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/EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF03-EM04-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/EF03-EM04-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/EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF03-EM04-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 EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF03-EM04-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 EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM04-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 EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM04-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:GW210315000:4:1101:26521:1047_1:N:0:GGCTTAAG+GGTCACGA/1 77 * 0 0 * * 0 0 TNTAAAATTAAATATATAAAATATAATTTTTTATTATATTTTAAATTTATTATTAATATTTTTAAAATTAATTTTTTTTTTTTATTTTTTTTAATTATTTTTTATAAAATATATTTTTAATTATA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFF,FFFFFFFFFFFF:FFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26521:1047_2:N:0:GGCTTAAG+GGTCACGA/2 141 * 0 0 * * 0 0 TATAATTAAAAATATATTTTATAAAAAATAATTAAAAAAAATAAAAAAAAAAAATTAATTTTAAAAATATTAATAATAAATTTAATATATAATAAAAAATTATATTTTATATATTTAATTTTAAA FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFF,F:FF:FFFFFFFFFFFFFF: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM04-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:GW210315000:4:1101:26521:1047_1:N:0:GGCTTAAG+GGTCACGA/1 77 * 0 0 * * 0 0 TNTAAAACTAAACACATAAAATATAATTTTTCATCATATTTTAAATCTACTATTAATATTTCTAAAATCAATTTTTTTTCTTCATTTTCTCTAACTATTTTCCATAAAATACATTTTTAATTATA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFF,FFFFFFFFFFFF:FFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26521:1047_2:N:0:GGCTTAAG+GGTCACGA/2 141 * 0 0 * * 0 0 TATAATTAAAAATGTATTTTATGGAAAATAGTTAGAGAAAATGAAGAAAAAAAATTGATTTTAGAAATATTAATAGTAGATTTAATATATGATGAAAAATTATATTTTATGTGTTTAGTTTTAAA FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFF,F:FF:FFFFFFFFFFFFFF: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM04-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:GW210315000:4:1101:26521:1047_1:N:0:GGCTTAAG+GGTCACGA/1 77 * 0 0 * * 0 0 TNTAAAACTAAACACATAAAATATAATTTTTCATCATATTTTAAATCTACTATTAATATTTCTAAAATCAATTTTTTTTCTTCATTTTCTCTAACTATTTTCCATAAAATACATTTTTAATTATA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFF,FFFFFFFFFFFF:FFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26521:1047_2:N:0:GGCTTAAG+GGTCACGA/2 141 * 0 0 * * 0 0 TATAATTAAAAATGTATTTTATGGAAAATAGTTAGAGAAAATGAAGAAAAAAAATTGATTTTAGAAATATTAATAGTAGATTTAATATATGATGAAAAATTATATTTTATGTGTTTAGTTTTAAA FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFF,F:FF:FFFFFFFFFFFFFF: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM04-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:GW210315000:4:1101:26521:1047_1:N:0:GGCTTAAG+GGTCACGA/1 77 * 0 0 * * 0 0 TNTAAAATTAAATATATAAAATATAATTTTTTATTATATTTTAAATTTATTATTAATATTTTTAAAATTAATTTTTTTTTTTTATTTTTTTTAATTATTTTTTATAAAATATATTTTTAATTATA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFF,FFFFFFFFFFFF:FFFFFFFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26521:1047_2:N:0:GGCTTAAG+GGTCACGA/2 141 * 0 0 * * 0 0 TATAATTAAAAATATATTTTATAAAAAATAATTAAAAAAAATAAAAAAAAAAAATTAATTTTAAAAATATTAATAATAAATTTAATATATAATAAAAAATTATATTTTATATATTTAATTTTAAA FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFF:FFFFFFFFFF:FFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFF,FFFFFFFFFFFFFFFFFF,F:FF:FFFFFFFFFFFFFF: YT:Z:UP >>> Writing bisulfite mapping results to EF03-EM04-Larvae_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these:10000 reads; of these: 10000 10000 reads; of these: ( 10000 (100.0010000% (100.00) were paired; of these:% 100.00) were paired; of these: % 7418) were paired; of these: ( 750074.18 (%776175.00) aligned concordantly 0 times (% 77.61) aligned concordantly 0 times % 1094) aligned concordantly 0 times ( 106210.94 (%90510.62) aligned concordantly exactly 1 time (% 9.05) aligned concordantly exactly 1 time % 1488) aligned concordantly exactly 1 time ( 143814.88 (%133414.38) aligned concordantly >1 times (% 13.34) aligned concordantly >1 times25.82% %) aligned concordantly >1 times25.00 overall alignment rate % 22.39 overall alignment rate% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7855 (78.55%) aligned concordantly 0 times 837 (8.37%) aligned concordantly exactly 1 time 1308 (13.08%) aligned concordantly >1 times 21.45% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF03-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF03-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM04-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: 159555 Total methylated C's in CpG context: 4566 Total methylated C's in CHG context: 1854 Total methylated C's in CHH context: 7690 Total methylated C's in Unknown context: 537 Total unmethylated C's in CpG context: 17634 Total unmethylated C's in CHG context: 31295 Total unmethylated C's in CHH context: 96516 Total unmethylated C's in Unknown context: 2177 C methylated in CpG context: 20.6% C methylated in CHG context: 5.6% C methylated in CHH context: 7.4% C methylated in Unknown context (CN or CHN): 19.8% Bismark completed in 0d 0h 0m 25s ==================== Bismark run complete ====================