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/1D3_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/1D3_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/1D3_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/1D3_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1D3_R1_001.fastp-trim.fq.gz to 1D3_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1D3_R1_001.fastp-trim.fq.gz (39335770 sequences in total) Writing a G -> A converted version of the input file 1D3_R2_001.fastp-trim.fq.gz to 1D3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1D3_R2_001.fastp-trim.fq.gz (39335770 sequences in total) Input files are 1D3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D3_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 1D3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D3_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: LH00526:197:22KFKVLT4:3:1101:38637:1070_1:N:0:TCCACGTT+GTTCAACC/1 99 ptg000007l_CT_converted 2557100 0 60M = 2557075 -85 ATTTTTTGGTAAAATTGTGATTTTTTAATTAATTTTTGTTGTTTTATAGAGTTTTTGGTA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-18 XS:i:-36 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:9A8A8G32 YS:i:-30 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:38637:1070_2:N:0:TCCACGTT+GTTCAACC/2 147 ptg000007l_CT_converted 2557075 0 60M = 2557100 85 TTGGTTAAAATTGTTTATTTTGAGTATTTTTTGGTAAAATTGTGATTTTTTAATTAATTT IIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-30 XS:i:-30 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:2A12A18A8A8G7 YS:i:-18 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1D3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D3_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: LH00526:197:22KFKVLT4:3:1101:38637:1070_1:N:0:TCCACGTT+GTTCAACC/1 83 ptg000011l_GA_converted 4055331 1 60M = 4055356 85 TACCAAAAACTCTATAAAACAACAAAAATTAATTAAAAAATCACAATTTTACCAAAAAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:60 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:38637:1070_2:N:0:TCCACGTT+GTTCAACC/2 163 ptg000011l_GA_converted 4055356 1 60M = 4055331 -85 AAATTAATTAAAAAATCACAATTTTACCAAAAAATACTCAAAATAAACAATTTTAACCAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:60 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to 1D3_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/1D3_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/1D3_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 LH00526:197:22KFKVLT4:3:1201:33458:16927_1:N:0:TCCACGTT+GTTCAACC ptg000006l 3 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 LH00526:197:22KFKVLT4:3:1315:13422:4936_1:N:0:TCCACGTT+GTTCAACC ptg000097l 1 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1333:23804:8928_1:N:0:TCCACGTT+GTTCAACC ptg000066l 66350 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1365:44981:5300_1:N:0:TCCACGTT+GTTCAACC ptg000066l 66348 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 LH00526:197:22KFKVLT4:3:1491:8914:8536_1:N:0:TCCACGTT+GTTCAACC ptg000012l 1 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2172:14773:4418_1:N:0:TCCACGTT+GTTCAACC ptg000006l 1 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2211:38629:9993_1:N:0:TCCACGTT+GTTCAACC ptg000007l 12295855 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2310:31645:4320_1:N:0:TCCACGTT+GTTCAACC ptg000060c 1 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2354:47652:18692_1:N:0:TCCACGTT+GTTCAACC ptg000007l 12295855 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2389:46559:25514_1:N:0:TCCACGTT+GTTCAACC ptg000015l 14997108 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 LH00526:197:22KFKVLT4:4:1162:35683:26242_1:N:0:TCCACGTT+GTTCAACC ptg000083l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1171:6697:7107_1:N:0:TCCACGTT+GTTCAACC ptg000171l 41130 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1172:11900:18804_1:N:0:TCCACGTT+GTTCAACC ptg000006l 1 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1249:23861:17683_1:N:0:TCCACGTT+GTTCAACC ptg000066l 66350 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1361:14490:8046_1:N:0:TCCACGTT+GTTCAACC ptg000006l 1 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1376:18625:28707_1:N:0:TCCACGTT+GTTCAACC ptg000066l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1382:27381:6015_1:N:0:TCCACGTT+GTTCAACC ptg000146l 38991 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1423:36541:12066_1:N:0:TCCACGTT+GTTCAACC ptg000006l 1 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1446:8752:12318_1:N:0:TCCACGTT+GTTCAACC ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1451:43695:7416_1:N:0:TCCACGTT+GTTCAACC ptg000066l 66349 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1453:2837:18412_1:N:0:TCCACGTT+GTTCAACC ptg000095l 40230 Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2114:11520:28511_1:N:0:TCCACGTT+GTTCAACC ptg000011l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2123:12531:14293_1:N:0:TCCACGTT+GTTCAACC ptg000106l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2133:32908:18692_1:N:0:TCCACGTT+GTTCAACC ptg000066l 66354 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 LH00526:197:22KFKVLT4:4:2260:51649:10988_1:N:0:TCCACGTT+GTTCAACC ptg000128l 44525 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2278:18188:22348_1:N:0:TCCACGTT+GTTCAACC ptg000012l 2 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2401:14126:21563_1:N:0:TCCACGTT+GTTCAACC ptg000066l 66347 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2475:23189:22124_1:N:0:TCCACGTT+GTTCAACC ptg000088l 96896 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2496:21028:1210_1:N:0:TCCACGTT+GTTCAACC ptg000083l 1 39335770 reads; of these: 39335770 (100.00%) were paired; of these: 13285741 (33.78%) aligned concordantly 0 times 8279124 (21.05%) aligned concordantly exactly 1 time 17770905 (45.18%) aligned concordantly >1 times 66.22% overall alignment rate 39335770 reads; of these: 39335770 (100.00%) were paired; of these: 13299252 (33.81%) aligned concordantly 0 times 8279043 (21.05%) aligned concordantly exactly 1 time 17757475 (45.14%) aligned concordantly >1 times 66.19% overall alignment rate Processed 39335770 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 157343080. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 157343080. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 157343080. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 157343080. Successfully deleted the temporary files 1D3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 39335770 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1212357954 Total methylated C's in CpG context: 21706961 Total methylated C's in CHG context: 1360393 Total methylated C's in CHH context: 5406472 Total methylated C's in Unknown context: 69229 Total unmethylated C's in CpG context: 168787788 Total unmethylated C's in CHG context: 204621341 Total unmethylated C's in CHH context: 810474999 Total unmethylated C's in Unknown context: 3198923 C methylated in CpG context: 11.4% C methylated in CHG context: 0.7% C methylated in CHH context: 0.7% C methylated in unknown context (CN or CHN): 2.1% Bismark completed in 0d 2h 49m 46s ==================== Bismark run complete ====================