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/1F5_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/1F5_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/1F5_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/1F5_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1F5_R1_001.fastp-trim.fq.gz to 1F5_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1F5_R1_001.fastp-trim.fq.gz (45838591 sequences in total) Writing a G -> A converted version of the input file 1F5_R2_001.fastp-trim.fq.gz to 1F5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1F5_R2_001.fastp-trim.fq.gz (45838591 sequences in total) Input files are 1F5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F5_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 1F5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F5_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: LH00526:197:22KFKVLT4:3:1101:50735:1056_1:N:0:CGCAACTA+GAATCCGA/1 99 ptg000001l_CT_converted 11325627 6 8M1I43M1I72M = 11325684 182 AGTTTTTTATGTTTGTGTATTTTATAATTATGTTTATTGTTAGATTGTTATTGTTTTTTTTTTAATTATATATTTATTATTATTTTTTTTTTGATAAATTTGAGTAATTTGTTGTGAAGATGAAG IIIIIIIIIIII9II-IIII-IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII9IIIIIIIIII9IIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIII99IIIIII9IIIIIIIIII AS:i:-52 XS:i:-68 XN:i:0 XM:i:6 XO:i:2 XG:i:2 NM:i:8 MD:Z:2A1A0A41A21G20T32 YS:i:-24 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:50735:1056_2:N:0:CGCAACTA+GAATCCGA/2 147 ptg000001l_CT_converted 11325684 6 125M = 11325627 -182 TTTTAATTATATATTTATTATTATTTTTTTTTTGATAAATTTGAGTAATTTGTTGTGAAGATGAAGAAATTTTGGGTGAAATATAGGATTTTTTTGTTAGTAAGTGTTTAATAAATGATATTTAG IIIIII9IIIIIIIIIIIIIIIIII9II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II AS:i:-24 XS:i:-30 XN:i:0 XM:i:4 XO:i:0 XG:i:0 NM:i:4 MD:Z:12G20T43A32T14 YS:i:-52 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1F5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F5_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: LH00526:197:22KFKVLT4:3:1101:50735:1056_1:N:0:CGCAACTA+GAATCCGA/1 83 ptg000001l_GA_converted 7044444 1 71M2I52M = 7044385 -182 CTTCATCTTCACAACAAATTACTCAAATTTATCAAAAAAAAAATAATAATAAATATATAATTAAAAAAAAAACAATAACAATCTAACAATAAACATAATTATAAAATACACAAACATAAAAAACT IIIIIIIIII9IIIIII99IIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIII9IIIIIIIIII9IIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIII-IIII-II9IIIIIIIIIIII AS:i:-35 XS:i:-35 XN:i:0 XM:i:4 XO:i:1 XG:i:2 NM:i:6 MD:Z:53C20T40T6C0 YS:i:-12 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:50735:1056_2:N:0:CGCAACTA+GAATCCGA/2 163 ptg000001l_GA_converted 7044385 1 125M = 7044444 182 CTAAATATCATTTATTAAACACTTACTAACAAAAAAATCCTATATTTCACCCAAAATTTCTTCATCTTCACAACAAATTACTCAAATTTATCAAAAAAAAAATAATAATAAATATATAATTAAAA II9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9II9IIIIIIIIIIIIIIIIII9IIIIII AS:i:-12 XS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:14A97C12 YS:i:-35 YT:Z:CP >>> Writing bisulfite mapping results to 1F5_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/1F5_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/1F5_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far 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 Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1431:37836:18174_1:N:0:CGCAACTA+GAATCCGA ptg000096l 1 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1455:48380:11688_1:N:0:CGCAACTA+GAATCCGA ptg000063l 30362 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1455:48404:11702_1:N:0:CGCAACTA+GAATCCGA ptg000063l 30362 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1493:4423:14518_1:N:0:CGCAACTA+GAATCCGA ptg000015l 14997092 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 LH00526:197:22KFKVLT4:3:2283:30796:8004_1:N:0:CGCAACTA+GAATCCGA ptg000125l 18897 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 LH00526:197:22KFKVLT4:3:2459:21384:8354_1:N:0:CGCAACTA+GAATCCGA ptg000075l 54162 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1133:37569:3003_1:N:0:CGCAACTA+GAATCCGA ptg000056l 2 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1184:2295:3689_1:N:0:CGCAACTA+GAATCCGA ptg000066l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1191:43954:27839_1:N:0:CGCAACTA+GAATCCGA ptg000133l 37099 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1221:41858:14265_1:N:0:CGCAACTA+GAATCCGA ptg000102l 1 Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1294:27947:25710_1:N:0:CGCAACTA+GAATCCGA ptg000083l 1 Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1383:41372:1434_1:N:0:CGCAACTA+GAATCCGA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1396:32981:20667_1:N:0:CGCAACTA+GAATCCGA ptg000069l 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 LH00526:197:22KFKVLT4:4:1494:25746:11198_1:N:0:CGCAACTA+GAATCCGA ptg000160l 1 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 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2407:33814:22194_1:N:0:CGCAACTA+GAATCCGA ptg000066l 2 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far 45838591 reads; of these: 45838591 (100.00%) were paired; of these: 17403749 (37.97%) aligned concordantly 0 times 9338719 (20.37%) aligned concordantly exactly 1 time 19096123 (41.66%) aligned concordantly >1 times 62.03% overall alignment rate 45838591 reads; of these: 45838591 (100.00%) were paired; of these: 17387272 (37.93%) aligned concordantly 0 times 9338771 (20.37%) aligned concordantly exactly 1 time 19112548 (41.70%) aligned concordantly >1 times 62.07% overall alignment rate Processed 45838591 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 183354364. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 183354364. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 183354364. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 183354364. Successfully deleted the temporary files 1F5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1F5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 45838591 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1360396266 Total methylated C's in CpG context: 20605660 Total methylated C's in CHG context: 1424600 Total methylated C's in CHH context: 5719187 Total methylated C's in Unknown context: 67746 Total unmethylated C's in CpG context: 193970435 Total unmethylated C's in CHG context: 229803206 Total unmethylated C's in CHH context: 908873178 Total unmethylated C's in Unknown context: 3429555 C methylated in CpG context: 9.6% 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 3h 16m 2s ==================== Bismark run complete ====================