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/2G2_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/2G2_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/2G2_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/2G2_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 2G2_R1_001.fastp-trim.fq.gz to 2G2_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 2G2_R1_001.fastp-trim.fq.gz (43630012 sequences in total) Writing a G -> A converted version of the input file 2G2_R2_001.fastp-trim.fq.gz to 2G2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 2G2_R2_001.fastp-trim.fq.gz (43630012 sequences in total) Input files are 2G2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2G2_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 2G2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2G2_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: LH00652:104:22KG7JLT4:7:1101:21128:1084_1:N:0:CCGCTTAA+ACGATGAC/1 77 * 0 0 * * 0 0 TATATTTATGGTGTTATTATATATGATTTTGTATGAAGTGGAAAGAGTTTGTTAATATGTATGATTGGTTTTTTATATTTATGAATAATTAAATATGTAGGGGAGGATTTAGGGGATAGGTGTAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00652:104:22KG7JLT4:7:1101:21128:1084_2:N:0:CCGCTTAA+ACGATGAC/2 141 * 0 0 * * 0 0 TTATTTATTTTATTATTATTTATTTATTTATTTCTTTATTTTTACAAAATACTACTTATTTTAAATTTAAAAAACTACAAATCACCTCAAAAAAATACCCACCCCCTACACCTATCCCCTAAATC IIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 2G2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2G2_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: LH00652:104:22KG7JLT4:7:1101:21128:1084_1:N:0:CCGCTTAA+ACGATGAC/1 83 ptg000035l_GA_converted 9038303 42 125M = 9038198 -230 CTACACCTATCCCCTAAATCCTCCCCTACATATTTAATTATTCATAAATATAAAAAACCAATCATACATATTAACAAACTCTTTCCACTTCATACAAAATCATATATAATAACACCATAAATATA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:125 YS:i:-6 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:21128:1084_2:N:0:CCGCTTAA+ACGATGAC/2 163 ptg000035l_GA_converted 9038198 42 125M = 9038303 230 TTATTTATTTTATTATTATTTATTTATTTATTTCTTTATTTTTACAAAATACTACTTATTTTAAATTTAAAAAACTACAAATCACCTCAAAAAAATACCCACCCCCTACACCTATCCCCTAAATC IIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:42A82 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to 2G2_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/2G2_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/2G2_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1132:30407:3901_1:N:0:CCGCTTAA+ACGATGAC ptg000048l 2 Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1182:27309:15629_1:N:0:CCGCTTAA+ACGATGAC ptg000066l 66350 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1204:42202:4629_1:N:0:CCGCTTAA+ACGATGAC ptg000011l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1205:45875:9898_1:N:0:CCGCTTAA+ACGATGAC ptg000055l 50533 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1229:19510:15096_1:N:0:CCGCTTAA+ACGATGAC ptg000146l 1 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1243:30116:8721_1:N:0:CCGCTTAA+ACGATGAC ptg000009l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1255:7213:2822_1:N:0:CCGCTTAA+ACGATGAC ptg000065l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1264:19437:18025_1:N:0:CCGCTTAA+ACGATGAC ptg000011l 1 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1302:37761:11986_1:N:0:CCGCTTAA+ACGATGAC ptg000126l 31081 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1311:8143:14242_1:N:0:CCGCTTAA+ACGATGAC ptg000006l 1 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1346:37915:7067_1:N:0:CCGCTTAA+ACGATGAC ptg000057l 39953 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1348:7076:8973_1:N:0:CCGCTTAA+ACGATGAC ptg000117l 30023 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1352:5951:1813_1:N:0:CCGCTTAA+ACGATGAC ptg000109l 39600 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1352:5951:1841_1:N:0:CCGCTTAA+ACGATGAC ptg000109l 39600 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1392:39088:13247_1:N:0:CCGCTTAA+ACGATGAC ptg000133l 37106 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1392:39080:13261_1:N:0:CCGCTTAA+ACGATGAC ptg000133l 37106 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1422:4325:1434_1:N:0:CCGCTTAA+ACGATGAC ptg000039l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1437:20068:24499_1:N:0:CCGCTTAA+ACGATGAC ptg000025l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1443:11760:6801_1:N:0:CCGCTTAA+ACGATGAC ptg000160l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1444:31847:13401_1:N:0:CCGCTTAA+ACGATGAC ptg000065l 49677 Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2108:43748:13527_1:N:0:CCGCTTAA+ACGATGAC ptg000057l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2116:28344:21038_1:N:0:CCGCTTAA+ACGATGAC ptg000185l 34486 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2131:23280:26811_1:N:0:CCGCTTAA+ACGATGAC ptg000092l 1 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2144:11104:14522_1:N:0:CCGCTTAA+ACGATGAC ptg000090l 49492 Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2177:14980:4391_1:N:0:CCGCTTAA+ACGATGAC ptg000063l 30363 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2185:39088:15461_1:N:0:CCGCTTAA+ACGATGAC ptg000122l 1 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2244:40414:13443_1:N:0:CCGCTTAA+ACGATGAC ptg000056l 59123 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2244:40431:13443_1:N:0:CCGCTTAA+ACGATGAC ptg000056l 59123 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2269:23701:5456_1:N:0:CCGCTTAA+ACGATGAC ptg000159l 1 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2310:44023:5596_1:N:0:CCGCTTAA+ACGATGAC ntLink_3 131396 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2320:21597:28100_1:N:0:CCGCTTAA+ACGATGAC ptg000026l 14741947 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2326:22924:3606_1:N:0:CCGCTTAA+ACGATGAC ptg000121l 50739 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2326:22940:3606_1:N:0:CCGCTTAA+ACGATGAC ptg000121l 50739 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2336:21977:10206_1:N:0:CCGCTTAA+ACGATGAC ptg000031l 15849141 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2362:15166:19286_1:N:0:CCGCTTAA+ACGATGAC ptg000052l 2 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2417:30812:28478_1:N:0:CCGCTTAA+ACGATGAC ptg000174l 27224 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2422:2845:11537_1:N:0:CCGCTTAA+ACGATGAC ptg000094l 36827 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2443:36418:10809_1:N:0:CCGCTTAA+ACGATGAC ptg000120l 1 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2481:28741:24919_1:N:0:CCGCTTAA+ACGATGAC ptg000065l 49672 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2481:28773:24919_1:N:0:CCGCTTAA+ACGATGAC ptg000065l 49672 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2481:28749:24933_1:N:0:CCGCTTAA+ACGATGAC ptg000065l 49672 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2495:24688:23672_1:N:0:CCGCTTAA+ACGATGAC ptg000125l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2495:24679:23686_1:N:0:CCGCTTAA+ACGATGAC ptg000125l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1102:5538:1462_1:N:0:CCGCTTAA+ACGATGAC ptg000046l 39583 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1173:22131:19945_1:N:0:CCGCTTAA+ACGATGAC ptg000016l 13007871 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1184:17123:15671_1:N:0:CCGCTTAA+ACGATGAC ptg000094l 36816 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1184:17148:15685_1:N:0:CCGCTTAA+ACGATGAC ptg000094l 36816 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1308:24607:15349_1:N:0:CCGCTTAA+ACGATGAC ptg000025l 1 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1346:12706:14101_1:N:0:CCGCTTAA+ACGATGAC ptg000002l 1 Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1368:23053:16918_1:N:0:CCGCTTAA+ACGATGAC ptg000066l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1370:25618:25760_1:N:0:CCGCTTAA+ACGATGAC ptg000092l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1375:6372:28436_1:N:0:CCGCTTAA+ACGATGAC ptg000133l 37113 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1398:25950:24989_1:N:0:CCGCTTAA+ACGATGAC ptg000099l 2 Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1465:15902:27483_1:N:0:CCGCTTAA+ACGATGAC ptg000048l 76457 Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2118:28797:2317_1:N:0:CCGCTTAA+ACGATGAC ptg000052l 52623 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2149:33465:16736_1:N:0:CCGCTTAA+ACGATGAC ptg000022l 9976728 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2240:17309:10668_1:N:0:CCGCTTAA+ACGATGAC ptg000048l 1 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2276:35528:15321_1:N:0:CCGCTTAA+ACGATGAC ptg000108l 1 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2295:25836:10416_1:N:0:CCGCTTAA+ACGATGAC ptg000120l 1 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2315:32850:19762_1:N:0:CCGCTTAA+ACGATGAC ptg000118l 34380 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2326:39225:11523_1:N:0:CCGCTTAA+ACGATGAC ptg000054l 36039 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2370:18199:24316_1:N:0:CCGCTTAA+ACGATGAC ptg000159l 21849 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2371:28506:27203_1:N:0:CCGCTTAA+ACGATGAC ptg000096l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2403:9859:3144_1:N:0:CCGCTTAA+ACGATGAC ptg000070l 1 Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2427:13370:21444_1:N:0:CCGCTTAA+ACGATGAC ptg000027l 1 Processed 42000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2447:32850:17352_1:N:0:CCGCTTAA+ACGATGAC ptg000048l 1 Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2497:51312:19790_1:N:0:CCGCTTAA+ACGATGAC ptg000155l 36784 43630012 reads; of these: 43630012 (100.00%) were paired; of these: 16705144 (38.29%) aligned concordantly 0 times 9257028 (21.22%) aligned concordantly exactly 1 time 17667840 (40.49%) aligned concordantly >1 times 61.71% overall alignment rate 43630012 reads; of these: 43630012 (100.00%) were paired; of these: 16687577 (38.25%) aligned concordantly 0 times 9255925 (21.21%) aligned concordantly exactly 1 time 17686510 (40.54%) aligned concordantly >1 times 61.75% overall alignment rate Processed 43630012 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 174520048. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 174520048. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 174520048. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 174520048. Successfully deleted the temporary files 2G2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 2G2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 43630012 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1337046916 Total methylated C's in CpG context: 21622123 Total methylated C's in CHG context: 1287207 Total methylated C's in CHH context: 5275226 Total methylated C's in Unknown context: 72498 Total unmethylated C's in CpG context: 185408722 Total unmethylated C's in CHG context: 225644216 Total unmethylated C's in CHH context: 897809422 Total unmethylated C's in Unknown context: 3579148 C methylated in CpG context: 10.4% 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 3h 2m 41s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe