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/EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF05-EM06-Larvae_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/EF05-EM06-Larvae_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/EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF05-EM06-Larvae_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 EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF05-EM06-Larvae_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 EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM06-Larvae_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 EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM06-Larvae_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:GW210315000:4:1101:26630:1047_1:N:0:TTACAGGA+GCTTGTCA/1 77 * 0 0 * * 0 0 ANTATAATAAAAATTTTTTTATTAATATTAATATTAATAAAATTATAATTTTAAATATTTTAATTAAAATATATTAATAAATTATTTTATTATTTTATTATTTAAATTTATGG F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26630:1047_2:N:0:TTACAGGA+GCTTGTCA/2 141 * 0 0 * * 0 0 CCATAAATTTAAATAATAAAATAATAAAATAATTTATTAATATATTTTAATTAAAATATTTAAAATTATAATTTTATTAATATTAATATTAATAAAAAAATTTTTATTATAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFF:FF:FFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF05-EM06-Larvae_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:GW210315000:4:1101:26630:1047_1:N:0:TTACAGGA+GCTTGTCA/1 99 NC_035784.1_GA_converted 19813649 1 113M = 19813649 113 ANTATAATAAAAACCCCTCCATTAATATTAACATTAATAAAATCACAACTCTAAACATTTCAACTAAAACACACCAACAAACTACTTTATTACTTTATTATCTAAACCTACAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-13 XS:i:-13 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:1T108T1T0 YS:i:-12 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26630:1047_2:N:0:TTACAGGA+GCTTGTCA/2 147 NC_035784.1_GA_converted 19813649 1 113M = 19813649 -113 ATTATAATAAAAACCCCTCCATTAATATTAACATTAATAAAATCACAACTCTAAACATTTCAACTAAAACACACCAACAAACTACTTTATTACTTTATTATCTAAACCTACAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFF:FF:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-12 XS:i:-12 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:110T1T0 YS:i:-13 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF05-EM06-Larvae_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:GW210315000:4:1101:26630:1047_1:N:0:TTACAGGA+GCTTGTCA/1 77 * 0 0 * * 0 0 ANTATAATAAAAACCCCTCCATTAATATTAACATTAATAAAATCACAACTCTAAACATTTCAACTAAAACACACCAACAAACTACTTTATTACTTTATTATCTAAACCTACAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26630:1047_2:N:0:TTACAGGA+GCTTGTCA/2 141 * 0 0 * * 0 0 TTGTAGGTTTAGATAATAAAGTAATAAAGTAGTTTGTTGGTGTGTTTTAGTTGAAATGTTTAGAGTTGTGATTTTATTAATGTTAATATTAATGGAGGGGTTTTTATTATAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFF:FF:FFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM06-Larvae_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:GW210315000:4:1101:26630:1047_1:N:0:TTACAGGA+GCTTGTCA/1 77 * 0 0 * * 0 0 ANTATAATAAAAATTTTTTTATTAATATTAATATTAATAAAATTATAATTTTAAATATTTTAATTAAAATATATTAATAAATTATTTTATTATTTTATTATTTAAATTTATGG F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26630:1047_2:N:0:TTACAGGA+GCTTGTCA/2 141 * 0 0 * * 0 0 CCATAAATTTAAATAATAAAATAATAAAATAATTTATTAATATATTTTAATTAAAATATTTAAAATTATAATTTTATTAATATTAATATTAATAAAAAAATTTTTATTATAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFF:FF:FFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF05-EM06-Larvae_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 7765 (77.65%) aligned concordantly 0 times 915 (9.15%) aligned concordantly exactly 1 time 1320 (13.20%) aligned concordantly >1 times 22.35% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7683 (76.83%) aligned concordantly 0 times 911 (9.11%) aligned concordantly exactly 1 time 1406 (1000014.06 reads; of these:% ) aligned concordantly >1 times 1000023.17 (% overall alignment rate 100.00%) were paired; of these: 7709 (77.09%) aligned concordantly 0 times 926 (9.26%) aligned concordantly exactly 1 time 1365 (13.65%) aligned concordantly >1 times 22.91% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7766 (77.66%) aligned concordantly 0 times 886 (8.86%) aligned concordantly exactly 1 time 1348 (13.48%) aligned concordantly >1 times 22.34% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF05-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF05-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM06-Larvae_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: 201154 Total methylated C's in CpG context: 4914 Total methylated C's in CHG context: 1933 Total methylated C's in CHH context: 8239 Total methylated C's in Unknown context: 414 Total unmethylated C's in CpG context: 23195 Total unmethylated C's in CHG context: 36879 Total unmethylated C's in CHH context: 125994 Total unmethylated C's in Unknown context: 1955 C methylated in CpG context: 17.5% C methylated in CHG context: 5.0% C methylated in CHH context: 6.1% C methylated in Unknown context (CN or CHN): 17.5% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ====================