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/EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF06-EM01-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/EF06-EM01-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/EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF06-EM01-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 EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF06-EM01-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 EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-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 EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 77 * 0 0 * * 0 0 TNTAAAATAAATAATTAATTATTAAATAATAAATATTATAATTAATTTTTATTTTTTATTAAATTAAATTTTTTAAAATAAAATTTTTATAATAAAAATTTTATTAAATATTTAAAAATTAAATATATAAAATAATTAAAAAATTATAAA F#FFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFF:FF::FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 141 * 0 0 * * 0 0 AATTAATTTTTTTTTTAAAATTATTATTTATTTTTTTTATAATTTTTTAATTATTTTATATATTTAATTTTTAAATATTTAATAAAATTTTTATTATAAAAATTTTATTTTAAAAAATTTAATTTAATAAAAAATAAAAATTAATTATAA FF,FFFFFFFFFFFFFFFFFFFF,FFFFFF,FFFFFFFF:FFFFFFFFF::F,FFFFF:F:F:FF:FFFFFFFF:FFFFFFF,:FFFFFFFFFFF,FFFFFFFFFFFFF::FFFFFFFFF:FF:FFFF:FFFF,:FFF:FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM01-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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 99 NC_035783.1_GA_converted 33762010 40 150M = 33762045 186 CNTAAAATAAACAATCAACCACCAAATAACAAATATTACAACCAATCTCTACCCTTCATTAAATTAAATTCTTTAAAACAAAACTTTTACAACAAAAATCTCACTAAATATCCAAAAATCAAACACACAAAACAACTAAAAAATTATAAA F#FFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFF:FF::FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-7 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:1C101A46 YS:i:-20 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 147 NC_035783.1_GA_converted 33762045 40 134M1D16M = 33762010 -186 TTACAACCAATCTCTACCCTTCATTAAATTAAATTCTTTAAAACAAAACTTTTACAACAAAAATCTCACTAAATATCCAAAAATCAAACACACAAAACAACTAAAAAATTATAAAAAAAATAAACAACAATCCCAAAAAAAAAACTAACC FFFFFFFFFFF:FFF:,FFFF:FFFF:FF:FFFFFFFFF::FFFFFFFFFFFFF,FFFFFFFFFFF:,FFFFFFF:FFFFFFFF:FF:F:F:FFFFF,F::FFFFFFFFF:FFFFFFFF,FFFFFF,FFFFFFFFFFFFFFFFFFFF,FF AS:i:-20 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:68A50C14^A16 YS:i:-7 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM01-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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 83 NC_035783.1_CT_converted 21935408 24 150M = 21935372 -186 TTTATAATTTTTTAGTTGTTTTGTGTGTTTGATTTTTGGATATTTAGTGAGATTTTTGTTGTAAAAGTTTTGTTTTAAAGAATTTAATTTAATGAAGGGTAGAGATTGGTTGTAATATTTGTTATTTGGTGGTTGATTGTTTATTTTANG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF::FF:FFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFF#F AS:i:-13 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:48A1A97G1 YS:i:-32 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 163 NC_035783.1_CT_converted 21935372 24 6M1D144M = 21935408 186 GGTTAGTTTTTTTTTTGGGATTGTTGTTTATTTTTTTTATAATTTTTTAGTTGTTTTGTGTGTTTGATTTTTGGATATTTAGTGAGATTTTTGTTGTAAAAGTTTTGTTTTAAAGAATTTAATTTAATGAAGGGTAGAGATTGGTTGTAA FF,FFFFFFFFFFFFFFFFFFFF,FFFFFF,FFFFFFFF:FFFFFFFFF::F,FFFFF:F:F:FF:FFFFFFFF:FFFFFFF,:FFFFFFFFFFF,FFFFFFFFFFFFF::FFFFFFFFF:FF:FFFF:FFFF,:FFF:FFFFFFFFFFF AS:i:-32 XN:i:0 XM:i:4 XO:i:1 XG:i:1 NM:i:5 MD:Z:6^T16T7G52A1A64 YS:i:-13 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 77 * 0 0 * * 0 0 TNTAAAATAAATAATTAATTATTAAATAATAAATATTATAATTAATTTTTATTTTTTATTAAATTAAATTTTTTAAAATAAAATTTTTATAATAAAAATTTTATTAAATATTTAAAAATTAAATATATAAAATAATTAAAAAATTATAAA F#FFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFF:FF::FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 141 * 0 0 * * 0 0 AATTAATTTTTTTTTTAAAATTATTATTTATTTTTTTTATAATTTTTTAATTATTTTATATATTTAATTTTTAAATATTTAATAAAATTTTTATTATAAAAATTTTATTTTAAAAAATTTAATTTAATAAAAAATAAAAATTAATTATAA FF,FFFFFFFFFFFFFFFFFFFF,FFFFFF,FFFFFFFF:FFFFFFFFF::F,FFFFF:F:F:FF:FFFFFFFF:FFFFFFF,:FFFFFFFFFFF,FFFFFFFFFFFFF::FFFFFFFFF:FF:FFFF:FFFF,:FFF:FFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF06-EM01-Larvae_L0-0.4_pe.bam <<< Reading in the sequence files ../../data/EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 8570 (85.70%) aligned concordantly 0 times 696 (6.96%) aligned concordantly exactly 1 time 734 (7.34%) aligned concordantly >1 times 14.30% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8772 (87.72%) aligned concordantly 0 times 603 (6.03%) aligned concordantly exactly 1 time 625 (6.25%) aligned concordantly >1 times 12.28% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8756 (87.56%) aligned concordantly 0 times 632 (6.32%) aligned concordantly exactly 1 time 612 (6.12%) aligned concordantly >1 times 12.44% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8602 (86.02%) aligned concordantly 0 times 658 (6.58%) aligned concordantly exactly 1 time 740 (7.40%) aligned concordantly >1 times 13.98% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-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: 116089 Total methylated C's in CpG context: 3144 Total methylated C's in CHG context: 449 Total methylated C's in CHH context: 1482 Total methylated C's in Unknown context: 64 Total unmethylated C's in CpG context: 13694 Total unmethylated C's in CHG context: 24020 Total unmethylated C's in CHH context: 73300 Total unmethylated C's in Unknown context: 301 C methylated in CpG context: 18.7% C methylated in CHG context: 1.8% C methylated in CHH context: 2.0% C methylated in Unknown context (CN or CHN): 17.5% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ====================