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_L-1-0.6/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,-1,-0.6 -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,-1,-0.6 -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,-1,-0.6 -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,-1,-0.6 -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,-1,-0.6 -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,-1,-0.6 -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_L-1-0.6_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%10000) were paired; of these:10000 reads; of these: 10000 reads; of these: reads; of these: 8985 10000 ( 10000 (89.8510000 (% (100.00) aligned concordantly 0 times100.00%100.00 %) were paired; of these:% ) were paired; of these: ) were paired; of these:453 ( 9009 4.539215 (9216% (90.09 () aligned concordantly exactly 1 time92.15%) aligned concordantly 0 times92.16 % % ) aligned concordantly 0 times ) aligned concordantly 0 times562 444 ( ( 5.623504.44373% (% () aligned concordantly >1 times3.50) aligned concordantly exactly 1 time3.73 % %10.15) aligned concordantly exactly 1 time ) aligned concordantly exactly 1 time% 547 overall alignment rate ( 4355.47411 (% (4.35) aligned concordantly >1 times4.11%% ) aligned concordantly >1 times) aligned concordantly >1 times9.91 %7.847.85 overall alignment rate%% overall alignment rate 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: 43705 Total methylated C's in CpG context: 1023 Total methylated C's in CHG context: 389 Total methylated C's in CHH context: 2025 Total methylated C's in Unknown context: 83 Total unmethylated C's in CpG context: 3973 Total unmethylated C's in CHG context: 8149 Total unmethylated C's in CHH context: 28146 Total unmethylated C's in Unknown context: 356 C methylated in CpG context: 20.5% C methylated in CHG context: 4.6% C methylated in CHH context: 6.7% C methylated in Unknown context (CN or CHN): 18.9% Bismark completed in 0d 0h 0m 23s ==================== Bismark run complete ====================