Bowtie 2 seems to be working fine (tested command 'bowtie2 --version' [2.5.4]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools found here: '/srlab/programs/samtools-1.20/samtools' Reference genome folder provided is ../../data/genome/ (absolute path is '/mmfs1/gscratch/scrubbed/sr320/github/ceasmallr/data/genome/)' FastQ format assumed (by default) Processing sequences up to read no. 10000 from the input file Attention: early reports suggested that high values of -p to have diminishing returns. Please test different values using a small subset of data for your hardware setting. 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 '/mmfs1/gscratch/scrubbed/sr320/github/ceasmallr/output/04.2-bismark-align'): ../../data/CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz Library was specified to be not strand-specific (non-directional), therefore alignments to all four possible bisulfite strands (OT, CTOT, OB and CTOB) will be reported Output will be written into the directory: /mmfs1/gscratch/scrubbed/sr320/github/ceasmallr/output/04.2-bismark-align/CF02-CM02-Zygote_score_L0-0.4/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /mmfs1/gscratch/scrubbed/sr320/github/ceasmallr/output/04.2-bismark-align Now reading in and storing sequence information of the genome specified in: /mmfs1/gscratch/scrubbed/sr320/github/ceasmallr/data/genome/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../../data/CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq Created C -> T as well as G -> A converted versions of the FastQ file CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq (FastQ) Now running 4 individual instances of Bowtie 2 against the bisulfite genome of /mmfs1/gscratch/scrubbed/sr320/github/ceasmallr/data/genome/ with the specified options: -q --score-min L,0,-0.4 -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 CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25672:1047_1:N:0:TTGGACTC+CTGCTTCC/1 77 * 0 0 * * 0 0 TNGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTGATTTTATGTTATTTTTTTTGGGATTGTTAATAGATGGAAGAGTATATGTTTGAATTTTATTTATTTGGATTTATTTTGTATTTTGTTATTAGTATGAAAAAAGGGGGGGGGGGGG F#FFFFFFFFF:FFFFFFFF,FFFFF,FF,FF,FFFF:,FFF,FFF:F,FFFFF,,:F:FFFFFFFF,F::FFF,F,::FFF::F,FFF,,,FF,FFF:,:,FFF,:FF::,,FFF,F,::F:,F,,:,:,FFFF,,,,:,,FF,FFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25672:1047_2:N:0:TTGGACTC+CTGCTTCC/2 141 * 0 0 * * 0 0 AAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAATA F:,,,,:FFFF::::FFF:FFFFFFFFFFFFF:,F:,FF:::,:F,,,,:,,,F,:,,,:::,,,:,:FF,F,F:FF:::F:,::,F,,,:,FFFF:::,:::,:FF,F,,F:F:FFF::F:,,,,,,,,,,:FFF:FFFFFFFF::,,, YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25672:1047_1:N:0:TTGGACTC+CTGCTTCC/1 77 * 0 0 * * 0 0 CNATTATTATTATTATTATTATTATTATTATTATAATTTTATATTATTTTTTTTAAAATTATTAATAAATAAAAAAACACACATCTAAACTCCATTCACTTAAACTCATCTCATATTCCATCATCAACATAAAAAAAAAAAAAAAAAAAA F#FFFFFFFFF:FFFFFFFF,FFFFF,FF,FF,FFFF:,FFF,FFF:F,FFFFF,,:F:FFFFFFFF,F::FFF,F,::FFF::F,FFF,,,FF,FFF:,:,FFF,:FF::,,FFF,F,::F:,F,,:,:,FFFF,,,,:,,FF,FFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25672:1047_2:N:0:TTGGACTC+CTGCTTCC/2 141 * 0 0 * * 0 0 AGGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAATA F:,,,,:FFFF::::FFF:FFFFFFFFFFFFF:,F:,FF:::,:F,,,,:,,,F,:,,,:::,,,:,:FF,F,F:FF:::F:,::,F,,,:,FFFF:::,:::,:FF,F,,F:F:FFF::F:,,,,,,,,,,:FFF:FFFFFFFF::,,, YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25672:1047_1:N:0:TTGGACTC+CTGCTTCC/1 77 * 0 0 * * 0 0 CNATTATTATTATTATTATTATTATTATTATTATAATTTTATATTATTTTTTTTAAAATTATTAATAAATAAAAAAACACACATCTAAACTCCATTCACTTAAACTCATCTCATATTCCATCATCAACATAAAAAAAAAAAAAAAAAAAA F#FFFFFFFFF:FFFFFFFF,FFFFF,FF,FF,FFFF:,FFF,FFF:F,FFFFF,,:F:FFFFFFFF,F::FFF,F,::FFF::F,FFF,,,FF,FFF:,:,FFF,:FF::,,FFF,F,::F:,F,,:,:,FFFF,,,,:,,FF,FFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25672:1047_2:N:0:TTGGACTC+CTGCTTCC/2 141 * 0 0 * * 0 0 AGGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAATA F:,,,,:FFFF::::FFF:FFFFFFFFFFFFF:,F:,FF:::,:F,,,,:,,,F,:,,,:::,,,:,:FF,F,F:FF:::F:,::,F,,,:,FFFF:::,:::,:FF,F,,F:F:FFF::F:,,,,,,,,,,:FFF:FFFFFFFF::,,, YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25672:1047_1:N:0:TTGGACTC+CTGCTTCC/1 77 * 0 0 * * 0 0 TNGTTGTTGTTGTTGTTGTTGTTGTTGTTGTTGTGATTTTATGTTATTTTTTTTGGGATTGTTAATAGATGGAAGAGTATATGTTTGAATTTTATTTATTTGGATTTATTTTGTATTTTGTTATTAGTATGAAAAAAGGGGGGGGGGGGG F#FFFFFFFFF:FFFFFFFF,FFFFF,FF,FF,FFFF:,FFF,FFF:F,FFFFF,,:F:FFFFFFFF,F::FFF,F,::FFF::F,FFF,,,FF,FFF:,:,FFF,:FF::,,FFF,F,::F:,F,,:,:,FFFF,,,,:,,FF,FFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25672:1047_2:N:0:TTGGACTC+CTGCTTCC/2 141 * 0 0 * * 0 0 AAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAAAAAAAAAAAAAAAAAAAAAAAATA F:,,,,:FFFF::::FFF:FFFFFFFFFFFFF:,F:,FF:::,:F,,,,:,,,F,:,,,:::,,,:,:FF,F,F:FF:::F:,::,F,,,:,FFFF:::,:::,:FF,F,,F:F:FFF::F:,,,,,,,,,,:FFF:FFFFFFFF::,,, YT:Z:UP >>> Writing bisulfite mapping results to CF02-CM02-Zygote_L0-0.4_pe.bam <<< Reading in the sequence files ../../data/CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz 1000010000 reads; of these: reads; of these: 1000010000 ( (100.00%100.00) were paired; of these:% ) were paired; of these: 9326 (956793.26 (%95.67) aligned concordantly 0 times% ) aligned concordantly 0 times 327 (2003.27 (%2.00) aligned concordantly exactly 1 time% ) aligned concordantly exactly 1 time 347 (2333.47 (%2.33) aligned concordantly >1 times% ) aligned concordantly >1 times6.74 %4.33 overall alignment rate% overall alignment rate 10000 reads; of these: 10000 reads; of these:10000 ( 10000100.00 (%) were paired; of these:100.00 % ) were paired; of these:9321 ( 93.219555% () aligned concordantly 0 times95.55 % ) aligned concordantly 0 times309 ( 3.09221% () aligned concordantly exactly 1 time2.21 % ) aligned concordantly exactly 1 time370 ( 3.70224% () aligned concordantly >1 times2.24 %6.79) aligned concordantly >1 times% overall alignment rate4.45 % overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, CF02-CM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF02-CM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 10000 Final Cytosine Methylation Report ================================= Total number of C's analysed: 23701 Total methylated C's in CpG context: 643 Total methylated C's in CHG context: 237 Total methylated C's in CHH context: 980 Total methylated C's in Unknown context: 31 Total unmethylated C's in CpG context: 1853 Total unmethylated C's in CHG context: 4267 Total unmethylated C's in CHH context: 15721 Total unmethylated C's in Unknown context: 82 C methylated in CpG context: 25.8% C methylated in CHG context: 5.3% C methylated in CHH context: 5.9% C methylated in Unknown context (CN or CHN): 27.4% Bismark completed in 0d 0h 0m 14s ==================== Bismark run complete ====================