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/CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/CF08-CM05-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/CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to CF08-CM05-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 CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to CF08-CM05-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 CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF08-CM05-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 CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF08-CM05-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:GW210315000:4:1101:25780:1047_1:N:0:CGTTAGAA+GACCTGAA/1 77 * 0 0 * * 0 0 TNTTAATTTTAGAGGTATTATTGTGATGGTTTGTATTAATGTTTTAAATTTGGTGATTTTGTAGTTTTTTATTAATATTTTAAATAATTTTTAGTTTGATTATAGTGTTAGTGGTGTTGGTTATGTGGTGTTTGGTTGAGTGATTGTGGG F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF:FFFFFFFFF:FFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25780:1047_2:N:0:CGTTAGAA+GACCTGAA/2 141 * 0 0 * * 0 0 ACCACTTCCATACCCACAATCACTCAACCAAACACCACATAACCAACACCACTAACACTATAATCAAACTAAAAATTATTTAAAATATTAATAAAAAACTACAAAATCACCAAATTTAAAACATTAATACAAACCATCACAATAATACCT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF08-CM05-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:GW210315000:4:1101:25780:1047_1:N:0:CGTTAGAA+GACCTGAA/1 77 * 0 0 * * 0 0 CNCTAATTTTAAAAATATTATTATAATAATTTATATTAATATTTTAAATTTAATAATTTTATAATTTTTTATTAATATTTTAAATAATTTTTAATTTAATTATAATATTAATAATATTAATTATATAATATTTAATTAAATAATTATAAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF:FFFFFFFFF:FFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25780:1047_2:N:0:CGTTAGAA+GACCTGAA/2 141 * 0 0 * * 0 0 ATTATTTTTATATTTATAATTATTTAATTAAATATTATATAATTAATATTATTAATATTATAATTAAATTAAAAATTATTTAAAATATTAATAAAAAATTATAAAATTATTAAATTTAAAATATTAATATAAATTATTATAATAATATTT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF08-CM05-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:GW210315000:4:1101:25780:1047_1:N:0:CGTTAGAA+GACCTGAA/1 77 * 0 0 * * 0 0 CNCTAATTTTAAAAATATTATTATAATAATTTATATTAATATTTTAAATTTAATAATTTTATAATTTTTTATTAATATTTTAAATAATTTTTAATTTAATTATAATATTAATAATATTAATTATATAATATTTAATTAAATAATTATAAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF:FFFFFFFFF:FFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25780:1047_2:N:0:CGTTAGAA+GACCTGAA/2 141 * 0 0 * * 0 0 ATTATTTTTATATTTATAATTATTTAATTAAATATTATATAATTAATATTATTAATATTATAATTAAATTAAAAATTATTTAAAATATTAATAAAAAATTATAAAATTATTAAATTTAAAATATTAATATAAATTATTATAATAATATTT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF08-CM05-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:GW210315000:4:1101:25780:1047_1:N:0:CGTTAGAA+GACCTGAA/1 77 * 0 0 * * 0 0 TNTTAATTTTAGAGGTATTATTGTGATGGTTTGTATTAATGTTTTAAATTTGGTGATTTTGTAGTTTTTTATTAATATTTTAAATAATTTTTAGTTTGATTATAGTGTTAGTGGTGTTGGTTATGTGGTGTTTGGTTGAGTGATTGTGGG F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF:FFFFFFFFF:FFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25780:1047_2:N:0:CGTTAGAA+GACCTGAA/2 141 * 0 0 * * 0 0 ACCACTTCCATACCCACAATCACTCAACCAAACACCACATAACCAACACCACTAACACTATAATCAAACTAAAAATTATTTAAAATATTAATAAAAAACTACAAAATCACCAAATTTAAAACATTAATACAAACCATCACAATAATACCT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to CF08-CM05-Larvae_pe.bam <<< Reading in the sequence files ../../data/CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.0010000% reads; of these:) were paired; of these: 100009906 ( (99.06%) aligned concordantly 0 times100.00 % ) were paired; of these:38 ( 0.389912% () aligned concordantly exactly 1 time99.12 % ) aligned concordantly 0 times56 ( 0.5642% () aligned concordantly >1 times0.42 %0.94) aligned concordantly exactly 1 time% overall alignment rate 46 (0.46%) aligned concordantly >1 times 0.88% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 9909 (99.09%) aligned concordantly 0 times 47 (0.47%) aligned concordantly exactly 1 time 44 (0.44%) aligned concordantly >1 times 0.91% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 9892 (98.92%) aligned concordantly 0 times 52 (0.52%) aligned concordantly exactly 1 time 56 (0.56%) aligned concordantly >1 times 1.08% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, CF08-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, CF08-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF08-CM05-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: 8450 Total methylated C's in CpG context: 105 Total methylated C's in CHG context: 21 Total methylated C's in CHH context: 184 Total methylated C's in Unknown context: 14 Total unmethylated C's in CpG context: 945 Total unmethylated C's in CHG context: 1508 Total unmethylated C's in CHH context: 5687 Total unmethylated C's in Unknown context: 53 C methylated in CpG context: 10.0% C methylated in CHG context: 1.4% C methylated in CHH context: 3.1% C methylated in Unknown context (CN or CHN): 20.9% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ====================