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/1A3_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/1A3_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/1A3_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/1A3_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1A3_R1_001.fastp-trim.fq.gz to 1A3_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1A3_R1_001.fastp-trim.fq.gz (47045069 sequences in total) Writing a G -> A converted version of the input file 1A3_R2_001.fastp-trim.fq.gz to 1A3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1A3_R2_001.fastp-trim.fq.gz (47045069 sequences in total) Input files are 1A3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A3_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 1A3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A3_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: LH00526:197:22KFKVLT4:3:1101:39025:1070_1:N:0:TTCCAGGT+AAGCACTG/1 99 ptg000047l_CT_converted 9625691 40 37M1D88M = 9625749 179 AATTTGGTATATAGTAATAGTTTTATAATAGATTTAATTTTTTTTTTGTTTGTTTTTGTTTGTTTTTAGGGATAGTAATTAGGTTAGTAATTAGGTTAAGGTAAATTTATTTTAAGTGGTTAAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII AS:i:-26 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:6A8T21^T78A9 YS:i:-29 YT:Z:CP LH00526:197:22KFKVLT4:3:1101:39025:1070_2:N:0:TTCCAGGT+AAGCACTG/2 147 ptg000047l_CT_converted 9625749 40 100M4I21M = 9625691 -179 GTTTGTTTTTAGGGATAGTAATTAGGTTAGTAATTAGGTTAAGGTAAATTTATTTTAAGTGGTTAAATTGTTGAATAGTTAAAGATGGATTTAGTAATAAAATTAATGTTTTGTTTTTTGAAGTA 9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-29 XN:i:0 XM:i:2 XO:i:1 XG:i:4 NM:i:6 MD:Z:58A56A5 YS:i:-26 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1A3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A3_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: LH00526:197:22KFKVLT4:3:1101:39025:1070_1:N:0:TTCCAGGT+AAGCACTG/1 77 * 0 0 * * 0 0 AATTTGGTATATAGTAATAGTTTTATAATAGATTTAATTTTTTTTTTGTTTGTTTTTGTTTGTTTTTAGGGATAGTAATTAGGTTAGTAATTAGGTTAAGGTAAATTTATTTTAAGTGGTTAAAT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:39025:1070_2:N:0:TTCCAGGT+AAGCACTG/2 141 * 0 0 * * 0 0 TACTTCAAAAAACAAAACATTAATTTTATTACTAAATCCATCTTTAACTATTCAACAATTTAACCACTTAAAATAAATTTACCTTAACCTAATTACTAACCTAATTACTATCCCTAAAAACAAAC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9 YT:Z:UP >>> Writing bisulfite mapping results to 1A3_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/1A3_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/1A3_R2_001.fastp-trim.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1179:11512:27293_1:N:0:TTCCAGGT+AAGCACTG ptg000090l 2 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1238:9837:24561_1:N:0:TTCCAGGT+AAGCACTG ptg000017l 1 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1392:23278:12528_1:N:0:TTCCAGGT+AAGCACTG ptg000002l 1 Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2105:47668:28105_1:N:0:TTCCAGGT+AAGCACTG ptg000114l 51134 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2169:22355:24239_1:N:0:TTCCAGGT+AAGCACTG ptg000066l 66345 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2185:6114:19686_1:N:0:TTCCAGGT+AAGCACTG ptg000015l 14997092 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:3:2445:16917:11128_1:N:0:TTCCAGGT+AAGCACTG ptg000075l 1 Processed 23000000 sequence pairs so far 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 LH00526:197:22KFKVLT4:4:1228:44528:16563_1:N:0:TTCCAGGT+AAGCACTG ptg000007l 12295855 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1232:22833:10049_1:N:0:TTCCAGGT+AAGCACTG ptg000006l 3 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far 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 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1491:8841:19056_1:N:0:TTCCAGGT+AAGCACTG ptg000024l 3 Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2167:33612:11030_1:N:0:TTCCAGGT+AAGCACTG ptg000121l 50737 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2259:34712:12767_1:N:0:TTCCAGGT+AAGCACTG ptg000066l 66345 Processed 42000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2307:15517:2681_1:N:0:TTCCAGGT+AAGCACTG ptg000077l 42461 Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2424:4884:5903_1:N:0:TTCCAGGT+AAGCACTG ptg000018l 2 Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far 47045069 reads; of these: 47045069 (100.00%) were paired; of these: 18322981 (38.95%) aligned concordantly 0 times 8902165 (18.92%) aligned concordantly exactly 1 time 19819923 (42.13%) aligned concordantly >1 times 61.05% overall alignment rate 47045069 reads; of these: 47045069 (100.00%) were paired; of these: 18329428 (38.96%) aligned concordantly 0 times 8909765 (18.94%) aligned concordantly exactly 1 time 19805876 (42.10%) aligned concordantly >1 times 61.04% overall alignment rate Processed 47045069 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 188180276. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 188180276. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 188180276. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 188180276. Successfully deleted the temporary files 1A3_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1A3_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 47045069 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1320944720 Total methylated C's in CpG context: 18411742 Total methylated C's in CHG context: 1366977 Total methylated C's in CHH context: 5384791 Total methylated C's in Unknown context: 65893 Total unmethylated C's in CpG context: 195176818 Total unmethylated C's in CHG context: 225349826 Total unmethylated C's in CHH context: 875254566 Total unmethylated C's in Unknown context: 3320910 C methylated in CpG context: 8.6% C methylated in CHG context: 0.6% C methylated in CHH context: 0.6% C methylated in unknown context (CN or CHN): 1.9% Bismark completed in 0d 3h 6m 1s ==================== Bismark run complete ====================