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/EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/EF03-EM03-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/EF03-EM03-Zygote_score_L0-1.0/ 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: /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/EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF03-EM03-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 EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF03-EM03-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 EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-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,-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 EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.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: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANATTATTATAAAATATATATATAAATAAAAATTAAATAAAAAAAATTGATTAATTAATTAATAAATTATTATAAATTATTATAATAATAAATAAATATTATTATATAAAATAAAATTATTAATAAAATTTATATTTATTAAAATAATAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTAATATTTTTATATTATTTTAATAAATATAAATTTTATTAATAATTTTATTTTATATAATAATATTTATTTATTATTATAATAATTTATAATAATTTATTAATTAATTAATCAATTTTTTTTATTTAATTTTTATTTATATATATATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.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: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANACCACTACAAAATACACATATAAATAAAAACCAAATAAAAAAAACTAACTAATTAACCAACAAATTATTATAAACCACCACAACAATAAACAAACACTACTACATAAAATAAAACCATCAATAAAACCTATACCCACCAAAATAATAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTGATATTTTTGTATTATTTTGGTGGGTATAGGTTTTATTGATGGTTTTATTTTATGTAGTAGTGTTTGTTTATTGTTGTGGTGGTTTATAATAATTTGTTGGTTAATTAGTTAGTTTTTTTTATTTGGTTTTTATTTATATGTGTATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.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: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANACCACTACAAAATACACATATAAATAAAAACCAAATAAAAAAAACTAACTAATTAACCAACAAATTATTATAAACCACCACAACAATAAACAAACACTACTACATAAAATAAAACCATCAATAAAACCTATACCCACCAAAATAATAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTGATATTTTTGTATTATTTTGGTGGGTATAGGTTTTATTGATGGTTTTATTTTATGTAGTAGTGTTTGTTTATTGTTGTGGTGGTTTATAATAATTTGTTGGTTAATTAGTTAGTTTTTTTTATTTGGTTTTTATTTATATGTGTATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.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: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANATTATTATAAAATATATATATAAATAAAAATTAAATAAAAAAAATTGATTAATTAATTAATAAATTATTATAAATTATTATAATAATAAATAAATATTATTATATAAAATAAAATTATTAATAAAATTTATATTTATTAAAATAATAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTAATATTTTTATATTATTTTAATAAATATAAATTTTATTAATAATTTTATTTTATATAATAATATTTATTTATTATTATAATAATTTATAATAATTTATTAATTAATTAATCAATTTTTTTTATTTAATTTTTATTTATATATATATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF03-EM03-Zygote_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 8419 (84.19%) aligned concordantly 0 times 631 (6.31%) aligned concordantly exactly 1 time 950 (9.50%) aligned concordantly >1 times 15.81% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8577 (85.77%) aligned concordantly 0 times 596 (5.96%) aligned concordantly exactly 1 time 827 (8.27%) aligned concordantly >1 times 14.23% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8385 (83.85%) aligned concordantly 0 times 648 (6.48%) aligned concordantly exactly 1 time 967 (9.67%) aligned concordantly >1 times 16.15% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8580 (85.80%) aligned concordantly 0 times 590 (5.90%) aligned concordantly exactly 1 time 830 (8.30%) aligned concordantly >1 times 14.20% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-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: 65611 Total methylated C's in CpG context: 1339 Total methylated C's in CHG context: 683 Total methylated C's in CHH context: 5005 Total methylated C's in Unknown context: 354 Total unmethylated C's in CpG context: 5814 Total unmethylated C's in CHG context: 11146 Total unmethylated C's in CHH context: 41624 Total unmethylated C's in Unknown context: 1071 C methylated in CpG context: 18.7% C methylated in CHG context: 5.8% C methylated in CHH context: 10.7% C methylated in Unknown context (CN or CHN): 24.8% Bismark completed in 0d 0h 0m 22s ==================== Bismark run complete ====================