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/1C8_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/1C8_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/1C8_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/1C8_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1C8_R1_001.fastp-trim.fq.gz to 1C8_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1C8_R1_001.fastp-trim.fq.gz (44096888 sequences in total) Writing a G -> A converted version of the input file 1C8_R2_001.fastp-trim.fq.gz to 1C8_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1C8_R2_001.fastp-trim.fq.gz (44096888 sequences in total) Input files are 1C8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C8_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 1C8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C8_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: LH00526:197:22KFKVLT4:3:1101:50185:1056_1:N:0:ATCTGACC+AAGTCGAG/1 77 * 0 0 * * 0 0 TGTTTGGTTGGTGTGAATAGGGGGGTTATATATTTATGGTTGTTATATAGATAATATATTGGTTTTTATATTTTGTTGATAAAAGTTTTTTAAGTTTGTTTTTGAATTAGTTTTTTTAAAATTGG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III9IIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:50185:1056_2:N:0:ATCTGACC+AAGTCGAG/2 141 * 0 0 * * 0 0 CCACTTCTAAAATTAAAAAAAAATCAAAATAACATATATATTTTTAAAAATTCAAAAATAAATATATAATAAACACACTAACCTTTAAATTATTATTAAAAAATTTCTTTTTATAAATTTTTTCA IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1C8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C8_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: LH00526:197:22KFKVLT4:3:1101:50185:1056_1:N:0:ATCTGACC+AAGTCGAG/1 83 ptg000025l_GA_converted 18483548 42 125M = 18483325 -348 CCAATTTTAAAAAAACTAATTCAAAAACAAACTTAAAAAACTTTTATCAACAAAATATAAAAACCAATATATTATCTATATAACAACCATAAATATATAACCCCCCTATTCACACCAACCAAACA IIIIIIIIIIIIIIII9III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:50185:1056_2:N:0:ATCTGACC+AAGTCGAG/2 163 ptg000025l_GA_converted 18483325 42 125M = 18483548 348 CCACTTCTAAAATTAAAAAAAAATCAAAATAACATATATATTTTTAAAAATTCAAAAATAAATATATAATAAACACACTAACCTTTAAATTATTATTAAAAAATTTCTTTTTATAAATTTTTTCA IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to 1C8_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/1C8_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/1C8_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1145:3298:23777_1:N:0:ATCTGACC+AAGTCGAG ptg000066l 66390 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1168:41275:20429_1:N:0:ATCTGACC+AAGTCGAG ptg000065l 2 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1242:17702:8200_1:N:0:ATCTGACC+AAGTCGAG ptg000007l 12295855 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1301:44293:22124_1:N:0:ATCTGACC+AAGTCGAG ptg000097l 2 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1332:27340:10091_1:N:0:ATCTGACC+AAGTCGAG ptg000039l 1139416 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1335:47725:5034_1:N:0:ATCTGACC+AAGTCGAG ptg000129l 1 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1361:30974:24281_1:N:0:ATCTGACC+AAGTCGAG ptg000040l 507174 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1385:28967:24505_1:N:0:ATCTGACC+AAGTCGAG ptg000083l 2 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1410:25285:13369_1:N:0:ATCTGACC+AAGTCGAG ptg000125l 1 Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1442:20543:23791_1:N:0:ATCTGACC+AAGTCGAG ptg000092l 1 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1479:26054:12598_1:N:0:ATCTGACC+AAGTCGAG ptg000048l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1479:4172:29016_1:N:0:ATCTGACC+AAGTCGAG ptg000117l 1 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2163:39090:3788_1:N:0:ATCTGACC+AAGTCGAG ptg000125l 18882 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2179:21918:16899_1:N:0:ATCTGACC+AAGTCGAG ptg000102l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2191:8340:26676_1:N:0:ATCTGACC+AAGTCGAG ptg000066l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2191:8356:26676_1:N:0:ATCTGACC+AAGTCGAG ptg000066l 1 Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2333:12321:27769_1:N:0:ATCTGACC+AAGTCGAG ptg000066l 66347 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2350:21619:15288_1:N:0:ATCTGACC+AAGTCGAG ptg000047l 1 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2383:40150:18482_1:N:0:ATCTGACC+AAGTCGAG ptg000101l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2388:9133:23426_1:N:0:ATCTGACC+AAGTCGAG ptg000160l 1 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2447:9003:4740_1:N:0:ATCTGACC+AAGTCGAG ptg000127l 1 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2470:44374:11030_1:N:0:ATCTGACC+AAGTCGAG ptg000077l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2482:7004:22516_1:N:0:ATCTGACC+AAGTCGAG ptg000096l 1 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1121:8137:2457_1:N:0:ATCTGACC+AAGTCGAG ptg000137l 40572 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1155:27769:24701_1:N:0:ATCTGACC+AAGTCGAG ntLink_4 304028 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1168:1324:18482_1:N:0:ATCTGACC+AAGTCGAG ptg000106l 40385 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1180:19604:2639_1:N:0:ATCTGACC+AAGTCGAG ptg000101l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1190:42351:22040_1:N:0:ATCTGACC+AAGTCGAG ptg000039l 1 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1218:39940:27671_1:N:0:ATCTGACC+AAGTCGAG ptg000160l 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1251:21142:11408_1:N:0:ATCTGACC+AAGTCGAG ptg000066l 1 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1287:28336:14644_1:N:0:ATCTGACC+AAGTCGAG ptg000174l 27225 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1330:45734:21872_1:N:0:ATCTGACC+AAGTCGAG ptg000092l 28991 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1356:16197:26102_1:N:0:ATCTGACC+AAGTCGAG ptg000025l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1378:6851:27573_1:N:0:ATCTGACC+AAGTCGAG ptg000046l 1 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1395:9788:27335_1:N:0:ATCTGACC+AAGTCGAG ptg000146l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1395:9796:27349_1:N:0:ATCTGACC+AAGTCGAG ptg000146l 1 Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1434:38402:11618_1:N:0:ATCTGACC+AAGTCGAG ptg000075l 54159 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:2231:45880:16016_1:N:0:ATCTGACC+AAGTCGAG ptg000093l 36770 Processed 38000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:2406:4520:23202_1:N:0:ATCTGACC+AAGTCGAG ptg000039l 1139399 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2459:34097:13943_1:N:0:ATCTGACC+AAGTCGAG ptg000120l 31326 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2490:31168:6771_1:N:0:ATCTGACC+AAGTCGAG ptg000121l 50749 Processed 44000000 sequence pairs so far 44096888 reads; of these: 44096888 (100.00%) were paired; of these: 16529992 (37.49%) aligned concordantly 0 times 9117454 (20.68%) aligned concordantly exactly 1 time 18449442 (41.84%) aligned concordantly >1 times 62.51% overall alignment rate 44096888 reads; of these: 44096888 (100.00%) were paired; of these: 16514399 (37.45%) aligned concordantly 0 times 9112763 (20.67%) aligned concordantly exactly 1 time 18469726 (41.88%) aligned concordantly >1 times 62.55% overall alignment rate Processed 44096888 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 176387552. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 176387552. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 176387552. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 176387552. Successfully deleted the temporary files 1C8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1C8_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 44096888 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1316936437 Total methylated C's in CpG context: 20815258 Total methylated C's in CHG context: 1351061 Total methylated C's in CHH context: 5428645 Total methylated C's in Unknown context: 67446 Total unmethylated C's in CpG context: 186526616 Total unmethylated C's in CHG context: 222088463 Total unmethylated C's in CHH context: 880726394 Total unmethylated C's in Unknown context: 3332726 C methylated in CpG context: 10.0% 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 3h 2m 55s ==================== Bismark run complete ====================