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/1D9_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/1D9_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/1D9_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/1D9_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1D9_R1_001.fastp-trim.fq.gz to 1D9_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1D9_R1_001.fastp-trim.fq.gz (45461427 sequences in total) Writing a G -> A converted version of the input file 1D9_R2_001.fastp-trim.fq.gz to 1D9_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1D9_R2_001.fastp-trim.fq.gz (45461427 sequences in total) Input files are 1D9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D9_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 1D9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D9_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: LH00526:197:22KFKVLT4:3:1101:50646:1070_1:N:0:TACCTGCA+TGATGTCC/1 99 ptg000016l_CT_converted 7561485 42 125M = 7561622 262 TAGTTGTTAGAGGGATGATTTGTGTGTTTTTTTTGGTTTGTTGTTTAGGTTGAGATAAGTTTGAATTTTTTGATGGAAATGTTGTAGTATTTAAGTTATTTGTGTTTTTAAATGTTGAATTTTTA IIIIIIIIII9I9II9-I9II9-IIIIIIIIII9IIIII99II-II-9IIII-I9II9IIIII9-IIIII9I-III9IIIIIIIIIIIIIIIIIII9IIIIIIIIIIII9II9IIIIIIIIII9I AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:43A81 YS:i:-12 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:50646:1070_2:N:0:TACCTGCA+TGATGTCC/2 147 ptg000016l_CT_converted 7561622 42 125M = 7561485 -262 GAAATGTGGGATTTTTTTGTGTTTTTATTATTTTTTTGAGATAAGTTTTGTATTTTTTGGGTTAGTAGGTTGTAAATATTTTTAAATTTGTTATTATTTTGGTTTGAAAAATATGTATTTAAGTA IIII9-I9-9I9II9II99II9IIII--IIII-IIIIII9III9IIIIIIIIII9II9II9IIIIIIIIIII9I-IIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9 AS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:2T5T116 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1D9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D9_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: LH00526:197:22KFKVLT4:3:1101:50646:1070_1:N:0:TACCTGCA+TGATGTCC/1 77 * 0 0 * * 0 0 TAGTTGTTAGAGGGATGATTTGTGTGTTTTTTTTGGTTTGTTGTTTAGGTTGAGATAAGTTTGAATTTTTTGATGGAAATGTTGTAGTATTTAAGTTATTTGTGTTTTTAAATGTTGAATTTTTA IIIIIIIIII9I9II9-I9II9-IIIIIIIIII9IIIII99II-II-9IIII-I9II9IIIII9-IIIII9I-III9IIIIIIIIIIIIIIIIIII9IIIIIIIIIIII9II9IIIIIIIIII9I YT:Z:UP LH00526:197:22KFKVLT4:3:1101:50646:1070_2:N:0:TACCTGCA+TGATGTCC/2 141 * 0 0 * * 0 0 TACTTAAATACATATTTTTCAAACCAAAATAATAACAAATTTAAAAATATTTACAACCTACTAACCCAAAAAATACAAAACTTATCTCAAAAAAATAATAAAAACACAAAAAAATCCCACATTTC 9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIII-I9IIIIIIIIIII9II9II9IIIIIIIIII9III9IIIIII-IIII--IIII9II99II9II9I9-9I-9IIII YT:Z:UP >>> Writing bisulfite mapping results to 1D9_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/1D9_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/1D9_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1141:13503:8438_1:N:0:TACCTGCA+TGATGTCC ptg000097l 1 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 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1329:45782:15736_1:N:0:TACCTGCA+TGATGTCC ptg000083l 1 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1360:8307:24855_1:N:0:TACCTGCA+TGATGTCC ptg000039l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1363:32778:17879_1:N:0:TACCTGCA+TGATGTCC ptg000066l 66400 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1411:30205:8382_1:N:0:TACCTGCA+TGATGTCC ptg000075l 1 Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1444:30043:7766_1:N:0:TACCTGCA+TGATGTCC ptg000015l 14997091 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1472:25592:29422_1:N:0:TACCTGCA+TGATGTCC ptg000066l 66351 Processed 12000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:3:2288:22250:2653_1:N:0:TACCTGCA+TGATGTCC ptg000039l 1 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2374:24467:15764_1:N:0:TACCTGCA+TGATGTCC ptg000128l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2386:47676:17193_1:N:0:TACCTGCA+TGATGTCC ptg000096l 1 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2418:5378:3647_1:N:0:TACCTGCA+TGATGTCC ptg000006l 3 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2430:4698:12781_1:N:0:TACCTGCA+TGATGTCC ptg000066l 66353 Processed 21000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:1141:23157:11282_1:N:0:TACCTGCA+TGATGTCC ptg000066l 66345 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1152:18698:17375_1:N:0:TACCTGCA+TGATGTCC ptg000066l 66346 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1195:16343:13383_1:N:0:TACCTGCA+TGATGTCC ptg000083l 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1212:45208:22586_1:N:0:TACCTGCA+TGATGTCC ptg000118l 2 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1234:21490:20527_1:N:0:TACCTGCA+TGATGTCC ptg000066l 66355 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1309:28773:4614_1:N:0:TACCTGCA+TGATGTCC ptg000117l 30023 Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:1458:39851:4124_1:N:0:TACCTGCA+TGATGTCC ptg000093l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1476:19313:24323_1:N:0:TACCTGCA+TGATGTCC ptg000052l 52599 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2158:4787:16997_1:N:0:TACCTGCA+TGATGTCC ptg000015l 14997092 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2182:38459:4880_1:N:0:TACCTGCA+TGATGTCC ptg000083l 2 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2191:5257:27195_1:N:0:TACCTGCA+TGATGTCC ptg000046l 39580 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2220:23464:23076_1:N:0:TACCTGCA+TGATGTCC ptg000080l 40109 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 LH00526:197:22KFKVLT4:4:2408:23861:21802_1:N:0:TACCTGCA+TGATGTCC ptg000066l 66405 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far 45461427 reads; of these: 45461427 (100.00%) were paired; of these: 17497867 (38.49%) aligned concordantly 0 times 8102009 (17.82%) aligned concordantly exactly 1 time 19861551 (43.69%) aligned concordantly >1 times 61.51% overall alignment rate 45461427 reads; of these: 45461427 (100.00%) were paired; of these: 17513411 (38.52%) aligned concordantly 0 times 8100262 (17.82%) aligned concordantly exactly 1 time 19847754 (43.66%) aligned concordantly >1 times 61.48% overall alignment rate Processed 45461427 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 181845708. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 181845708. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 181845708. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 181845708. Successfully deleted the temporary files 1D9_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1D9_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 45461427 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1190435988 Total methylated C's in CpG context: 16228288 Total methylated C's in CHG context: 1247389 Total methylated C's in CHH context: 4895570 Total methylated C's in Unknown context: 62964 Total unmethylated C's in CpG context: 184257531 Total unmethylated C's in CHG context: 203330966 Total unmethylated C's in CHH context: 780476244 Total unmethylated C's in Unknown context: 3087205 C methylated in CpG context: 8.1% 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 36s ==================== Bismark run complete ====================