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/1B1_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/1B1_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/1B1_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/1B1_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1B1_R1_001.fastp-trim.fq.gz to 1B1_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1B1_R1_001.fastp-trim.fq.gz (40296833 sequences in total) Writing a G -> A converted version of the input file 1B1_R2_001.fastp-trim.fq.gz to 1B1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1B1_R2_001.fastp-trim.fq.gz (40296833 sequences in total) Input files are 1B1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B1_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 1B1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B1_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: LH00526:197:22KFKVLT4:3:1101:43023:1070_1:N:0:TTGCGAGA+GTGCCATA/1 99 ptg000023l_CT_converted 32070714 6 125M = 32070738 149 TGATTTAAAGTGAGGATTTTGTATGGGTTATAGTTTTTGTATGATTGTTGATTTTTAAAATGTTTTTATTTTTTGAATATTTGGTTTTTTTTTGAGTGGTTTTTTGTATAGAATAATTGTAAATG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIII--IIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-18 XS:i:-30 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:62G1G8G51 YS:i:-42 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:43023:1070_2:N:0:TTGCGAGA+GTGCCATA/2 147 ptg000023l_CT_converted 32070738 6 125M = 32070714 -149 GGGTTATAGTTTTTGTATGATTGTTGATTTTTAAAATGTTTTTATTTTTTGAATATTTGGTTTTTTTTTGAGTGGTTTTTTGTATAGAATAATTGTAAATGATGTGTTAAATGGATTTTGATATA IIIIIIIIIIIIIIIII9I-IIIIIIIIIIIIIIIIIIIIIIIIII-I9IIII-IIIIIIIIII9II-III9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-42 XS:i:-48 XN:i:0 XM:i:7 XO:i:0 XG:i:0 NM:i:7 MD:Z:38G1G8G53T3A0G15G0 YS:i:-18 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1B1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B1_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: LH00526:197:22KFKVLT4:3:1101:43023:1070_1:N:0:TTGCGAGA+GTGCCATA/1 83 ptg000004l_GA_converted 1636678 1 125M = 1636654 -149 CATTTACAATTATTCTATACAAAAAACCACTCAAAAAAAAACCAAATATTCAAAAAATAAAAACATTTTAAAAATCAACAATCATACAAAAACTATAACCCATACAAAATCCTCACTTTAAATCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIII--IIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:39C85 YS:i:-30 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:43023:1070_2:N:0:TTGCGAGA+GTGCCATA/2 163 ptg000004l_GA_converted 1636654 1 125M = 1636678 149 TATATCAAAATCCATTTAACACATCATTTACAATTATTCTATACAAAAAACCACTCAAAAAAAAACCAAATATTCAAAAAATAAAAACATTTTAAAAATCAACAATCATACAAAAACTATAACCC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III-II9IIIIIIIIII-IIII9I-IIIIIIIIIIIIIIIIIIIIIIIIII-I9IIIIIIIIIIIIIIIII AS:i:-30 XS:i:-30 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:0C15C0T3A41C61 YS:i:-6 YT:Z:CP >>> Writing bisulfite mapping results to 1B1_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/1B1_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/1B1_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1131:43889:23300_1:N:0:TTGCGAGA+GTGCCATA ptg000025l 21443081 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1143:4213:18047_1:N:0:TTGCGAGA+GTGCCATA ntLink_7 182831 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1143:4221:18061_1:N:0:TTGCGAGA+GTGCCATA ntLink_7 182831 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1208:52013:15876_1:N:0:TTGCGAGA+GTGCCATA ptg000116l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1213:22752:10609_1:N:0:TTGCGAGA+GTGCCATA ptg000121l 50742 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1236:39414:20261_1:N:0:TTGCGAGA+GTGCCATA ptg000008l 2 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 LH00526:197:22KFKVLT4:3:1412:5103:18075_1:N:0:TTGCGAGA+GTGCCATA ptg000118l 1 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2124:42837:16885_1:N:0:TTGCGAGA+GTGCCATA ptg000002l 1 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2130:45540:29352_1:N:0:TTGCGAGA+GTGCCATA ptg000113l 1 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2175:2991:23076_1:N:0:TTGCGAGA+GTGCCATA ptg000055l 50545 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2205:25584:22152_1:N:0:TTGCGAGA+GTGCCATA ptg000017l 3 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2260:48526:1378_1:N:0:TTGCGAGA+GTGCCATA ptg000144l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2266:12386:5244_1:N:0:TTGCGAGA+GTGCCATA ptg000050l 26324 Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2340:47368:7023_1:N:0:TTGCGAGA+GTGCCATA ptg000160l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2340:47344:7037_1:N:0:TTGCGAGA+GTGCCATA ptg000160l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2352:30448:9167_1:N:0:TTGCGAGA+GTGCCATA ptg000095l 1 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2364:4925:27153_1:N:0:TTGCGAGA+GTGCCATA ptg000012l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2386:23432:9461_1:N:0:TTGCGAGA+GTGCCATA ptg000066l 66412 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2391:13349:2317_1:N:0:TTGCGAGA+GTGCCATA ptg000020l 17861297 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2415:6503:24197_1:N:0:TTGCGAGA+GTGCCATA ptg000007l 12295912 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2425:31524:5679_1:N:0:TTGCGAGA+GTGCCATA ptg000096l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2432:40158:26508_1:N:0:TTGCGAGA+GTGCCATA ptg000048l 1 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2469:12766:5511_1:N:0:TTGCGAGA+GTGCCATA ptg000055l 50534 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2472:13996:12879_1:N:0:TTGCGAGA+GTGCCATA ptg000092l 28980 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2485:20737:15974_1:N:0:TTGCGAGA+GTGCCATA ptg000006l 3 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2497:32689:13859_1:N:0:TTGCGAGA+GTGCCATA ptg000066l 66399 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2498:18423:14966_1:N:0:TTGCGAGA+GTGCCATA ptg000015l 14997091 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1253:9610:18258_1:N:0:TTGCGAGA+GTGCCATA ptg000089l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1258:23100:18916_1:N:0:TTGCGAGA+GTGCCATA ptg000083l 2 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1331:14417:1420_1:N:0:TTGCGAGA+GTGCCATA ptg000048l 76455 Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1392:6697:23356_1:N:0:TTGCGAGA+GTGCCATA ptg000056l 59103 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1404:13859:14770_1:N:0:TTGCGAGA+GTGCCATA ptg000114l 51156 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1429:31661:1182_1:N:0:TTGCGAGA+GTGCCATA ptg000048l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1434:25285:15890_1:N:0:TTGCGAGA+GTGCCATA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1442:51026:11758_1:N:0:TTGCGAGA+GTGCCATA ptg000053l 1 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1453:23043:17669_1:N:0:TTGCGAGA+GTGCCATA ptg000174l 27225 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1466:42966:17613_1:N:0:TTGCGAGA+GTGCCATA ptg000066l 66409 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1471:21409:2877_1:N:0:TTGCGAGA+GTGCCATA ptg000051l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1477:24209:21031_1:N:0:TTGCGAGA+GTGCCATA ptg000082l 1 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2129:9133:16535_1:N:0:TTGCGAGA+GTGCCATA ptg000098l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2132:47223:25906_1:N:0:TTGCGAGA+GTGCCATA ptg000066l 66389 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2133:47482:8060_1:N:0:TTGCGAGA+GTGCCATA ptg000039l 1 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2311:26766:14027_1:N:0:TTGCGAGA+GTGCCATA ptg000124l 54830 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2350:11358:20919_1:N:0:TTGCGAGA+GTGCCATA ptg000018l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2355:18471:5328_1:N:0:TTGCGAGA+GTGCCATA ptg000066l 66358 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2366:22647:17039_1:N:0:TTGCGAGA+GTGCCATA ptg000134l 83972 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2368:29444:4516_1:N:0:TTGCGAGA+GTGCCATA ptg000118l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2378:13163:20653_1:N:0:TTGCGAGA+GTGCCATA ptg000077l 42482 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2385:7563:27068_1:N:0:TTGCGAGA+GTGCCATA ptg000155l 2 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2389:33628:24197_1:N:0:TTGCGAGA+GTGCCATA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2412:12928:25177_1:N:0:TTGCGAGA+GTGCCATA ptg000055l 50537 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2414:20041:19672_1:N:0:TTGCGAGA+GTGCCATA ptg000046l 2 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far 40296833 reads; of these: 40296833 (100.00%) were paired; of these: 15241916 (37.82%) aligned concordantly 0 times 8280486 (20.55%) aligned concordantly exactly 1 time 16774431 (41.63%) aligned concordantly >1 times 62.18% overall alignment rate 40296833 reads; of these: 40296833 (100.00%) were paired; of these: 15227803 (37.79%) aligned concordantly 0 times 8280525 (20.55%) aligned concordantly exactly 1 time 16788505 (41.66%) aligned concordantly >1 times 62.21% overall alignment rate Processed 40296833 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 161187332. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 161187332. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 161187332. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 161187332. Successfully deleted the temporary files 1B1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 40296833 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1195065801 Total methylated C's in CpG context: 22107991 Total methylated C's in CHG context: 1336144 Total methylated C's in CHH context: 5407154 Total methylated C's in Unknown context: 68018 Total unmethylated C's in CpG context: 164233733 Total unmethylated C's in CHG context: 200652764 Total unmethylated C's in CHH context: 801328015 Total unmethylated C's in Unknown context: 3147027 C methylated in CpG context: 11.9% 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 46m 6s ==================== Bismark run complete ====================