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/1F9_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/1F9_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/1F9_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/1F9_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1F9_R1_001.fastp-trim.fq.gz to 1F9_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1F9_R1_001.fastp-trim.fq.gz (37898911 sequences in total) Writing a G -> A converted version of the input file 1F9_R2_001.fastp-trim.fq.gz to 1F9_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1F9_R2_001.fastp-trim.fq.gz (37898911 sequences in total) Input files are 1F9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F9_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 1F9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F9_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: LH00526:197:22KFKVLT4:3:1101:50201:1056_1:N:0:CTCGAACA+GGAATTGC/1 77 * 0 0 * * 0 0 TTGGTGTTTATGTATATGTGAGAAGAAATAGGTTGTTGTTTAAGTATATATTATGAAGATAATAGGTTATTTTTGAATTATTTTTGGTTTTTTTGTTAAATTTTGTTAAATTTAATATTTATATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIII-IIIII9IIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII-IIIIII9IIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:50201:1056_2:N:0:CTCGAACA+GGAATTGC/2 141 * 0 0 * * 0 0 AAATTATAAATTAAATAACCATATTACAAAAAACAAAAAATCATCAAACTACTACACCATTCATTAACCAAAAAACTATAAAAAATAAATTCATTCAAACTTTAAATCAAAATAAACTTATTATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIII-IIIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1F9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F9_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: LH00526:197:22KFKVLT4:3:1101:50201:1056_1:N:0:CTCGAACA+GGAATTGC/1 77 * 0 0 * * 0 0 TTGGTGTTTATGTATATGTGAGAAGAAATAGGTTGTTGTTTAAGTATATATTATGAAGATAATAGGTTATTTTTGAATTATTTTTGGTTTTTTTGTTAAATTTTGTTAAATTTAATATTTATATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIII-IIIII9IIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII-IIIIII9IIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:50201:1056_2:N:0:CTCGAACA+GGAATTGC/2 141 * 0 0 * * 0 0 AAATTATAAATTAAATAACCATATTACAAAAAACAAAAAATCATCAAACTACTACACCATTCATTAACCAAAAAACTATAAAAAATAAATTCATTCAAACTTTAAATCAAAATAAACTTATTATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIII-IIIIIII YT:Z:UP >>> Writing bisulfite mapping results to 1F9_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/1F9_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/1F9_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1149:32430:22824_1:N:0:CTCGAACA+GGAATTGC ptg000121l 50744 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1224:4876:17767_1:N:0:CTCGAACA+GGAATTGC ptg000022l 9976765 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1248:19062:19126_1:N:0:CTCGAACA+GGAATTGC ptg000048l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1249:31046:27797_1:N:0:CTCGAACA+GGAATTGC ptg000113l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1249:31022:27811_1:N:0:CTCGAACA+GGAATTGC ptg000113l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1272:47474:17403_1:N:0:CTCGAACA+GGAATTGC ptg000066l 66392 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 LH00526:197:22KFKVLT4:3:1407:30885:23623_1:N:0:CTCGAACA+GGAATTGC ptg000004l 15492347 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1414:5499:14868_1:N:0:CTCGAACA+GGAATTGC ptg000054l 36041 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1416:40482:3732_1:N:0:CTCGAACA+GGAATTGC ptg000114l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1432:40288:2163_1:N:0:CTCGAACA+GGAATTGC ptg000108l 75357 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1437:42659:21367_1:N:0:CTCGAACA+GGAATTGC ptg000159l 21841 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1461:25147:21704_1:N:0:CTCGAACA+GGAATTGC ptg000066l 66380 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1468:45006:13439_1:N:0:CTCGAACA+GGAATTGC ptg000082l 1 Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2116:48178:29548_1:N:0:CTCGAACA+GGAATTGC ptg000117l 1 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2216:11075:21689_1:N:0:CTCGAACA+GGAATTGC ptg000092l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2243:46033:10707_1:N:0:CTCGAACA+GGAATTGC ptg000007l 12295855 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2266:37229:27937_1:N:0:CTCGAACA+GGAATTGC ptg000117l 30003 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2273:31144:21858_1:N:0:CTCGAACA+GGAATTGC ptg000066l 66411 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2317:44520:28231_1:N:0:CTCGAACA+GGAATTGC ptg000048l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2323:35732:14055_1:N:0:CTCGAACA+GGAATTGC ptg000089l 1 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2485:49327:21647_1:N:0:CTCGAACA+GGAATTGC ptg000066l 1 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1111:2910:24897_1:N:0:CTCGAACA+GGAATTGC ptg000113l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1111:2926:24897_1:N:0:CTCGAACA+GGAATTGC ptg000113l 2 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1153:10500:12598_1:N:0:CTCGAACA+GGAATTGC ptg000046l 2 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1213:42821:23384_1:N:0:CTCGAACA+GGAATTGC ptg000052l 52620 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1238:44172:14125_1:N:0:CTCGAACA+GGAATTGC ptg000007l 12295855 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1239:11010:22278_1:N:0:CTCGAACA+GGAATTGC ptg000065l 49680 Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1346:4010:19967_1:N:0:CTCGAACA+GGAATTGC ptg000102l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1350:17816:20471_1:N:0:CTCGAACA+GGAATTGC ptg000117l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1350:17808:20485_1:N:0:CTCGAACA+GGAATTGC ptg000117l 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1363:32455:2835_1:N:0:CTCGAACA+GGAATTGC ptg000096l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1388:4706:21984_1:N:0:CTCGAACA+GGAATTGC ptg000048l 2 Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:2427:16958:5595_1:N:0:CTCGAACA+GGAATTGC ptg000077l 1 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2473:30342:25177_1:N:0:CTCGAACA+GGAATTGC ptg000121l 50718 37898911 reads; of these: 37898911 (100.00%) were paired; of these: 14510708 (38.29%) aligned concordantly 0 times 7989235 (21.08%) aligned concordantly exactly 1 time 15398968 (40.63%) aligned concordantly >1 times 61.71% overall alignment rate 37898911 reads; of these: 37898911 (100.00%) were paired; of these: 14494338 (38.24%) aligned concordantly 0 times 7994720 (21.09%) aligned concordantly exactly 1 time 15409853 (40.66%) aligned concordantly >1 times 61.76% overall alignment rate Processed 37898911 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 151595644. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 151595644. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 151595644. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 151595644. Successfully deleted the temporary files 1F9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F9_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 37898911 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1150729048 Total methylated C's in CpG context: 17982433 Total methylated C's in CHG context: 1130568 Total methylated C's in CHH context: 4558452 Total methylated C's in Unknown context: 56956 Total unmethylated C's in CpG context: 160984889 Total unmethylated C's in CHG context: 193669130 Total unmethylated C's in CHH context: 772403576 Total unmethylated C's in Unknown context: 2924699 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): 1.9% Bismark completed in 0d 2h 42m 5s ==================== Bismark run complete ====================