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/1B10_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/1B10_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/1B10_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/1B10_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1B10_R1_001.fastp-trim.fq.gz to 1B10_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1B10_R1_001.fastp-trim.fq.gz (56882972 sequences in total) Writing a G -> A converted version of the input file 1B10_R2_001.fastp-trim.fq.gz to 1B10_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1B10_R2_001.fastp-trim.fq.gz (56882972 sequences in total) Input files are 1B10_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B10_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 1B10_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B10_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: LH00526:197:22KFKVLT4:3:1101:51512:1056_1:N:0:CTCAGAAG+AACTTGCC/1 99 ptg000016l_CT_converted 9858894 6 125M = 9858948 179 TTTAAGAGTTTTAATGTAATTTGTTTATTGATAGAAGAAAATGTTAGATTTTTAATTTAAGGTTTTTATTATGGATAAAAAAATATAAAGTTAAATAATTGAAGGTTTTAATTTAAAGGTTGTGT III9IIIIIII9IIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-12 XS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:41A74T8 YS:i:-6 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:51512:1056_2:N:0:CTCAGAAG+AACTTGCC/2 147 ptg000016l_CT_converted 9858948 6 125M = 9858894 -179 ATTTAAGGTTTTTATTATGGATAAAAAAATATAAAGTTAAATAATTGAAGGTTTTAATTTAAAGGTTGTGTGAGATATTTATTGATGTTTTGTTGTTATGTTGTTAATAATTGTTGTGAGTTGTT IIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-12 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:62T62 YS:i:-12 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1B10_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B10_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: LH00526:197:22KFKVLT4:3:1101:51512:1056_1:N:0:CTCAGAAG+AACTTGCC/1 83 ptg000002l_GA_converted 8626720 6 42M1D83M = 8626666 -180 ACACAACCTTTAAATTAAAACCTTCAATTATTTAACTTTATATTTTTTTATCCATAATAAAAACCTTAAATTAAAAATCTAACATTTTCTTCTATCAATAAACAAATTACATTAAAACTCTTAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIII9IIIIIII9III AS:i:-32 XS:i:-24 XN:i:0 XM:i:4 XO:i:1 XG:i:1 NM:i:5 MD:Z:3A4A33^T41T28C12 YS:i:-20 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:51512:1056_2:N:0:CTCAGAAG+AACTTGCC/2 163 ptg000002l_GA_converted 8626666 6 96M1D29M = 8626720 180 AACAACTCACAACAATTATTAACAACATAACAACAAAACATCAATAAATATCTCACACAACCTTTAAATTAAAACCTTCAATTATTTAACTTTATATTTTTTTATCCATAATAAAAACCTTAAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIII AS:i:-20 XS:i:-38 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:57A4A33^T29 YS:i:-32 YT:Z:CP >>> Writing bisulfite mapping results to 1B10_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/1B10_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/1B10_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1140:45159:27237_1:N:0:CTCAGAAG+AACTTGCC ptg000039l 1 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1262:31386:20681_1:N:0:CTCAGAAG+AACTTGCC ptg000066l 66348 Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1314:38758:9237_1:N:0:CTCAGAAG+AACTTGCC ptg000105l 1 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1355:46664:28469_1:N:0:CTCAGAAG+AACTTGCC ptg000083l 1 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1376:12451:17935_1:N:0:CTCAGAAG+AACTTGCC ptg000015l 14997113 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 Processed 17000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:3:2293:17929:6799_1:N:0:CTCAGAAG+AACTTGCC ptg000149l 81044 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2351:37536:10960_1:N:0:CTCAGAAG+AACTTGCC ptg000066l 66398 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2416:42044:23496_1:N:0:CTCAGAAG+AACTTGCC ptg000004l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2416:42052:23511_1:N:0:CTCAGAAG+AACTTGCC ptg000004l 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2434:30812:15596_1:N:0:CTCAGAAG+AACTTGCC ptg000024l 3 Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1143:23990:21213_1:N:0:CTCAGAAG+AACTTGCC ptg000018l 1 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1175:12467:8466_1:N:0:CTCAGAAG+AACTTGCC ptg000009l 19096021 Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1201:33223:27363_1:N:0:CTCAGAAG+AACTTGCC ptg000024l 1 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1255:44196:19126_1:N:0:CTCAGAAG+AACTTGCC ptg000155l 2 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1277:14255:12963_1:N:0:CTCAGAAG+AACTTGCC ptg000066l 66385 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1286:20802:24351_1:N:0:CTCAGAAG+AACTTGCC ptg000066l 66382 Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1347:47118:17487_1:N:0:CTCAGAAG+AACTTGCC ptg000024l 1 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1439:18487:26284_1:N:0:CTCAGAAG+AACTTGCC ptg000066l 66351 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1472:5548:10609_1:N:0:CTCAGAAG+AACTTGCC ptg000006l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1472:5556:10623_1:N:0:CTCAGAAG+AACTTGCC ptg000006l 1 Processed 44000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2122:22809:19140_1:N:0:CTCAGAAG+AACTTGCC ptg000066l 66361 Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2135:31022:15148_1:N:0:CTCAGAAG+AACTTGCC ptg000098l 55079 Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2201:7765:18146_1:N:0:CTCAGAAG+AACTTGCC ptg000080l 40112 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2212:50290:2779_1:N:0:CTCAGAAG+AACTTGCC ptg000083l 1 Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2222:15687:6505_1:N:0:CTCAGAAG+AACTTGCC ptg000092l 28970 Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2308:46988:10735_1:N:0:CTCAGAAG+AACTTGCC ptg000025l 1 Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2380:6834:5188_1:N:0:CTCAGAAG+AACTTGCC ptg000018l 1 Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2458:19580:9321_1:N:0:CTCAGAAG+AACTTGCC ptg000012l 2 Processed 56000000 sequence pairs so far 56882972 reads; of these: 56882972 (100.00%) were paired; of these: 21162963 (37.20%) aligned concordantly 0 times 10373770 (18.24%) aligned concordantly exactly 1 time 25346239 (44.56%) aligned concordantly >1 times 62.80% overall alignment rate 56882972 reads; of these: 56882972 (100.00%) were paired; of these: 21177588 (37.23%) aligned concordantly 0 times 10370657 (18.23%) aligned concordantly exactly 1 time 25334727 (44.54%) aligned concordantly >1 times 62.77% overall alignment rate Processed 56882972 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 227531888. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 227531888. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 227531888. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 227531888. Successfully deleted the temporary files 1B10_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B10_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 56882972 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1513283689 Total methylated C's in CpG context: 22384666 Total methylated C's in CHG context: 1980271 Total methylated C's in CHH context: 7597353 Total methylated C's in Unknown context: 87453 Total unmethylated C's in CpG context: 235158927 Total unmethylated C's in CHG context: 260403770 Total unmethylated C's in CHH context: 985758702 Total unmethylated C's in Unknown context: 3673547 C methylated in CpG context: 8.7% C methylated in CHG context: 0.8% C methylated in CHH context: 0.8% C methylated in unknown context (CN or CHN): 2.3% Bismark completed in 0d 3h 38m 47s ==================== Bismark run complete ====================