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/1B5_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/1B5_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/1B5_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/1B5_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1B5_R1_001.fastp-trim.fq.gz to 1B5_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1B5_R1_001.fastp-trim.fq.gz (40968727 sequences in total) Writing a G -> A converted version of the input file 1B5_R2_001.fastp-trim.fq.gz to 1B5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1B5_R2_001.fastp-trim.fq.gz (40968727 sequences in total) Input files are 1B5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B5_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 1B5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B5_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: LH00526:197:22KFKVLT4:3:1101:49521:1056_1:N:0:TCGGATTC+TAGTTGCG/1 77 * 0 0 * * 0 0 AAGGGGTTTGGTTTTTTTTTTTTTTGTGTTGATTTTTTTTAATAGAGTTGTAATAATTTTTTTAATTTGGGGAAAAAATATTAGTAATAAGTTTTATAATTTTTTTTAAATGAAAATATTGGTTT IIIIIIIIIIIIIIIIIIIIIIIII--9-I9I-9-9IIIII----9-9----9-----9-9I9-I999999999--99-9-99-9-9--99--9-9-9--999-99II99--99999-9999-99 YT:Z:UP LH00526:197:22KFKVLT4:3:1101:49521:1056_2:N:0:TCGGATTC+TAGTTGCG/2 141 * 0 0 * * 0 0 ATTTAATTTAATTTATATAAAATTATTATCTCCAATTAACTCCATAAAAAACTAAAATACCTATTATTATTAATAAACTTATAACCCTCAAACTAACATACATCAATATTTTCAAAATAAAAACC IIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIII9I9IIIIIIIIIIIIIIIII-IIIIIIIIIIII9IIIIIIIIIIIIIII9II9I-III9IIIII9II9I YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1B5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B5_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: LH00526:197:22KFKVLT4:3:1101:49521:1056_1:N:0:TCGGATTC+TAGTTGCG/1 83 ptg000027l_GA_converted 3715831 40 100M1I24M = 3715503 -452 AAACCAATATTTTCATTTAAAAAAAATTATAAAACTTATTACTAATATTTTTTCCCCAAATTAAAAAAATTATTACAACTCTATTAAAAAAAATCAACACAAAAAAAAAAAAAACCAAACCCCTT 99-9999-99999--99II99-999--9-9-9--99--9-9-99-9-99--999999999I-9I9-9-----9----9-9----IIIII9-9-I9I-9--IIIIIIIIIIIIIIIIIIIIIIIII AS:i:-62 XN:i:0 XM:i:9 XO:i:1 XG:i:1 NM:i:10 MD:Z:7C10T7C3T1T32C3C19T1T32 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:49521:1056_2:N:0:TCGGATTC+TAGTTGCG/2 163 ptg000027l_GA_converted 3715503 40 125M = 3715831 452 ATTTAATTTAATTTATATAAAATTATTATCTCCAATTAACTCCATAAAAAACTAAAATACCTATTATTATTAATAAACTTATAACCCTCAAACTAACATACATCAATATTTTCAAAATAAAAACC IIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIII9I9IIIIIIIIIIIIIIIII-IIIIIIIIIIII9IIIIIIIIIIIIIII9II9I-III9IIIII9II9I AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:-62 YT:Z:CP >>> Writing bisulfite mapping results to 1B5_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/1B5_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/1B5_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1172:47749:12388_1:N:0:TCGGATTC+TAGTTGCG ptg000134l 2 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1243:42246:3255_1:N:0:TCGGATTC+TAGTTGCG ptg000033l 2625610 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1313:44043:3928_1:N:0:TCGGATTC+TAGTTGCG ptg000045l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1313:44051:3942_1:N:0:TCGGATTC+TAGTTGCG ptg000045l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1320:37949:18678_1:N:0:TCGGATTC+TAGTTGCG ptg000066l 66346 Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1404:24678:26494_1:N:0:TCGGATTC+TAGTTGCG ptg000127l 1 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 LH00526:197:22KFKVLT4:3:2180:45175:6085_1:N:0:TCGGATTC+TAGTTGCG ptg000055l 50538 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2230:39357:11758_1:N:0:TCGGATTC+TAGTTGCG ptg000035l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2230:39333:11772_1:N:0:TCGGATTC+TAGTTGCG ptg000035l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2230:39349:11772_1:N:0:TCGGATTC+TAGTTGCG ptg000035l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2246:3573:17669_1:N:0:TCGGATTC+TAGTTGCG ptg000015l 14997091 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2277:40798:2345_1:N:0:TCGGATTC+TAGTTGCG ptg000033l 2625622 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2285:10565:24701_1:N:0:TCGGATTC+TAGTTGCG ptg000039l 1139412 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2285:10573:24715_1:N:0:TCGGATTC+TAGTTGCG ptg000039l 1139412 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 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 LH00526:197:22KFKVLT4:4:1253:25795:12879_1:N:0:TCGGATTC+TAGTTGCG ptg000004l 2 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1296:31419:11688_1:N:0:TCGGATTC+TAGTTGCG ptg000054l 36028 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1296:32544:27503_1:N:0:TCGGATTC+TAGTTGCG ptg000187l 17982 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1304:24152:3928_1:N:0:TCGGATTC+TAGTTGCG ptg000039l 1 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1333:6333:23314_1:N:0:TCGGATTC+TAGTTGCG ptg000007l 12295855 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2144:8105:26746_1:N:0:TCGGATTC+TAGTTGCG ptg000094l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2144:8121:26746_1:N:0:TCGGATTC+TAGTTGCG ptg000094l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2144:8113:26760_1:N:0:TCGGATTC+TAGTTGCG ptg000094l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2155:47919:26074_1:N:0:TCGGATTC+TAGTTGCG ptg000033l 2625597 Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2240:23278:25472_1:N:0:TCGGATTC+TAGTTGCG ptg000124l 54848 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2242:42893:6169_1:N:0:TCGGATTC+TAGTTGCG ptg000007l 12295855 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2262:16764:13439_1:N:0:TCGGATTC+TAGTTGCG ptg000023l 45111806 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2263:14733:27909_1:N:0:TCGGATTC+TAGTTGCG ptg000155l 2 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2283:2877:8956_1:N:0:TCGGATTC+TAGTTGCG ptg000027l 16268248 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2337:27421:27068_1:N:0:TCGGATTC+TAGTTGCG ptg000004l 1 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2398:23917:16296_1:N:0:TCGGATTC+TAGTTGCG ptg000033l 2625651 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far 40968727 reads; of these: 40968727 (100.00%) were paired; of these: 16248677 (39.66%) aligned concordantly 0 times 8657364 (21.13%) aligned concordantly exactly 1 time 16062686 (39.21%) aligned concordantly >1 times 60.34% overall alignment rate 40968727 reads; of these: 40968727 (100.00%) were paired; of these: 16261248 (39.69%) aligned concordantly 0 times 8652643 (21.12%) aligned concordantly exactly 1 time 16054836 (39.19%) aligned concordantly >1 times 60.31% overall alignment rate Processed 40968727 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 163874908. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 163874908. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 163874908. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 163874908. Successfully deleted the temporary files 1B5_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1B5_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 40968727 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1246507827 Total methylated C's in CpG context: 24585193 Total methylated C's in CHG context: 1505658 Total methylated C's in CHH context: 6073935 Total methylated C's in Unknown context: 71753 Total unmethylated C's in CpG context: 166543446 Total unmethylated C's in CHG context: 209353758 Total unmethylated C's in CHH context: 838445837 Total unmethylated C's in Unknown context: 3286127 C methylated in CpG context: 12.9% C methylated in CHG context: 0.7% C methylated in CHH context: 0.7% C methylated in unknown context (CN or CHN): 2.1% Bismark completed in 0d 2h 52m 9s ==================== Bismark run complete ====================