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/1H3_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/1H3_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/1H3_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/1H3_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1H3_R1_001.fastp-trim.fq.gz to 1H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1H3_R1_001.fastp-trim.fq.gz (39232241 sequences in total) Writing a G -> A converted version of the input file 1H3_R2_001.fastp-trim.fq.gz to 1H3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1H3_R2_001.fastp-trim.fq.gz (39232241 sequences in total) Input files are 1H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H3_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 1H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H3_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: LH00526:197:22KFKVLT4:3:1101:50023:1056_1:N:0:GGTTGAAC+AACGTGGA/1 77 * 0 0 * * 0 0 TTAATTGATGGATGGTTGTAATTGATGTATTTGTTAGTAATAAATATTTTTTGTAATTATTATTATATATTTTAAGTGTAATTTTGTAAATATGAAGGTTAAATGAGATGTGGAATTTGTTATTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:50023:1056_2:N:0:GGTTGAAC+AACGTGGA/2 141 * 0 0 * * 0 0 TAACACTTAACACTCATCAAACACTAAAAAATCTTTCTCTTTCTTAAATTTCAATATCTATAAACAATCTTCAATTTTCATACCAAAATAATAACAAAAAAACAAAAC IIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIII9IIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H3_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: LH00526:197:22KFKVLT4:3:1101:50023:1056_1:N:0:GGTTGAAC+AACGTGGA/1 83 ptg000016l_GA_converted 3837807 42 125M = 3837584 -348 AAATAACAAATTCCACATCTCATTTAACCTTCATATTTACAAAATTACACTTAAAATATATAATAATAATTACAAAAAATATTTATTACTAACAAATACATCAATTACAACCATCCATCAATTAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 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 LH00526:197:22KFKVLT4:3:1101:50023:1056_2:N:0:GGTTGAAC+AACGTGGA/2 163 ptg000016l_GA_converted 3837584 42 108M = 3837807 348 TAACACTTAACACTCATCAAACACTAAAAAATCTTTCTCTTTCTTAAATTTCAATATCTATAAACAATCTTCAATTTTCATACCAAAATAATAACAAAAAAACAAAAC IIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIII9IIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:108 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to 1H3_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/1H3_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/1H3_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1129:42853:5903_1:N:0:GGTTGAAC+AACGTGGA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1129:42861:5917_1:N:0:GGTTGAAC+AACGTGGA ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1129:49691:29142_1:N:0:GGTTGAAC+AACGTGGA ptg000118l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1129:49707:29142_1:N:0:GGTTGAAC+AACGTGGA ptg000118l 1 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1155:6778:28875_1:N:0:GGTTGAAC+AACGTGGA ptg000025l 21443081 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1182:33102:17207_1:N:0:GGTTGAAC+AACGTGGA ptg000025l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1195:37642:16353_1:N:0:GGTTGAAC+AACGTGGA ptg000030l 2 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1211:45434:5244_1:N:0:GGTTGAAC+AACGTGGA ptg000066l 66364 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1220:48696:23553_1:N:0:GGTTGAAC+AACGTGGA ptg000097l 1 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1263:12693:25892_1:N:0:GGTTGAAC+AACGTGGA ptg000066l 66405 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1381:10824:4782_1:N:0:GGTTGAAC+AACGTGGA ptg000117l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1383:14465:4698_1:N:0:GGTTGAAC+AACGTGGA ptg000096l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1383:8170:8452_1:N:0:GGTTGAAC+AACGTGGA ptg000066l 66353 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1402:44730:4166_1:N:0:GGTTGAAC+AACGTGGA ptg000012l 3 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1414:25981:3003_1:N:0:GGTTGAAC+AACGTGGA ptg000007l 12295855 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1434:47903:3633_1:N:0:GGTTGAAC+AACGTGGA ptg000024l 2 Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2127:7069:2205_1:N:0:GGTTGAAC+AACGTGGA ptg000066l 66410 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2129:35441:6239_1:N:0:GGTTGAAC+AACGTGGA ptg000160l 1 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2167:25180:10918_1:N:0:GGTTGAAC+AACGTGGA ptg000155l 36784 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2177:20599:7135_1:N:0:GGTTGAAC+AACGTGGA ptg000117l 1 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2207:16068:7948_1:N:0:GGTTGAAC+AACGTGGA ptg000016l 13007877 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2220:6228:19630_1:N:0:GGTTGAAC+AACGTGGA ntLink_3 131388 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2255:45103:6407_1:N:0:GGTTGAAC+AACGTGGA ptg000048l 2 Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2353:48097:27139_1:N:0:GGTTGAAC+AACGTGGA ptg000020l 2 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2438:20413:9335_1:N:0:GGTTGAAC+AACGTGGA ptg000025l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2438:20429:9335_1:N:0:GGTTGAAC+AACGTGGA ptg000025l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2449:25374:18538_1:N:0:GGTTGAAC+AACGTGGA ptg000024l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2466:12005:20303_1:N:0:GGTTGAAC+AACGTGGA ntLink_2 2 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2468:39212:5174_1:N:0:GGTTGAAC+AACGTGGA ptg000160l 32572 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2469:40943:29212_1:N:0:GGTTGAAC+AACGTGGA ptg000160l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2476:21570:1308_1:N:0:GGTTGAAC+AACGTGGA ntLink_0 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2493:21457:1756_1:N:0:GGTTGAAC+AACGTGGA ptg000022l 9976760 Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1183:51779:28693_1:N:0:GGTTGAAC+AACGTGGA ptg000065l 49673 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1183:51787:28707_1:N:0:GGTTGAAC+AACGTGGA ptg000065l 49673 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1192:9756:14419_1:N:0:GGTTGAAC+AACGTGGA ptg000097l 2 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1236:41218:15176_1:N:0:GGTTGAAC+AACGTGGA ptg000023l 45111808 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1236:41226:15190_1:N:0:GGTTGAAC+AACGTGGA ptg000023l 45111808 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1255:40425:5062_1:N:0:GGTTGAAC+AACGTGGA ptg000121l 50738 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1259:23003:1742_1:N:0:GGTTGAAC+AACGTGGA ntLink_0 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1268:33693:23945_1:N:0:GGTTGAAC+AACGTGGA ptg000048l 1 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1306:1623:1434_1:N:0:GGTTGAAC+AACGTGGA ptg000098l 55086 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1340:2165:19126_1:N:0:GGTTGAAC+AACGTGGA ptg000183l 26189 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1369:37229:1826_1:N:0:GGTTGAAC+AACGTGGA ptg000098l 55084 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1447:2627:13061_1:N:0:GGTTGAAC+AACGTGGA ptg000023l 45111799 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1455:51350:7500_1:N:0:GGTTGAAC+AACGTGGA ptg000066l 1 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1498:23666:1322_1:N:0:GGTTGAAC+AACGTGGA ptg000090l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2102:42206:2485_1:N:0:GGTTGAAC+AACGTGGA ptg000101l 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 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2308:18123:11058_1:N:0:GGTTGAAC+AACGTGGA ptg000048l 1 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2341:21312:28147_1:N:0:GGTTGAAC+AACGTGGA ptg000066l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2368:40652:6155_1:N:0:GGTTGAAC+AACGTGGA ntLink_3 131386 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2443:24646:18258_1:N:0:GGTTGAAC+AACGTGGA ptg000016l 13007879 Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far 39232241 reads; of these: 39232241 (100.00%) were paired; of these: 14734522 (37.56%) aligned concordantly 0 times 8397366 (21.40%) aligned concordantly exactly 1 time 16100353 (41.04%) aligned concordantly >1 times 62.44% overall alignment rate 39232241 reads; of these: 39232241 (100.00%) were paired; of these: 14745460 (37.59%) aligned concordantly 0 times 8394723 (21.40%) aligned concordantly exactly 1 time 16092058 (41.02%) aligned concordantly >1 times 62.41% overall alignment rate Processed 39232241 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 156928964. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 156928964. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 156928964. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 156928964. Successfully deleted the temporary files 1H3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 39232241 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1218104592 Total methylated C's in CpG context: 19182946 Total methylated C's in CHG context: 1287940 Total methylated C's in CHH context: 5201820 Total methylated C's in Unknown context: 62259 Total unmethylated C's in CpG context: 169775949 Total unmethylated C's in CHG context: 204453849 Total unmethylated C's in CHH context: 818202088 Total unmethylated C's in Unknown context: 3113556 C methylated in CpG context: 10.2% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 2.0% Bismark completed in 0d 2h 49m 59s ==================== Bismark run complete ====================