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_L0-0.8/ 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/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,-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 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,-0.8 -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,-0.8 -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 20135244 1 113M = 20135244 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 20135244 1 113M = 20135244 -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,-0.8 -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,-0.8 -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_L0-0.8_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: 8085 (80.85%) aligned concordantly 0 times 820 (8.20%) aligned concordantly exactly 1 time 1095 (10.95%) aligned concordantly >1 times 19.15% overall alignment rate 100001000010000 reads; of these: reads; of these: reads; of these: 100001000010000 ( ( (100.00%100.00) were paired; of these:100.00% %) were paired; of these: ) were paired; of these: 8039 ( 811180.398037 (% (81.11) aligned concordantly 0 times80.37% %) aligned concordantly 0 times ) aligned concordantly 0 times 813 ( 7818.13847 (% (7.81) aligned concordantly exactly 1 time8.47% %) aligned concordantly exactly 1 time ) aligned concordantly exactly 1 time 1148 ( 110811.481116 (% (11.08) aligned concordantly >1 times11.16% %) aligned concordantly >1 times19.61) aligned concordantly >1 times % 18.89 overall alignment rate19.63% % overall alignment rate 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: 180472 Total methylated C's in CpG context: 4607 Total methylated C's in CHG context: 1767 Total methylated C's in CHH context: 7113 Total methylated C's in Unknown context: 293 Total unmethylated C's in CpG context: 20635 Total unmethylated C's in CHG context: 33127 Total unmethylated C's in CHH context: 113223 Total unmethylated C's in Unknown context: 1220 C methylated in CpG context: 18.3% C methylated in CHG context: 5.1% C methylated in CHH context: 5.9% C methylated in Unknown context (CN or CHN): 19.4% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ====================