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/1F1_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/1F1_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/1F1_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/1F1_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1F1_R1_001.fastp-trim.fq.gz to 1F1_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1F1_R1_001.fastp-trim.fq.gz (46779251 sequences in total) Writing a G -> A converted version of the input file 1F1_R2_001.fastp-trim.fq.gz to 1F1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1F1_R2_001.fastp-trim.fq.gz (46779251 sequences in total) Input files are 1F1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F1_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 1F1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F1_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: LH00526:197:22KFKVLT4:3:1101:45192:1070_1:N:0:TATGGCAC+TCTCGCAA/1 99 ptg000020l_CT_converted 15097613 12 125M = 15097647 159 AGTAAATTTTATTAAGTGATTTTGTTATATAATATATTAAATATGATATTTTGTTTTTGTTTTTATGAAAATTTGTGAATTTTGTTTAAAGGAGGGTTTTTGTTTAGATATTTTTAGAAGGGGAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-36 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:18T106 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:45192:1070_2:N:0:TATGGCAC+TCTCGCAA/2 147 ptg000020l_CT_converted 15097647 12 125M = 15097613 -159 TATTAAATATGATATTTTGTTTTTGTTTTTATGAAAATTTGTGAATTTTGTTTAAAGGAGGGTTTTTGTTTAGATATTTTTAGAAGGGGAATGAAGGTTTTAGTTTGTTGTGATTTATTAAGATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-9IIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:-18 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1F1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F1_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: LH00526:197:22KFKVLT4:3:1101:45192:1070_1:N:0:TATGGCAC+TCTCGCAA/1 83 ptg000020l_GA_converted 15340804 12 125M = 15340770 -159 TTCCCCTTCTAAAAATATCTAAACAAAAACCCTCCTTTAAACAAAATTCACAAATTTTCATAAAAACAAAAACAAAATATCATATTTAATATATTATATAACAAAATCACTTAATAAAATTTACT IIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-12 XS:i:-26 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:72A33A18 YS:i:-6 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:45192:1070_2:N:0:TATGGCAC+TCTCGCAA/2 163 ptg000020l_GA_converted 15340770 12 125M = 15340804 159 AATCTTAATAAATCACAACAAACTAAAACCTTCATTCCCCTTCTAAAAATATCTAAACAAAAACCCTCCTTTAAACAAAATTCACAAATTTTCATAAAAACAAAAACAAAATATCATATTTAATA IIIIIIIIIIIIIIIIIIIIIIIIIII9-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-26 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:106A18 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to 1F1_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/1F1_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/1F1_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1105:17087:11198_1:N:0:TATGGCAC+TCTCGCAA ptg000066l 66350 Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1215:25002:3914_1:N:0:TATGGCAC+TCTCGCAA ptg000066l 66352 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1225:21457:2177_1:N:0:TATGGCAC+TCTCGCAA ptg000066l 66347 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1311:24087:11268_1:N:0:TATGGCAC+TCTCGCAA ptg000066l 66357 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1325:20947:4656_1:N:0:TATGGCAC+TCTCGCAA ptg000039l 1 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1369:49974:15764_1:N:0:TATGGCAC+TCTCGCAA ptg000029c 1802584 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1409:44552:12010_1:N:0:TATGGCAC+TCTCGCAA ptg000066l 66414 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1455:24467:2933_1:N:0:TATGGCAC+TCTCGCAA ptg000155l 1 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1469:19717:24827_1:N:0:TATGGCAC+TCTCGCAA ptg000185l 34487 Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2139:10460:15470_1:N:0:TATGGCAC+TCTCGCAA ptg000024l 2 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 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2395:18940:17151_1:N:0:TATGGCAC+TCTCGCAA ptg000126l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2412:41494:13495_1:N:0:TATGGCAC+TCTCGCAA ptg000026l 2 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2437:41558:6351_1:N:0:TATGGCAC+TCTCGCAA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2437:41574:6351_1:N:0:TATGGCAC+TCTCGCAA ptg000039l 1 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2488:30642:6449_1:N:0:TATGGCAC+TCTCGCAA ptg000033l 2625597 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1110:42165:4236_1:N:0:TATGGCAC+TCTCGCAA ptg000095l 2 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1155:2578:13733_1:N:0:TATGGCAC+TCTCGCAA ptg000092l 2 Processed 26000000 sequence pairs so far 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 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1365:7522:8760_1:N:0:TATGGCAC+TCTCGCAA ptg000006l 2 Processed 32000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:1470:5766:16703_1:N:0:TATGGCAC+TCTCGCAA ptg000149l 81044 Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far 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 46779251 reads; of these: 46779251 (100.00%) were paired; of these: 16367665 (34.99%) aligned concordantly 0 times 9893880 (21.15%) aligned concordantly exactly 1 time 20517706 (43.86%) aligned concordantly >1 times 65.01% overall alignment rate 46779251 reads; of these: 46779251 (100.00%) were paired; of these: 16375356 (35.01%) aligned concordantly 0 times 9898994 (21.16%) aligned concordantly exactly 1 time 20504901 (43.83%) aligned concordantly >1 times 64.99% overall alignment rate Processed 46779251 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 187117004. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 187117004. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 187117004. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 187117004. Successfully deleted the temporary files 1F1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 46779251 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1406805423 Total methylated C's in CpG context: 27867822 Total methylated C's in CHG context: 1846504 Total methylated C's in CHH context: 7224012 Total methylated C's in Unknown context: 84513 Total unmethylated C's in CpG context: 198064823 Total unmethylated C's in CHG context: 242265849 Total unmethylated C's in CHH context: 929536413 Total unmethylated C's in Unknown context: 3545008 C methylated in CpG context: 12.3% 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 24m 35s ==================== Bismark run complete ====================