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/CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/CF05-CM02-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/CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz to CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz to CF05-CM02-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 CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz to CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz to CF05-CM02-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 CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM02-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 CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM02-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:GW210315000:4:1101:26051:1047_1:N:0:ACTAAGAT+CCGCGGTT/1 77 * 0 0 * * 0 0 ANTTTAAATTTAATTATATATATTTTATTTTTAATATTAAATAATTATAATTAATTATTAAAAAATAAAAATATTATAATAAATTATATTTTTAATTTTTAATTTAATAATAAATTTTTTATATATATTAAATAATAAAAAAAATATATA F#F:FFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFF:FFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFF:FF:FF:FFF,FFFFFF,,FFFFFFF:FFFFFFFFFFFFFFFFF:FFF:FF,FFFFFFFFFF:FFFF,FFFF,FFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26051:1047_2:N:0:ACTAAGAT+CCGCGGTT/2 141 * 0 0 * * 0 0 TTATATTTAATTTTTATTAAAAATTTAATTAATTTTTTATTTTTTTAATAAAATTTTATTATAAATTTTTTTTATTATTTAATATATATAAAAAATTTATTATTAAATTAAAAATTAAAAATATAATTTATTATAATATTTTTATTTTTT :FFFFFFF:FFFFFFFF:FFFFF,FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFF:FFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF,F,FFFFFFFFFFFF,FFF:,FFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM02-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:GW210315000:4:1101:26051:1047_1:N:0:ACTAAGAT+CCGCGGTT/1 99 NC_035780.1_GA_converted 5568298 17 150M = 5568357 209 ANTTTAAATTTAACCACATACACCTTATTTCCAATATTAAACAATTATAATCAACTATTAAAAAACAAAAATACCATAATAAATTATACTTTCAATTTTCAATTCAATAATAAACTCTTTACATATACCAAACAATAAAAAAAATATACA F#F:FFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFF:FFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFF:FF:FF:FFF,FFFFFF,,FFFFFFF:FFFFFFFFFFFFFFFFF:FFF:FF,FFFFFFFFFF:FFFF,FFFF,FFFF AS:i:-13 XS:i:-42 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:1C11A131T4 YS:i:-6 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26051:1047_2:N:0:ACTAAGAT+CCGCGGTT/2 147 NC_035780.1_GA_converted 5568357 17 150M = 5568298 -209 AAAAAACAAAAATACCATAATAAATTATACTTTCAATTTTCAATTCAATAATAAACTCTTTACATATACCAAACAATAAAAAAAATTTACAATAAAATTCTATTAAAAAAACAAAAAATTAATTAAATTTTTAATAAAAATCAAACACAA FFFFFFFFFFFFF,:FFF,FFFFFFFFFFFF,F,FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFF:FFFF:FFFFF:FFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFF,FFFFF:FFFFFFFF:FFFFFFF: AS:i:-6 XS:i:-29 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:106T43 YS:i:-13 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM02-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:GW210315000:4:1101:26051:1047_1:N:0:ACTAAGAT+CCGCGGTT/1 77 * 0 0 * * 0 0 ANTTTAAATTTAACCACATACACCTTATTTCCAATATTAAACAATTATAATCAACTATTAAAAAACAAAAATACCATAATAAATTATACTTTCAATTTTCAATTCAATAATAAACTCTTTACATATACCAAACAATAAAAAAAATATACA F#F:FFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFF:FFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFF:FF:FF:FFF,FFFFFF,,FFFFFFF:FFFFFFFFFFFFFFFFF:FFF:FF,FFFFFFFFFF:FFFF,FFFF,FFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26051:1047_2:N:0:ACTAAGAT+CCGCGGTT/2 141 * 0 0 * * 0 0 TTGTGTTTGATTTTTATTAAAAATTTAATTAATTTTTTGTTTTTTTAATAGAATTTTATTGTAAATTTTTTTTATTGTTTGGTATATGTAAAGAGTTTATTATTGAATTGAAAATTGAAAGTATAATTTATTATGGTATTTTTGTTTTTT :FFFFFFF:FFFFFFFF:FFFFF,FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFF:FFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF,F,FFFFFFFFFFFF,FFF:,FFFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM02-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:GW210315000:4:1101:26051:1047_1:N:0:ACTAAGAT+CCGCGGTT/1 77 * 0 0 * * 0 0 ANTTTAAATTTAATTATATATATTTTATTTTTAATATTAAATAATTATAATTAATTATTAAAAAATAAAAATATTATAATAAATTATATTTTTAATTTTTAATTTAATAATAAATTTTTTATATATATTAAATAATAAAAAAAATATATA F#F:FFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFF:FFFFFFF:FFF:FFFFFFFFFFFFFFFFFFFFF:FF:FF:FFF,FFFFFF,,FFFFFFF:FFFFFFFFFFFFFFFFF:FFF:FF,FFFFFFFFFF:FFFF,FFFF,FFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26051:1047_2:N:0:ACTAAGAT+CCGCGGTT/2 141 * 0 0 * * 0 0 TTATATTTAATTTTTATTAAAAATTTAATTAATTTTTTATTTTTTTAATAAAATTTTATTATAAATTTTTTTTATTATTTAATATATATAAAAAATTTATTATTAAATTAAAAATTAAAAATATAATTTATTATAATATTTTTATTTTTT :FFFFFFF:FFFFFFFF:FFFFF,FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFF:FFFFF:FFFF:FFFFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF,F,FFFFFFFFFFFF,FFF:,FFFFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to CF05-CM02-Larvae_pe.bam <<< Reading in the sequence files ../../data/CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz 1000010000 reads; of these: reads; of these: 1000010000 ( (100.00100.00%%) were paired; of these:) were paired; of these: 81428107 ( (81.4281.07%%) aligned concordantly 0 times) aligned concordantly 0 times 803861 ( (8.038.61%%) aligned concordantly exactly 1 time) aligned concordantly exactly 1 time 10551032 ( (10.5510.32%%) aligned concordantly >1 times) aligned concordantly >1 times 18.5818.93%% overall alignment rate overall alignment rate 10000 reads; of these: 1000010000 ( reads; of these: 100.00 %10000) were paired; of these: ( 100.008306% () were paired; of these:83.06 % ) aligned concordantly 0 times8319 ( 83.19673% () aligned concordantly 0 times6.73 % ) aligned concordantly exactly 1 time724 ( 7.241021% () aligned concordantly exactly 1 time10.21 % ) aligned concordantly >1 times957 (16.949.57%% overall alignment rate) aligned concordantly >1 times 16.81% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, CF05-CM02-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, CF05-CM02-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM02-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: 136978 Total methylated C's in CpG context: 3575 Total methylated C's in CHG context: 456 Total methylated C's in CHH context: 1692 Total methylated C's in Unknown context: 155 Total unmethylated C's in CpG context: 17203 Total unmethylated C's in CHG context: 29492 Total unmethylated C's in CHH context: 84560 Total unmethylated C's in Unknown context: 597 C methylated in CpG context: 17.2% C methylated in CHG context: 1.5% C methylated in CHH context: 2.0% C methylated in Unknown context (CN or CHN): 20.6% Bismark completed in 0d 0h 0m 17s ==================== Bismark run complete ====================