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-bismark-align'): ../../data/EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF05-EM01-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-bismark-align/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-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-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-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF05-EM01-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-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF05-EM01-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-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM01-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,-0.6 -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 EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:28402:1047_1:N:0:CCGCGGTT+CTAGCGCT/1 77 * 0 0 * * 0 0 ANTTAAAATTATTAATTATTTAAATAAAAATTATATTAATAATAAAATTAATTTAATTATAATTTTTTTTAATTTATTAATATTTATTAATAATAATTTAATTTTAAATAAAATTATTAATATATTAATATATAATTTAATTTATTAAAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:28402:1047_2:N:0:CCGCGGTT+CTAGCGCT/2 141 * 0 0 * * 0 0 CAACCCATCTTATTAATTTAATAAATTAAATTATATATTAATATATTAATAATTTTATTTAAAATTAAATTATTATTAATAAATATTAATAAATTAAAAAAAATTATAATTAAATTAATTTTATTATTAATATAATTTTTATTTAAATAA FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFF:FFFFFFFFFFF:FFFFF::F,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFF:FF:FFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:28402:1047_1:N:0:CCGCGGTT+CTAGCGCT/1 77 * 0 0 * * 0 0 ANCCAAAACCATCAACCACCTAAATAAAAACTACATCAACAACAAAATTAACCTAATCACAACCTTCCTCAACCCACTAACATTCACCAACAACAACCCAACCCCAAACAAAACCACCAACACACTAACACACAACCTAACCCATCAAAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:28402:1047_2:N:0:CCGCGGTT+CTAGCGCT/2 141 * 0 0 * * 0 0 TAATTTATTTTATTGATTTGATGGGTTAGGTTGTGTGTTAGTGTGTTGGTGGTTTTGTTTGGGGTTGGGTTGTTGTTGGTGAATGTTAGTGGGTTGAGGAAGGTTGTGATTAGGTTAATTTTGTTGTTGATGTAGTTTTTATTTAGGTGG FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFF:FFFFFFFFFFF:FFFFF::F,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFF:FF:FFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:28402:1047_1:N:0:CCGCGGTT+CTAGCGCT/1 77 * 0 0 * * 0 0 ANCCAAAACCATCAACCACCTAAATAAAAACTACATCAACAACAAAATTAACCTAATCACAACCTTCCTCAACCCACTAACATTCACCAACAACAACCCAACCCCAAACAAAACCACCAACACACTAACACACAACCTAACCCATCAAAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:28402:1047_2:N:0:CCGCGGTT+CTAGCGCT/2 141 * 0 0 * * 0 0 TAATTTATTTTATTGATTTGATGGGTTAGGTTGTGTGTTAGTGTGTTGGTGGTTTTGTTTGGGGTTGGGTTGTTGTTGGTGAATGTTAGTGGGTTGAGGAAGGTTGTGATTAGGTTAATTTTGTTGTTGATGTAGTTTTTATTTAGGTGG FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFF:FFFFFFFFFFF:FFFFF::F,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFF:FF:FFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:28402:1047_1:N:0:CCGCGGTT+CTAGCGCT/1 77 * 0 0 * * 0 0 ANTTAAAATTATTAATTATTTAAATAAAAATTATATTAATAATAAAATTAATTTAATTATAATTTTTTTTAATTTATTAATATTTATTAATAATAATTTAATTTTAAATAAAATTATTAATATATTAATATATAATTTAATTTATTAAAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:28402:1047_2:N:0:CCGCGGTT+CTAGCGCT/2 141 * 0 0 * * 0 0 CAACCCATCTTATTAATTTAATAAATTAAATTATATATTAATATATTAATAATTTTATTTAAAATTAAATTATTATTAATAAATATTAATAAATTAAAAAAAATTATAATTAAATTAATTTTATTATTAATATAATTTTTATTTAAATAA FFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFF:FFFFFFFFFFF:FFFFF::F,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFF:FF:FFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF05-EM01-Larvae_pe.bam <<< Reading in the sequence files ../../data/EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 8475 (84.75%) aligned concordantly 0 times 657 (6.5710000% reads; of these:) aligned concordantly exactly 1 time 10000868 ( (8.68100.00%%) aligned concordantly >1 times) were paired; of these: 15.25 %8440 overall alignment rate ( 84.40%) aligned concordantly 0 times 698 (6.98%) aligned concordantly exactly 1 time 862 (8.62%) aligned concordantly >1 times 15.60% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8651 (86.51%) aligned concordantly 0 times 628 (6.28%) aligned concordantly exactly 1 time 721 (7.21%) aligned concordantly >1 times 13.49% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8688 (86.88%) aligned concordantly 0 times 611 (6.11%) aligned concordantly exactly 1 time 701 (7.01%) aligned concordantly >1 times 13.12% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF05-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF05-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF05-EM01-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: 115893 Total methylated C's in CpG context: 3294 Total methylated C's in CHG context: 621 Total methylated C's in CHH context: 2337 Total methylated C's in Unknown context: 232 Total unmethylated C's in CpG context: 14643 Total unmethylated C's in CHG context: 24691 Total unmethylated C's in CHH context: 70307 Total unmethylated C's in Unknown context: 561 C methylated in CpG context: 18.4% C methylated in CHG context: 2.5% C methylated in CHH context: 3.2% C methylated in Unknown context (CN or CHN): 29.3% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ====================