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_L0-0.8/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.8 -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,-0.8 -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,-0.8 -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,-0.8 -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 99 NC_035784.1_GA_converted 91080327 0 4M9I2M1I6M2I9M5I6M1I105M = 91080361 182 ANAAAAAAAATCTATCTCTATACCTACAAAAAAAATCTATCTCTATACATAAATTTTATCTCTCTCTTCTCTAACTTTTTTATTAAAATACCAACTCTACCAAATAATTTTTCAACATTTTTAAAAAAATAACCAACAATAAAAACTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, AS:i:-104 XN:i:0 XM:i:5 XO:i:5 XG:i:18 NM:i:23 MD:Z:1C5T6T1T11C103 YS:i:-80 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 147 NC_035784.1_GA_converted 91080361 0 113M1I15M1D4M1I6M1I9M = 91080327 -182 ATTTTATCTCTCTCTTCTCTAACTTTTTTATTAAAATACCAACTCTACCAAATAATTTTTCAACATTTTTAAAAAAATAACCAACAATAAAAACTAATAAAACTAATCTCATTAATTTTTATTATTATTATTTATTTTAATAATTTATAA F:FFFFFFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFF:FFFF,FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF,FF:FF:FFFFFFF:FFFFF,FFFF,:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-80 XN:i:0 XM:i:8 XO:i:4 XG:i:4 NM:i:12 MD:Z:115C1C2A3A3^C1A0A12C0C2 YS:i:-104 YT:Z:CP 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,-0.8 -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,-0.8 -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_L0-0.8_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 1000010000 reads; of these: reads; of these: 1000010000 (10000 (100.00 reads; of these:% ) were paired; of these:100.00 %10000 ) were paired; of these: (8221 (100.00 82.21%8550%) were paired; of these: () aligned concordantly 0 times 85.50 % 8562) aligned concordantly 0 times771 ( (85.62 7.71%585%) aligned concordantly 0 times () aligned concordantly exactly 1 time 5.85 % 577) aligned concordantly exactly 1 time1008 ( (5.77 10.08%865%) aligned concordantly exactly 1 time () aligned concordantly >1 times 8.65 %17.79861) aligned concordantly >1 times% ( overall alignment rate8.6114.50 %%) aligned concordantly >1 times overall alignment rate 14.38% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8251 (82.51%) aligned concordantly 0 times 781 (7.81%) aligned concordantly exactly 1 time 968 (9.68%) aligned concordantly >1 times 17.49% 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: 94386 Total methylated C's in CpG context: 2065 Total methylated C's in CHG context: 376 Total methylated C's in CHH context: 2343 Total methylated C's in Unknown context: 240 Total unmethylated C's in CpG context: 11476 Total unmethylated C's in CHG context: 19248 Total unmethylated C's in CHH context: 58878 Total unmethylated C's in Unknown context: 1049 C methylated in CpG context: 15.2% C methylated in CHG context: 1.9% C methylated in CHH context: 3.8% C methylated in Unknown context (CN or CHN): 18.6% Bismark completed in 0d 0h 0m 18s ==================== Bismark run complete ====================