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/EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/EF02-EM02-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/EF02-EM02-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/EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF02-EM02-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 EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF02-EM02-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 EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-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 EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 77 * 0 0 * * 0 0 ANAAAAAAAATTTATTTTTATATTTATAAAAAAAATTTATTTTTATATATAAATTTTATTTTTTTTTTTTTTAATTTTTTTATTAAAATATTAATTTTATTAAATAATTTTTTAATATTTTTAAAAAAATAATTAATAATAAAAATTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 141 * 0 0 * * 0 0 TCATAAATCATTAAAACAAATAATAACAATAAAAACTAATAAAATTAATTTTATTAATTTTTATTATTAATTATTTTTTTAAAAATATTAAAAAATTATTTAATAAAATTAATATTTTAATAAAAAAATTAAAAAAAAAAAAAATAAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:,FFFF,FFFFF:FFFFFFF:FF:FF,FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFFFFFF:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 77 * 0 0 * * 0 0 ANAAAAAAAATCTATCTCTATACCTACAAAAAAAATCTATCTCTATACATAAATTTTATCTCTCTCTTCTCTAACTTTTTTATTAAAATACCAACTCTACCAAATAATTTTTCAACATTTTTAAAAAAATAACCAACAATAAAAACTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 141 * 0 0 * * 0 0 TTATAAATTATTAAAATAAATAATAATAATAAAAATTAATGAGATTAGTTTTATTAGTTTTTATTGTTGGTTATTTTTTTAAAAATGTTGAAAAATTATTTGGTAGAGTTGGTATTTTAATAAAAAAGTTAGAGAAGAGAGAGATAAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:,FFFF,FFFFF:FFFFFFF:FF:FF,FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFFFFFF:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 77 * 0 0 * * 0 0 ANAAAAAAAATCTATCTCTATACCTACAAAAAAAATCTATCTCTATACATAAATTTTATCTCTCTCTTCTCTAACTTTTTTATTAAAATACCAACTCTACCAAATAATTTTTCAACATTTTTAAAAAAATAACCAACAATAAAAACTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 141 * 0 0 * * 0 0 TTATAAATTATTAAAATAAATAATAATAATAAAAATTAATGAGATTAGTTTTATTAGTTTTTATTGTTGGTTATTTTTTTAAAAATGTTGAAAAATTATTTGGTAGAGTTGGTATTTTAATAAAAAAGTTAGAGAAGAGAGAGATAAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:,FFFF,FFFFF:FFFFFFF:FF:FF,FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFFFFFF:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 77 * 0 0 * * 0 0 ANAAAAAAAATTTATTTTTATATTTATAAAAAAAATTTATTTTTATATATAAATTTTATTTTTTTTTTTTTTAATTTTTTTATTAAAATATTAATTTTATTAAATAATTTTTTAATATTTTTAAAAAAATAATTAATAATAAAAATTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 141 * 0 0 * * 0 0 TCATAAATCATTAAAACAAATAATAACAATAAAAACTAATAAAATTAATTTTATTAATTTTTATTATTAATTATTTTTTTAAAAATATTAAAAAATTATTTAATAAAATTAATATTTTAATAAAAAAATTAAAAAAAAAAAAAATAAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:,FFFF,FFFFF:FFFFFFF:FF:FF,FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFFFFFF:F YT:Z:UP >>> Writing bisulfite mapping results to EF02-EM02-Zygote_L0-0.4_pe.bam <<< Reading in the sequence files ../../data/EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 9231 (92.31%) aligned concordantly 0 times 341 (3.41%) aligned concordantly exactly 1 time 428 (4.28%) aligned concordantly >1 times 7.69% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 9019 (90.19%) aligned concordantly 0 times 493 (4.93%) aligned concordantly exactly 1 time 488 (4.88%) aligned concordantly >1 times 9.81% overall alignment rate 10000 reads; of these: 10000 (10000100.00 reads; of these:% ) were paired; of these: 10000 (9040 (100.0090.40%%) were paired; of these:) aligned concordantly 0 times 9203477 ( (92.034.77%%) aligned concordantly 0 times) aligned concordantly exactly 1 time 373483 ( (3.734.83%%) aligned concordantly exactly 1 time) aligned concordantly >1 times 9.60424% ( overall alignment rate4.24 %) aligned concordantly >1 times 7.97% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-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: 55056 Total methylated C's in CpG context: 1314 Total methylated C's in CHG context: 199 Total methylated C's in CHH context: 865 Total methylated C's in Unknown context: 51 Total unmethylated C's in CpG context: 6647 Total unmethylated C's in CHG context: 11492 Total unmethylated C's in CHH context: 34539 Total unmethylated C's in Unknown context: 182 C methylated in CpG context: 16.5% C methylated in CHG context: 1.7% C methylated in CHH context: 2.4% C methylated in Unknown context (CN or CHN): 21.9% Bismark completed in 0d 0h 0m 18s ==================== Bismark run complete ====================