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/1A8_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/1A8_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/1A8_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/1A8_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1A8_R1_001.fastp-trim.fq.gz to 1A8_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1A8_R1_001.fastp-trim.fq.gz (53054030 sequences in total) Writing a G -> A converted version of the input file 1A8_R2_001.fastp-trim.fq.gz to 1A8_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1A8_R2_001.fastp-trim.fq.gz (53054030 sequences in total) Input files are 1A8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A8_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 1A8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A8_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: LH00526:197:22KFKVLT4:3:1101:51674:1056_1:N:0:TACTCCAG+CCTATACC/1 77 * 0 0 * * 0 0 TAAAGGTTTTGTTTTATAGTAGGGGGAGTTGGAAGATTTGAAAGAGGTAATTTTGGTTTTTTTAATATTTATTTTTAATTAAAAAATATTATTAATGGTGAAGTATAGTTTTTTAAGGTAAATAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII9IIIIIIIIIII9I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:51674:1056_2:N:0:TACTCCAG+CCTATACC/2 141 * 0 0 * * 0 0 AAACTATACTTCACCATTAATAATATTTTTTAATTAAAAATAAATATTAAAAAAACCAAAATTACCTCTTTCAAATCTTCCAACTCCCCCTACTATAAAACAAAACCTTTAACTATCCCACCACA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII9I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1A8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A8_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: LH00526:197:22KFKVLT4:3:1101:51674:1056_1:N:0:TACTCCAG+CCTATACC/1 77 * 0 0 * * 0 0 TAAAGGTTTTGTTTTATAGTAGGGGGAGTTGGAAGATTTGAAAGAGGTAATTTTGGTTTTTTTAATATTTATTTTTAATTAAAAAATATTATTAATGGTGAAGTATAGTTTTTTAAGGTAAATAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII9IIIIIIIIIII9I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:51674:1056_2:N:0:TACTCCAG+CCTATACC/2 141 * 0 0 * * 0 0 AAACTATACTTCACCATTAATAATATTTTTTAATTAAAAATAAATATTAAAAAAACCAAAATTACCTCTTTCAAATCTTCCAACTCCCCCTACTATAAAACAAAACCTTTAACTATCCCACCACA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII9I-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP >>> Writing bisulfite mapping results to 1A8_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/1A8_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/1A8_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1102:43055:12164_1:N:0:TACTCCAG+CCTATACC ptg000066l 66347 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1135:2190:22306_1:N:0:TACTCCAG+CCTATACC ptg000090l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1135:2214:22320_1:N:0:TACTCCAG+CCTATACC ptg000090l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1150:33377:3844_1:N:0:TACTCCAG+CCTATACC ptg000012l 2 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1202:27308:28693_1:N:0:TACTCCAG+CCTATACC ptg000018l 3 Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1312:33790:2373_1:N:0:TACTCCAG+CCTATACC ptg000007l 12295855 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1330:26183:2149_1:N:0:TACTCCAG+CCTATACC ptg000018l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1341:30399:8438_1:N:0:TACTCCAG+CCTATACC ptg000149l 81053 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1479:24646:28063_1:N:0:TACTCCAG+CCTATACC ptg000015l 14997092 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2134:26491:17809_1:N:0:TACTCCAG+CCTATACC ptg000066l 66375 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 LH00526:197:22KFKVLT4:3:2339:35570:24897_1:N:0:TACTCCAG+CCTATACC ptg000066l 66412 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2364:27510:11786_1:N:0:TACTCCAG+CCTATACC ptg000102l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2386:41963:1672_1:N:0:TACTCCAG+CCTATACC ptg000077l 42461 Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2453:17063:6505_1:N:0:TACTCCAG+CCTATACC ptg000139l 18384 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2465:2740:28245_1:N:0:TACTCCAG+CCTATACC ptg000024l 1 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1120:45086:8396_1:N:0:TACTCCAG+CCTATACC ptg000066l 66408 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1145:51342:4656_1:N:0:TACTCCAG+CCTATACC ptg000024l 1 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1238:48898:8102_1:N:0:TACTCCAG+CCTATACC ptg000066l 66349 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1241:10953:5567_1:N:0:TACTCCAG+CCTATACC ptg000066l 66346 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1241:10945:5581_1:N:0:TACTCCAG+CCTATACC ptg000066l 66352 Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1269:19669:3956_1:N:0:TACTCCAG+CCTATACC ptg000154l 1 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1351:26814:27195_1:N:0:TACTCCAG+CCTATACC ptg000116l 48475 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1363:19127:10581_1:N:0:TACTCCAG+CCTATACC ptg000030l 2 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1369:29703:20093_1:N:0:TACTCCAG+CCTATACC ptg000033l 2625624 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1404:25066:5202_1:N:0:TACTCCAG+CCTATACC ptg000126l 1 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 LH00526:197:22KFKVLT4:4:2111:39535:22516_1:N:0:TACTCCAG+CCTATACC ptg000066l 66376 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2122:6252:1602_1:N:0:TACTCCAG+CCTATACC ptg000066l 66394 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2289:18188:20807_1:N:0:TACTCCAG+CCTATACC ptg000066l 66349 Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2368:29744:8872_1:N:0:TACTCCAG+CCTATACC ptg000007l 12295855 Processed 50000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2411:15024:19812_1:N:0:TACTCCAG+CCTATACC ptg000018l 2 Processed 51000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2436:28740:5062_1:N:0:TACTCCAG+CCTATACC ptg000154l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2444:30763:12879_1:N:0:TACTCCAG+CCTATACC ptg000083l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2453:6292:25514_1:N:0:TACTCCAG+CCTATACC ptg000066l 66381 Processed 52000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2474:20162:29548_1:N:0:TACTCCAG+CCTATACC ptg000027l 1 Processed 53000000 sequence pairs so far 53054030 reads; of these: 53054030 (100.00%) were paired; of these: 19360044 (36.49%) aligned concordantly 0 times 8754782 (16.50%) aligned concordantly exactly 1 time 24939204 (47.01%) aligned concordantly >1 times 63.51% overall alignment rate 53054030 reads; of these: 53054030 (100.00%) were paired; of these: 19356517 (36.48%) aligned concordantly 0 times 8760209 (16.51%) aligned concordantly exactly 1 time 24937304 (47.00%) aligned concordantly >1 times 63.52% overall alignment rate Processed 53054030 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 212216120. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 212216120. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 212216120. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 212216120. Successfully deleted the temporary files 1A8_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A8_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 53054030 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1246714180 Total methylated C's in CpG context: 15155779 Total methylated C's in CHG context: 2804361 Total methylated C's in CHH context: 10567362 Total methylated C's in Unknown context: 98979 Total unmethylated C's in CpG context: 204361694 Total unmethylated C's in CHG context: 213719500 Total unmethylated C's in CHH context: 800105484 Total unmethylated C's in Unknown context: 3426210 C methylated in CpG context: 6.9% C methylated in CHG context: 1.3% C methylated in CHH context: 1.3% C methylated in unknown context (CN or CHN): 2.8% Bismark completed in 0d 3h 6m 24s ==================== Bismark run complete ====================