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/1H2_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/1H2_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/1H2_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/1H2_R2_001.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 1H2_R1_001.fastp-trim.fq.gz to 1H2_R1_001.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 1H2_R1_001.fastp-trim.fq.gz (44192297 sequences in total) Writing a G -> A converted version of the input file 1H2_R2_001.fastp-trim.fq.gz to 1H2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 1H2_R2_001.fastp-trim.fq.gz (44192297 sequences in total) Input files are 1H2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H2_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 1H2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H2_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: LH00526:197:22KFKVLT4:3:1101:38750:1070_1:N:0:CAATGCGA+AGTTCGTC/1 77 * 0 0 * * 0 0 TTGGGTTTTTATTTTTTTTTTTATGAGAGTTGAATTGGGGGATTTTGTTTTGGAAGTTTTAAGTTGTGAAGGATTATTTGGAAATTTGATTTTGATGTTTTAAGTTGGTGGGTTTGTGAGAAAGG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIII-III-9IIIIIIIIIII9IIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:38750:1070_2:N:0:CAATGCGA+AGTTCGTC/2 141 * 0 0 * * 0 0 TTAAAAACTTAAATATACATATTAATATATAATATATATAAATAAACAACCCTTTCTCACAAACCCACCAACTTAAAACATCAAAATCAAATTTCCAAATAATCCTTCACAACTTAAAACTTCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 1H2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H2_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: LH00526:197:22KFKVLT4:3:1101:38750:1070_1:N:0:CAATGCGA+AGTTCGTC/1 77 * 0 0 * * 0 0 TTGGGTTTTTATTTTTTTTTTTATGAGAGTTGAATTGGGGGATTTTGTTTTGGAAGTTTTAAGTTGTGAAGGATTATTTGGAAATTTGATTTTGATGTTTTAAGTTGGTGGGTTTGTGAGAAAGG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIII-III-9IIIIIIIIIII9IIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00526:197:22KFKVLT4:3:1101:38750:1070_2:N:0:CAATGCGA+AGTTCGTC/2 141 * 0 0 * * 0 0 TTAAAAACTTAAATATACATATTAATATATAATATATATAAATAAACAACCCTTTCTCACAAACCCACCAACTTAAAACATCAAAATCAAATTTCCAAATAATCCTTCACAACTTAAAACTTCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP >>> Writing bisulfite mapping results to 1H2_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/1H2_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/1H2_R2_001.fastp-trim.fq.gz Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1126:40636:13887_1:N:0:CAATGCGA+AGTTCGTC ptg000066l 66384 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1139:33021:10932_1:N:0:CAATGCGA+AGTTCGTC ptg000090l 2 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1177:10622:13341_1:N:0:CAATGCGA+AGTTCGTC ptg000029c 1802544 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1196:2368:28441_1:N:0:CAATGCGA+AGTTCGTC ptg000018l 2 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 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:1364:6260:1252_1:N:0:CAATGCGA+AGTTCGTC ptg000015l 14997115 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2237:52013:16521_1:N:0:CAATGCGA+AGTTCGTC ptg000015l 14997091 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2287:42918:26046_1:N:0:CAATGCGA+AGTTCGTC ptg000093l 1 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2314:8429:2317_1:N:0:CAATGCGA+AGTTCGTC ptg000066l 66356 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2392:26612:3031_1:N:0:CAATGCGA+AGTTCGTC ptg000015l 14997111 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2455:12629:19448_1:N:0:CAATGCGA+AGTTCGTC ptg000129l 33362 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2470:36525:17837_1:N:0:CAATGCGA+AGTTCGTC ptg000144l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2472:8663:20905_1:N:0:CAATGCGA+AGTTCGTC ptg000009l 19095986 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2481:10727:27699_1:N:0:CAATGCGA+AGTTCGTC ptg000066l 66349 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:3:2495:5087:3339_1:N:0:CAATGCGA+AGTTCGTC ptg000024l 2 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 LH00526:197:22KFKVLT4:4:1172:37674:6379_1:N:0:CAATGCGA+AGTTCGTC ptg000012l 2 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:1385:34923:8704_1:N:0:CAATGCGA+AGTTCGTC ptg000059l 1573514 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 Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2310:24095:3409_1:N:0:CAATGCGA+AGTTCGTC ptg000009l 19095985 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2360:25293:12991_1:N:0:CAATGCGA+AGTTCGTC ptg000083l 1 Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2424:22720:27363_1:N:0:CAATGCGA+AGTTCGTC ptg000006l 3 Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2458:50168:20555_1:N:0:CAATGCGA+AGTTCGTC ptg000029c 1802552 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2469:15323:10609_1:N:0:CAATGCGA+AGTTCGTC ptg000024l 1 Chromosomal sequence could not be extracted for LH00526:197:22KFKVLT4:4:2470:3347:13271_1:N:0:CAATGCGA+AGTTCGTC ptg000006l 2 Processed 44000000 sequence pairs so far 44192297 reads; of these: 44192297 (100.00%) were paired; of these: 17126805 (38.76%) aligned concordantly 0 times 7910754 (17.90%) aligned concordantly exactly 1 time 19154738 (43.34%) aligned concordantly >1 times 61.24% overall alignment rate 44192297 reads; of these: 44192297 (100.00%) were paired; of these: 17136882 (38.78%) aligned concordantly 0 times 7909679 (17.90%) aligned concordantly exactly 1 time 19145736 (43.32%) aligned concordantly >1 times 61.22% overall alignment rate Processed 44192297 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 176769188. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 176769188. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 176769188. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 176769188. Successfully deleted the temporary files 1H2_R1_001.fastp-trim.fq.gz_C_to_T.fastq and 1H2_R2_001.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 44192297 Final Cytosine Methylation Report ================================= Total number of C's analysed: 1180742051 Total methylated C's in CpG context: 16371701 Total methylated C's in CHG context: 1382716 Total methylated C's in CHH context: 5223354 Total methylated C's in Unknown context: 64079 Total unmethylated C's in CpG context: 183099342 Total unmethylated C's in CHG context: 205765224 Total unmethylated C's in CHH context: 768899714 Total unmethylated C's in Unknown context: 2941454 C methylated in CpG context: 8.2% C methylated in CHG context: 0.7% C methylated in CHH context: 0.7% C methylated in unknown context (CN or CHN): 2.1% Bismark completed in 0d 2h 47m 3s ==================== Bismark run complete ====================