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/1G5_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/1G5_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/1G5_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/1G5_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1G5_R1_001.fastp-trim.fq.gz to 1G5_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1G5_R1_001.fastp-trim.fq.gz (42675276 sequences in total) Writing a G -> A converted version of the input file 1G5_R2_001.fastp-trim.fq.gz to 1G5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1G5_R2_001.fastp-trim.fq.gz (42675276 sequences in total) Input files are 1G5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1G5_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 1G5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1G5_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: LH00526:197:22KFKVLT4:3:1101:39414:1070_1:N:0:CAACTCCA+ACGGATTC/1 99 ptg000017l_CT_converted 1723 6 125M = 1729 131 ATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTTAATTTTAAATTTTAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIII9III9IIIIIIIIII99IIIII9II9IIIIIIIIIIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:104A20 YS:i:-6 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:39414:1070_2:N:0:CAACTCCA+ACGGATTC/2 147 ptg000017l_CT_converted 1729 6 125M = 1723 -131 ATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTAATTTTTAATTTTAAATTTTAATTTTAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-16 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:98A26 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1G5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1G5_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: LH00526:197:22KFKVLT4:3:1101:39414:1070_1:N:0:CAACTCCA+ACGGATTC/1 83 ptg000010l_GA_converted 2424136 6 125M = 2424130 -131 ATTAAAATTTAAAATTAAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAAT IIIIIIIIIIII9II9IIIII99IIIIIIIIII9III9IIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:9A115 YS:i:-12 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:39414:1070_2:N:0:CAACTCCA+ACGGATTC/2 163 ptg000010l_GA_converted 2424130 6 125M = 2424136 131 ATTAAAATTAAAATTTAAAATTAAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTTAAAATTAAAATTAAAATTAAAATTAAAATTAAAATTAAAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-12 XS:i:-14 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:2C12A109 YS:i:-6 YT:Z:CP >>> Writing bisulfite mapping results to 1G5_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/1G5_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/1G5_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1132:4552:6393_1:N:0:CAACTCCA+ACGGATTC ptg000009l 19096022 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1132:4544:6407_1:N:0:CAACTCCA+ACGGATTC ptg000009l 19096022 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1134:46778:1602_1:N:0:CAACTCCA+ACGGATTC ptg000017l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1135:42983:10189_1:N:0:CAACTCCA+ACGGATTC ptg000006l 1 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1323:35093:20093_1:N:0:CAACTCCA+ACGGATTC ptg000066l 66351 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1365:22873:27096_1:N:0:CAACTCCA+ACGGATTC ptg000009l 19095996 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1379:29962:2947_1:N:0:CAACTCCA+ACGGATTC ptg000009l 19095996 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 Processed 14000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:3:2322:4366:21844_1:N:0:CAACTCCA+ACGGATTC ptg000066l 66345 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2412:48655:12388_1:N:0:CAACTCCA+ACGGATTC ptg000066l 66351 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2471:17087:17585_1:N:0:CAACTCCA+ACGGATTC ptg000006l 3 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:1247:10436:25626_1:N:0:CAACTCCA+ACGGATTC ptg000066l 66348 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1339:35165:15764_1:N:0:CAACTCCA+ACGGATTC ptg000015l 14997092 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1341:9934:20079_1:N:0:CAACTCCA+ACGGATTC ptg000024l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1361:48817:10791_1:N:0:CAACTCCA+ACGGATTC ptg000024l 1 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 LH00526:197:22KFKVLT4:4:1454:41437:7822_1:N:0:CAACTCCA+ACGGATTC ptg000018l 2 Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1497:36039:29660_1:N:0:CAACTCCA+ACGGATTC ptg000027l 1 Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2205:4116:4824_1:N:0:CAACTCCA+ACGGATTC ptg000033l 2625610 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2230:48097:11394_1:N:0:CAACTCCA+ACGGATTC ptg000025l 1 Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2337:33620:15050_1:N:0:CAACTCCA+ACGGATTC ptg000092l 1 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2364:39697:20499_1:N:0:CAACTCCA+ACGGATTC ptg000066l 66380 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2370:49950:13285_1:N:0:CAACTCCA+ACGGATTC ptg000095l 2 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2395:15760:2205_1:N:0:CAACTCCA+ACGGATTC ptg000004l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2401:31160:4292_1:N:0:CAACTCCA+ACGGATTC ptg000024l 1 Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far 42675276 reads; of these: 42675276 (100.00%) were paired; of these: 17264420 (40.46%) aligned concordantly 0 times 7249852 (16.99%) aligned concordantly exactly 1 time 18161004 (42.56%) aligned concordantly >1 times 59.54% overall alignment rate 42675276 reads; of these: 42675276 (100.00%) were paired; of these: 17258879 (40.44%) aligned concordantly 0 times 7252045 (16.99%) aligned concordantly exactly 1 time 18164352 (42.56%) aligned concordantly >1 times 59.56% overall alignment rate Processed 42675276 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 170701104. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 170701104. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 170701104. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 170701104. Successfully deleted the temporary files 1G5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1G5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 42675276 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1068406519 Total methylated C's in CpG context: 14305475 Total methylated C's in CHG context: 1227593 Total methylated C's in CHH context: 4615374 Total methylated C's in Unknown context: 60523 Total unmethylated C's in CpG context: 169756139 Total unmethylated C's in CHG context: 187703187 Total unmethylated C's in CHH context: 690798751 Total unmethylated C's in Unknown context: 2652126 C methylated in CpG context: 7.8% C methylated in CHG context: 0.6% C methylated in CHH context: 0.7% C methylated in unknown context (CN or CHN): 2.2% Bismark completed in 0d 2h 33m 10s ==================== Bismark run complete ====================