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-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF03-EM05-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-EM05-Larvae_score_L0-0.4/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.4 -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-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF03-EM05-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-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF03-EM05-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-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM05-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,-0.4 -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-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26775:1047_1:N:0:AATCCGGA+AACTGTAG/1 99 NC_035782.1_CT_converted 29879545 11 4M3I143M = 29879574 179 GNATTTATTAAATTTATTAAATTTAATGAATGGAGGAGGATGTGTTATTTTATTTGTTTTTAGTTGTAAAATAGTAATGTTTAATTTTTTATTGTGATAAAGAATTTTAGGTTTTAGAGAAAATTTTAAGGAATTAATTTGATAGTGTTT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,F:FFFF:FF AS:i:-27 XS:i:-45 XN:i:0 XM:i:3 XO:i:1 XG:i:3 NM:i:6 MD:Z:1T0G1G142 YS:i:0 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26775:1047_2:N:0:AATCCGGA+AACTGTAG/2 147 NC_035782.1_CT_converted 29879574 11 150M = 29879545 -179 GAGGAGGATGTGTTATTTTATTTGTTTTTAGTTGTAAAATAGTAATGTTTAATTTTTTATTGTGATAAAGAATTTTAGGTTTTAGAGAAAATTTTAAGGAATTAATTTGATAGTGTTTTTTAAATTTAAAATTGATAAATATTTTAGGAT FFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XS:i:-18 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:150 YS:i:-27 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26775:1047_1:N:0:AATCCGGA+AACTGTAG/1 77 * 0 0 * * 0 0 ANATTCATTAAATTTATTAAATTTAATAAATAAAAAAAAATATATTATTTTATTTATTTTTAATTATAAAATAATAATATTTAATTTTTTATTATAATAAAAAATTTTAAATTTTAAAAAAAATTTTAAAAAATTAATTTAATAATATTC F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,F:FFFF:FF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26775:1047_2:N:0:AATCCGGA+AACTGTAG/2 141 * 0 0 * * 0 0 ATTTTAAAATATTTATTAATTTTAAATTTAAAGAATATTATTAAATTAATTTTTTAAAATTTTTTTTAAAATTTAAAATTTTTTATTATAATAAAAAATTAAATATTATTATTTTATAATTAAAAATAAATAAAATAATATATTTTTTTT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26775:1047_1:N:0:AATCCGGA+AACTGTAG/1 77 * 0 0 * * 0 0 ANATTCATTAAATTTATTAAATTTAATAAATAAAAAAAAATATATTATTTTATTTATTTTTAATTATAAAATAATAATATTTAATTTTTTATTATAATAAAAAATTTTAAATTTTAAAAAAAATTTTAAAAAATTAATTTAATAATATTC F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,F:FFFF:FF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26775:1047_2:N:0:AATCCGGA+AACTGTAG/2 141 * 0 0 * * 0 0 ATTTTAAAATATTTATTAATTTTAAATTTAAAGAATATTATTAAATTAATTTTTTAAAATTTTTTTTAAAATTTAAAATTTTTTATTATAATAAAAAATTAAATATTATTATTTTATAATTAAAAATAAATAAAATAATATATTTTTTTT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.4 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26775:1047_1:N:0:AATCCGGA+AACTGTAG/1 77 * 0 0 * * 0 0 GNATTTATTAAATTTATTAAATTTAATGAATGGAGGAGGATGTGTTATTTTATTTGTTTTTAGTTGTAAAATAGTAATGTTTAATTTTTTATTGTGATAAAGAATTTTAGGTTTTAGAGAAAATTTTAAGGAATTAATTTGATAGTGTTT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,F:FFFF:FF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26775:1047_2:N:0:AATCCGGA+AACTGTAG/2 141 * 0 0 * * 0 0 ATCCTAAAATATTTATCAATTTTAAATTTAAAAAACACTATCAAATTAATTCCTTAAAATTTTCTCTAAAACCTAAAATTCTTTATCACAATAAAAAATTAAACATTACTATTTTACAACTAAAAACAAATAAAATAACACATCCTCCTC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:FFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF03-EM05-Larvae_L0-0.4_pe.bam <<< Reading in the sequence files ../../data/EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz 1000010000 reads; of these: reads; of these: 1000010000 ( (1000010000100.00100.00 reads; of these: reads; of these:%% ) were paired; of these:) were paired; of these: 1000010000 ( (89869050100.00 (100.00 (%89.86%90.50) were paired; of these:%) were paired; of these:% ) aligned concordantly 0 times ) aligned concordantly 0 times 9045 9040 (530 (49690.45 (90.40 (%5.30%4.96) aligned concordantly 0 times%) aligned concordantly 0 times% ) aligned concordantly exactly 1 time ) aligned concordantly exactly 1 time 497 472 (484 (4544.97 (4.72 (%4.84%4.54) aligned concordantly exactly 1 time%) aligned concordantly exactly 1 time% ) aligned concordantly >1 times ) aligned concordantly >1 times 45810.144889.50 (% (%4.58 overall alignment rate4.88 overall alignment rate% % ) aligned concordantly >1 times) aligned concordantly >1 times 9.559.60%% overall alignment rate overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF03-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF03-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM05-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: 78689 Total methylated C's in CpG context: 2467 Total methylated C's in CHG context: 528 Total methylated C's in CHH context: 2046 Total methylated C's in Unknown context: 84 Total unmethylated C's in CpG context: 9006 Total unmethylated C's in CHG context: 16086 Total unmethylated C's in CHH context: 48556 Total unmethylated C's in Unknown context: 261 C methylated in CpG context: 21.5% C methylated in CHG context: 3.2% C methylated in CHH context: 4.0% C methylated in Unknown context (CN or CHN): 24.3% Bismark completed in 0d 0h 0m 14s ==================== Bismark run complete ====================