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/1A1_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/1A1_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/1A1_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/1A1_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1A1_R1_001.fastp-trim.fq.gz to 1A1_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1A1_R1_001.fastp-trim.fq.gz (39273285 sequences in total) Writing a G -> A converted version of the input file 1A1_R2_001.fastp-trim.fq.gz to 1A1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1A1_R2_001.fastp-trim.fq.gz (39273285 sequences in total) Input files are 1A1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A1_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 1A1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A1_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: LH00526:197:22KFKVLT4:3:1101:43557:1070_1:N:0:TTACCGAC+CGTATTCG/1 99 ptg000016l_CT_converted 10323632 42 125M = 10323723 216 TTTTTGATTTGTAGTTTTTATTTAAGAATATTTGAAAGTTTAGTTTTTTTATTTAGTTAATTTAAGATTTTGAGTGTTGGTTTGGTTGTAGTTGAATTTATGATTTTTTGTGTTATAGTTTGAAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIII9IIII9IIIIIII9IIIIIIIIIIII-IIIIIIIIIIIIIIII9I-IIIIIIIIIIIIIIII9II-IIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:43557:1070_2:N:0:TTACCGAC+CGTATTCG/2 147 ptg000016l_CT_converted 10323723 42 125M = 10323632 -216 TTGAATTTATGATTTTTTGTGTTATAGTTTGAAGTTTAATGAATTGAGTTAATGGTGTGTAGTTAAAGTTTAAAGAGTAAATGGTTTTGTTTTTTTTAAGATGTGAAAAAAGTGTATTTAGTAAT -IIIII9IIIIIIIII9IIIIIIII-II9III-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:0 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1A1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A1_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: LH00526:197:22KFKVLT4:3:1101:43557:1070_1:N:0:TTACCGAC+CGTATTCG/1 77 * 0 0 * * 0 0 TTTTTGATTTGTAGTTTTTATTTAAGAATATTTGAAAGTTTAGTTTTTTTATTTAGTTAATTTAAGATTTTGAGTGTTGGTTTGGTTGTAGTTGAATTTATGATTTTTTGTGTTATAGTTTGAAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIII9IIII9IIIIIII9IIIIIIIIIIII-IIIIIIIIIIIIIIII9I-IIIIIIIIIIIIIIII9II-IIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:43557:1070_2:N:0:TTACCGAC+CGTATTCG/2 141 * 0 0 * * 0 0 ATTACTAAATACACTTTTTTCACATCTTAAAAAAAACAAAACCATTTACTCTTTAAACTTTAACTACACACCATTAACTCAATTCATTAAACTTCAAACTATAACACAAAAAATCATAAATTCAA IIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-III9II-IIIIIIII9IIIIIIIII9IIIII- YT:Z:UP >>> Writing bisulfite mapping results to 1A1_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/1A1_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/1A1_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1105:33838:25093_1:N:0:TTACCGAC+CGTATTCG ptg000160l 32598 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1113:45993:14363_1:N:0:TTACCGAC+CGTATTCG ptg000184l 1 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1141:3581:10763_1:N:0:TTACCGAC+CGTATTCG ptg000060c 88492 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1191:7174:19812_1:N:0:TTACCGAC+CGTATTCG ntLink_0 1 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1242:27721:20667_1:N:0:TTACCGAC+CGTATTCG ptg000090l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1249:43856:2065_1:N:0:TTACCGAC+CGTATTCG ptg000098l 55091 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1278:43055:11464_1:N:0:TTACCGAC+CGTATTCG ptg000066l 66393 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1376:24548:10889_1:N:0:TTACCGAC+CGTATTCG ptg000052l 52619 Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2149:41364:11226_1:N:0:TTACCGAC+CGTATTCG ptg000025l 21443081 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2203:12774:3956_1:N:0:TTACCGAC+CGTATTCG ptg000048l 1 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2260:46689:15036_1:N:0:TTACCGAC+CGTATTCG ptg000128l 44510 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2318:35206:14461_1:N:0:TTACCGAC+CGTATTCG ptg000017l 2 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2381:9772:28147_1:N:0:TTACCGAC+CGTATTCG ptg000055l 50535 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2482:46147:2415_1:N:0:TTACCGAC+CGTATTCG ptg000098l 55094 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2484:40199:9433_1:N:0:TTACCGAC+CGTATTCG ptg000083l 2 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1125:20438:10077_1:N:0:TTACCGAC+CGTATTCG ptg000083l 120266 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1128:24573:23735_1:N:0:TTACCGAC+CGTATTCG ptg000009l 19095986 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1136:49173:21269_1:N:0:TTACCGAC+CGTATTCG ptg000096l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1148:12928:10189_1:N:0:TTACCGAC+CGTATTCG ptg000047l 1 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1194:22792:25247_1:N:0:TTACCGAC+CGTATTCG ptg000048l 76441 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1225:35902:28918_1:N:0:TTACCGAC+CGTATTCG ptg000125l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1233:4811:13481_1:N:0:TTACCGAC+CGTATTCG ptg000096l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1233:7838:13649_1:N:0:TTACCGAC+CGTATTCG ptg000088l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1240:51682:3928_1:N:0:TTACCGAC+CGTATTCG ptg000113l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1245:43039:10203_1:N:0:TTACCGAC+CGTATTCG ptg000054l 1 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1263:44261:15484_1:N:0:TTACCGAC+CGTATTCG ptg000098l 55089 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1320:40433:21830_1:N:0:TTACCGAC+CGTATTCG ptg000125l 18887 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1349:4059:27727_1:N:0:TTACCGAC+CGTATTCG ptg000066l 66343 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1354:16391:4950_1:N:0:TTACCGAC+CGTATTCG ptg000125l 18898 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1381:3161:28805_1:N:0:TTACCGAC+CGTATTCG ptg000080l 40104 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1394:29323:21647_1:N:0:TTACCGAC+CGTATTCG ptg000125l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1397:15097:4810_1:N:0:TTACCGAC+CGTATTCG ptg000024l 3 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1461:29525:23595_1:N:0:TTACCGAC+CGTATTCG ptg000185l 34486 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1497:25034:7388_1:N:0:TTACCGAC+CGTATTCG ptg000094l 36813 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1497:25042:7402_1:N:0:TTACCGAC+CGTATTCG ptg000094l 36813 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2116:19823:17753_1:N:0:TTACCGAC+CGTATTCG ptg000039l 1 Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2248:46664:9755_1:N:0:TTACCGAC+CGTATTCG ptg000055l 1 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2292:4771:2177_1:N:0:TTACCGAC+CGTATTCG ptg000160l 1 Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2426:13859:21830_1:N:0:TTACCGAC+CGTATTCG ptg000018l 1 Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2498:15388:7752_1:N:0:TTACCGAC+CGTATTCG ptg000160l 1 39273285 reads; of these: 39273285 (100.00%) were paired; of these: 14926436 (38.01%) aligned concordantly 0 times 8109353 (20.65%) aligned concordantly exactly 1 time 16237496 (41.34%) aligned concordantly >1 times 61.99% overall alignment rate 39273285 reads; of these: 39273285 (100.00%) were paired; of these: 14915610 (37.98%) aligned concordantly 0 times 8110094 (20.65%) aligned concordantly exactly 1 time 16247581 (41.37%) aligned concordantly >1 times 62.02% overall alignment rate Processed 39273285 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 157093140. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 157093140. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 157093140. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 157093140. Successfully deleted the temporary files 1A1_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A1_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 39273285 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1173067945 Total methylated C's in CpG context: 20689882 Total methylated C's in CHG context: 1239195 Total methylated C's in CHH context: 4986097 Total methylated C's in Unknown context: 63480 Total unmethylated C's in CpG context: 164449978 Total unmethylated C's in CHG context: 196847249 Total unmethylated C's in CHH context: 784855544 Total unmethylated C's in Unknown context: 2975382 C methylated in CpG context: 11.2% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 2.1% Bismark completed in 0d 2h 42m 20s ==================== Bismark run complete ====================