCGTTAC+GCTTAGCT ptg000115l 42069 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2179:34493:28240_1:N:0:GTCGTTAC+GCTTAGCT ptg000105l 83250 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2179:34509:28240_1:N:0:GTCGTTAC+GCTTAGCT ptg000105l 83250 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2387:20554:10234_1:N:0:GTCGTTAC+GCTTAGCT ptg000030l 2 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2415:3556:22999_1:N:0:GTCGTTAC+GCTTAGCT ptg000048l 76432 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2440:50171:29529_1:N:0:GTCGTTAC+GCTTAGCT ptg000066l 66413 Processed 18000000 sequence pairs so far 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 LH00652:104:22KG7JLT4:8:1196:36458:20239_1:N:0:GTCGTTAC+GCTTAGCT ptg000093l 1 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1229:45357:17184_1:N:0:GTCGTTAC+GCTTAGCT ptg000146l 38989 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1271:32252:18473_1:N:0:GTCGTTAC+GCTTAGCT ptg000048l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1274:41644:17955_1:N:0:GTCGTTAC+GCTTAGCT ptg000017l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1291:3516:10430_1:N:0:GTCGTTAC+GCTTAGCT ptg000095l 2 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1363:32268:21024_1:N:0:GTCGTTAC+GCTTAGCT ptg000094l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1363:32284:21024_1:N:0:GTCGTTAC+GCTTAGCT ptg000094l 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1395:16039:8413_1:N:0:GTCGTTAC+GCTTAGCT ptg000066l 66348 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1426:50098:25900_1:N:0:GTCGTTAC+GCTTAGCT ntLink_3 131400 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1440:12164:10528_1:N:0:GTCGTTAC+GCTTAGCT ptg000018l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1440:12172:10542_1:N:0:GTCGTTAC+GCTTAGCT ptg000018l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1452:9761:19202_1:N:0:GTCGTTAC+GCTTAGCT ptg000094l 2 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2117:45511:6128_1:N:0:GTCGTTAC+GCTTAGCT ptg000066l 66404 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2187:40819:8623_1:N:0:GTCGTTAC+GCTTAGCT ptg000059l 1573514 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2259:23425:6997_1:N:0:GTCGTTAC+GCTTAGCT ptg000066l 66345 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2324:21557:20491_1:N:0:GTCGTTAC+GCTTAGCT ptg000160l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2361:17075:10094_1:N:0:GTCGTTAC+GCTTAGCT ptg000046l 2 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2432:13208:3985_1:N:0:GTCGTTAC+GCTTAGCT ptg000105l 83250 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2439:35051:17549_1:N:0:GTCGTTAC+GCTTAGCT ptg000017l 2 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2482:1582:6997_1:N:0:GTCGTTAC+GCTTAGCT ptg000039l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2484:15206:4251_1:N:0:GTCGTTAC+GCTTAGCT ptg000125l 1 Processed 38000000 sequence pairs so far 38150783 reads; of these: 38150783 (100.00%) were paired; of these: 13417969 (35.17%) aligned concordantly 0 times 7893172 (20.69%) aligned concordantly exactly 1 time 16839642 (44.14%) aligned concordantly >1 times 64.83% overall alignment rate 38150783 reads; of these: 38150783 (100.00%) were paired; of these: 13410459 (35.15%) aligned concordantly 0 times 7892559 (20.69%) aligned concordantly exactly 1 time 16847765 (44.16%) aligned concordantly >1 times 64.85% overall alignment rate Processed 38150783 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 152603132. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 152603132. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 152603132. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 152603132. Successfully deleted the temporary files 2D1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2D1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 38150783 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1155923227 Total methylated C's in CpG context: 18423285 Total methylated C's in CHG context: 1201592 Total methylated C's in CHH context: 4841725 Total methylated C's in Unknown context: 58290 Total unmethylated C's in CpG context: 167592742 Total unmethylated C's in CHG context: 196095633 Total unmethylated C's in CHH context: 767768250 Total unmethylated C's in Unknown context: 2819897 C methylated in CpG context: 9.9% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 2.0% Bismark completed in 0d 2h 40m 36s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe