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/1F7_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/1F7_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/1F7_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/1F7_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1F7_R1_001.fastp-trim.fq.gz to 1F7_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1F7_R1_001.fastp-trim.fq.gz (35878442 sequences in total) Writing a G -> A converted version of the input file 1F7_R2_001.fastp-trim.fq.gz to 1F7_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1F7_R2_001.fastp-trim.fq.gz (35878442 sequences in total) Input files are 1F7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F7_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 1F7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F7_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: LH00526:197:22KFKVLT4:3:1101:52127:1056_1:N:0:TGGAAGCA+GTACTCTC/1 99 ptg000011l_CT_converted 13540569 6 125M = 13540772 324 AGTATTGTTTAGTTTTTGAAAAAAAGTTAGAAATTTTTGATTTTTTTTTTTTTGTAAAGTTAGTAGTTTGTAAAGTGTTAGTTTTTTTGTTTTTGTTATTATAAATTATGTGAGTTTTTTTGTTT IIIIIII-II9I99II--IIIIIIII9II9III-IIIIII9I9I9IIIII-I-9I-I-I99--9-9-99--9-99-9-I-9---99999999---II--99--99-9-9999-99-99999--9- AS:i:-36 XS:i:-48 XN:i:0 XM:i:6 XO:i:0 XG:i:0 NM:i:6 MD:Z:10T5A35G18G20A19T12 YS:i:-23 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:52127:1056_2:N:0:TGGAAGCA+GTACTCTC/2 147 ptg000011l_CT_converted 13540772 6 111M4I10M = 13540569 -324 ATTGTTTTGGTAAATAAAGTATGTTTAGTAAAAGTTTATTTAAGTTTATTAAGTAATGAAATTTTATTATTTTTATGTTTTTTTAATTTAATGGTGTTTAGAAATATTGTTTTAATTAAGAGTTA IIIIIIIII9IIIIII9IIIIIII9IIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-23 XS:i:-23 XN:i:0 XM:i:1 XO:i:1 XG:i:4 NM:i:5 MD:Z:81A39 YS:i:-36 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1F7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F7_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: LH00526:197:22KFKVLT4:3:1101:52127:1056_1:N:0:TGGAAGCA+GTACTCTC/1 83 ptg000031l_GA_converted 13336538 18 125M = 13336335 -328 AAACAAAAAAACTCACATAATTTATAATAACAAAAACAAAAAAACTAACACTTTACAAACTACTAACTTTACAAAAAAAAAAAAATCAAAAATTTCTAACTTTTTTTCAAAAACTAAACAATACT -9--99999-99-9999-9-99--99--II---99999999---9-I-9-99-9--99-9-9--99I-I-I9-I-IIIII9I9I9IIIIII-III9II9IIIIIIII--II99I9II-IIIIIII AS:i:-30 XS:i:-36 XN:i:0 XM:i:5 XO:i:0 XG:i:0 NM:i:5 MD:Z:12A19T34C4C35T16 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:52127:1056_2:N:0:TGGAAGCA+GTACTCTC/2 163 ptg000031l_GA_converted 13336335 18 125M = 13336538 328 TAACTCTTAATTAAAACAATATTTCTAAACACCATTAAATTAAAAAAACATAAAAATAATAAAATTTCATTACTTAATAAACTTAAATAAACTTTTACTAAACATACTTTATTTACCAAAACAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIII9IIIIIII9IIIIII9IIIIIIIII AS:i:0 XS:i:-94 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:-30 YT:Z:CP >>> Writing bisulfite mapping results to 1F7_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/1F7_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/1F7_R2_001.fastp-trim.fq.gz 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 LH00526:197:22KFKVLT4:3:1245:14126:8844_1:N:0:TGGAAGCA+GTACTCTC ptg000093l 36773 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1257:51860:25668_1:N:0:TGGAAGCA+GTACTCTC ptg000067l 1 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1305:10387:28007_1:N:0:TGGAAGCA+GTACTCTC ptg000012l 1 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1388:31710:17347_1:N:0:TGGAAGCA+GTACTCTC ptg000007l 12295855 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1435:5839:13915_1:N:0:TGGAAGCA+GTACTCTC ptg000066l 66349 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 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2412:40077:7626_1:N:0:TGGAAGCA+GTACTCTC ptg000066l 66348 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1127:16019:1224_1:N:0:TGGAAGCA+GTACTCTC ptg000174l 27224 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far 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 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1447:5588:19700_1:N:0:TGGAAGCA+GTACTCTC ptg000020l 1 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1463:17079:6645_1:N:0:TGGAAGCA+GTACTCTC ptg000012l 2 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2123:16408:8956_1:N:0:TGGAAGCA+GTACTCTC ptg000125l 18893 Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2212:17646:28189_1:N:0:TGGAAGCA+GTACTCTC ptg000018l 2 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 35878442 reads; of these: 35878442 (100.00%) were paired; of these: 12860019 (35.84%) aligned concordantly 0 times 7610556 (21.21%) aligned concordantly exactly 1 time 15407867 (42.94%) aligned concordantly >1 times 64.16% overall alignment rate 35878442 reads; of these: 35878442 (100.00%) were paired; of these: 12847707 (35.81%) aligned concordantly 0 times 7612313 (21.22%) aligned concordantly exactly 1 time 15418422 (42.97%) aligned concordantly >1 times 64.19% overall alignment rate Processed 35878442 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 143513768. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 143513768. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 143513768. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 143513768. Successfully deleted the temporary files 1F7_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F7_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 35878442 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1110825400 Total methylated C's in CpG context: 16407394 Total methylated C's in CHG context: 1204696 Total methylated C's in CHH context: 4842553 Total methylated C's in Unknown context: 54664 Total unmethylated C's in CpG context: 159938671 Total unmethylated C's in CHG context: 187620834 Total unmethylated C's in CHH context: 740811252 Total unmethylated C's in Unknown context: 2767293 C methylated in CpG context: 9.3% 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 40m 27s ==================== Bismark run complete ====================