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/EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF08-EM03-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/EF08-EM03-Larvae_score_L0-0.8/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.8 -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/EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF08-EM03-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 EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF08-EM03-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 EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM03-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,-0.8 -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 EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25400:1047_1:N:0:AATGCCTC+TGGATCGA/1 77 * 0 0 * * 0 0 TNATAAATTAATTAAATTTTTATTAATTTTTAAATTTTTTAAAATTTTTATTTTATATAATAATAAATTTTTTTTAATAATAATAAATTATATTATTAAATAAATAAATATATTTTTTTATTATTTTTAAATAATTAAAATTATTTT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25400:1047_2:N:0:AATGCCTC+TGGATCGA/2 141 * 0 0 * * 0 0 AAAATAATTTTAATTATTTAAAAATAATAAAAAAATATATTTATTTATTTAATAATATAATTTATTATTATTAAAAAAAATTTATTATTATATAAAATAAAAATTTTAAAAAATTTAAAAATTAATAAAAATTTAATTAATTTATTA FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF,FFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFF:F:FFFFFFFFFFFFFFFFFFFF:FFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25400:1047_1:N:0:AATGCCTC+TGGATCGA/1 99 NC_035787.1_GA_converted 40960853 3 105M1D17M1I24M = 40960853 147 CNACAAATCAACTAAACCTTTACTAATCTCTAAATTTTCTAAAACTCTTACCCCATACAATAATAAATTTTTCCTAATAATAATAAACCATATCACCAAATAAATAAATACATCCCCTTACCATTCCTAAACAACTAAAATTACCCC F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-71 XN:i:0 XM:i:10 XO:i:2 XG:i:2 NM:i:12 MD:Z:1T0C42C23C0A19A0T13^A17C3C17A1 YS:i:-76 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25400:1047_2:N:0:AATGCCTC+TGGATCGA/2 147 NC_035787.1_GA_converted 40960853 3 105M1D17M1I24M = 40960853 -147 CAACAAATCAACTAAACCTTTACTAATCTCTAAATTTTCTAAAACTCTTACCCCATACAATAATAAATTTTTCCTAATAATAATAAACCATATCACCAAATAAATAAATACATCCCCTTACCATTCCTAAACAACTAAAATTACCCC FFFFFF:FFFFFFFFFFFFFFFFFFFF:F:FFFFFFF::FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFF AS:i:-76 XN:i:0 XM:i:10 XO:i:2 XG:i:2 NM:i:12 MD:Z:1T0C42C23C0A19A0T13^A17C3C17A1 YS:i:-71 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25400:1047_1:N:0:AATGCCTC+TGGATCGA/1 83 NC_035787.1_CT_converted 43773939 2 19M1I19M1D108M = 43773939 -147 GGGGTAATTTTAGTTGTTTAGGAATGGTAAGGGGATGTATTTATTTATTTGGTGATATGGTTTATTATTATTAGGAAAAATTTATTATTGTATGGGGTAAGAGTTTTAGAAAATTTAGAGATTAGTAAAGGTTTAGTTGATTTGTNG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF#F AS:i:-95 XS:i:-101 XN:i:0 XM:i:14 XO:i:2 XG:i:2 NM:i:16 MD:Z:1T19G1G5T8^T6G7A1A0T6T12T0G11T11G43A1 YS:i:-100 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25400:1047_2:N:0:AATGCCTC+TGGATCGA/2 163 NC_035787.1_CT_converted 43773939 2 19M1I19M1D108M = 43773939 147 GGGGTAATTTTAGTTGTTTAGGAATGGTAAGGGGATGTATTTATTTATTTGGTGATATGGTTTATTATTATTAGGAAAAATTTATTATTGTATGGGGTAAGAGTTTTAGAAAATTTAGAGATTAGTAAAGGTTTAGTTGATTTGTTG FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF,FFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFF:F:FFFFFFFFFFFFFFFFFFFF:FFFFFF AS:i:-100 XS:i:-106 XN:i:0 XM:i:14 XO:i:2 XG:i:2 NM:i:16 MD:Z:1T19G1G5T8^T6G7A1A0T6T12T0G11T11G43A1 YS:i:-95 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25400:1047_1:N:0:AATGCCTC+TGGATCGA/1 77 * 0 0 * * 0 0 TNATAAATTAATTAAATTTTTATTAATTTTTAAATTTTTTAAAATTTTTATTTTATATAATAATAAATTTTTTTTAATAATAATAAATTATATTATTAAATAAATAAATATATTTTTTTATTATTTTTAAATAATTAAAATTATTTT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25400:1047_2:N:0:AATGCCTC+TGGATCGA/2 141 * 0 0 * * 0 0 AAAATAATTTTAATTATTTAAAAATAATAAAAAAATATATTTATTTATTTAATAATATAATTTATTATTATTAAAAAAAATTTATTATTATATAAAATAAAAATTTTAAAAAATTTAAAAATTAATAAAAATTTAATTAATTTATTA FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF,FFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF::FFFFFFF:F:FFFFFFFFFFFFFFFFFFFF:FFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF08-EM03-Larvae_L0-0.8_pe.bam <<< Reading in the sequence files ../../data/EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 7634 (76.34%) aligned concordantly 0 times 967 (9.67%) aligned concordantly exactly 1 time 1399 (13.99%) aligned concordantly >1 times 23.66% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7783 (1000077.83 reads; of these:% ) aligned concordantly 0 times 10000 (861 (100.008.61%%) were paired; of these:) aligned concordantly exactly 1 time 76471356 ( (76.4713.56%%) aligned concordantly 0 times) aligned concordantly >1 times 22.17939% ( overall alignment rate9.39 %) aligned concordantly exactly 1 time 1414 (14.14%) aligned concordantly >1 times 23.53% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7748 (77.48%) aligned concordantly 0 times 857 (8.57%) aligned concordantly exactly 1 time 1395 (13.95%) aligned concordantly >1 times 22.52% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF08-EM03-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF08-EM03-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM03-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: 158861 Total methylated C's in CpG context: 4195 Total methylated C's in CHG context: 616 Total methylated C's in CHH context: 2959 Total methylated C's in Unknown context: 214 Total unmethylated C's in CpG context: 19673 Total unmethylated C's in CHG context: 33862 Total unmethylated C's in CHH context: 97556 Total unmethylated C's in Unknown context: 920 C methylated in CpG context: 17.6% C methylated in CHG context: 1.8% C methylated in CHH context: 2.9% C methylated in Unknown context (CN or CHN): 18.9% Bismark completed in 0d 0h 0m 21s ==================== Bismark run complete ====================