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/39--2E2_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/39--2E2_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/39--2E2_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/39--2E2_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 39--2E2_R1_001.fastp-trim.fq.gz to 39--2E2_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 39--2E2_R1_001.fastp-trim.fq.gz (38813143 sequences in total) Writing a G -> A converted version of the input file 39--2E2_R2_001.fastp-trim.fq.gz to 39--2E2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 39--2E2_R2_001.fastp-trim.fq.gz (38813143 sequences in total) Input files are 39--2E2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 39--2E2_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 39--2E2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 39--2E2_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: LH00652:104:22KG7JLT4:7:1101:4560:1084_1:N:0:CTTAGGAC+CAGTGAAG/1 77 * 0 0 * * 0 0 TGTTGAAAAGTGTTTATTTTTGTTGAGATGTATAAATGGGTTTTAGATGAAGGAGGATGATGGTGATTTTTTTTAGAGTTTTTTGGGGTATTTTGGGTAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00652:104:22KG7JLT4:7:1101:4560:1084_2:N:0:CTTAGGAC+CAGTGAAG/2 141 * 0 0 * * 0 0 TAAAAAAAATCACCATCATCCTCCTTCATCTAAAACCCATTTATACATCTCAACAAAAATAAACACTTTTCAACATAATCTAACCACAAATTTACTTAAC IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIII-IIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 39--2E2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 39--2E2_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: LH00652:104:22KG7JLT4:7:1101:4560:1084_1:N:0:CTTAGGAC+CAGTGAAG/1 83 ptg000018l_GA_converted 8565526 42 100M = 8565551 125 ATACCCAAAATACCCCAAAAAACTCTAAAAAAAATCACCATCATCCTCCTTCATCTAAAACCCATTTATACATCTCAACAAAAATAAACACTTTTCAACA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YS:i:-6 YT:Z:CP LH00652:104:22KG7JLT4:7:1101:4560:1084_2:N:0:CTTAGGAC+CAGTGAAG/2 163 ptg000018l_GA_converted 8565551 42 100M = 8565526 -125 TAAAAAAAATCACCATCATCCTCCTTCATCTAAAACCCATTTATACATCTCAACAAAAATAAACACTTTTCAACATAATCTAACCACAAATTTACTTAAC IIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIII-IIIII AS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:75C24 YS:i:0 YT:Z:CP >>> Writing bisulfite mapping results to 39--2E2_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/39--2E2_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/39--2E2_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1169:6809:19300_1:N:0:CTTAGGAC+CAGTGAAG ptg000066l 66370 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:1319:31111:29557_1:N:0:CTTAGGAC+CAGTGAAG ptg000018l 2 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far 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 LH00652:104:22KG7JLT4:7:2187:33522:2121_1:N:0:CTTAGGAC+CAGTGAAG ptg000120l 1 Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2387:20465:23812_1:N:0:CTTAGGAC+CAGTGAAG ptg000017l 2 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2396:50478:10444_1:N:0:CTTAGGAC+CAGTGAAG ptg000059l 1573514 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:7:2403:51797:21920_1:N:0:CTTAGGAC+CAGTGAAG ptg000024l 2 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 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1231:5077:19524_1:N:0:CTTAGGAC+CAGTGAAG ptg000025l 21443080 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1246:31435:5596_1:N:0:CTTAGGAC+CAGTGAAG ptg000133l 37112 Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:1270:51635:16988_1:N:0:CTTAGGAC+CAGTGAAG ptg000015l 14997092 Processed 24000000 sequence pairs so far 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 LH00652:104:22KG7JLT4:8:1420:20796:17717_1:N:0:CTTAGGAC+CAGTGAAG ptg000066l 66407 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2121:30351:15461_1:N:0:CTTAGGAC+CAGTGAAG ptg000116l 48475 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 LH00652:104:22KG7JLT4:8:2329:47396:21276_1:N:0:CTTAGGAC+CAGTGAAG ptg000033l 2625623 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2413:17245:6913_1:N:0:CTTAGGAC+CAGTGAAG ptg000082l 2 Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00652:104:22KG7JLT4:8:2482:10983:28464_1:N:0:CTTAGGAC+CAGTGAAG ptg000018l 2 38813143 reads; of these: 38813143 (100.00%) were paired; of these: 13590426 (35.02%) aligned concordantly 0 times 7726747 (19.91%) aligned concordantly exactly 1 time 17495970 (45.08%) aligned concordantly >1 times 64.98% overall alignment rate 38813143 reads; of these: 38813143 (100.00%) were paired; of these: 13582154 (34.99%) aligned concordantly 0 times 7720707 (19.89%) aligned concordantly exactly 1 time 17510282 (45.11%) aligned concordantly >1 times 65.01% overall alignment rate Processed 38813143 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 155252572. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 155252572. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 155252572. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 155252572. Successfully deleted the temporary files 39--2E2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 39--2E2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 38813143 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1154045774 Total methylated C's in CpG context: 15124793 Total methylated C's in CHG context: 1273827 Total methylated C's in CHH context: 4989383 Total methylated C's in Unknown context: 57130 Total unmethylated C's in CpG context: 173060432 Total unmethylated C's in CHG context: 199516557 Total unmethylated C's in CHH context: 760080782 Total unmethylated C's in Unknown context: 2853301 C methylated in CpG context: 8.0% C methylated in CHG context: 0.6% C methylated in CHH context: 0.7% C methylated in unknown context (CN or CHN): 2.0% Bismark completed in 0d 2h 39m 18s ==================== Bismark run complete ==================== Unable to flush stdout: Broken pipe