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-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF06-EM06-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-EM06-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/EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF06-EM06-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-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF06-EM06-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-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM06-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 EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM06-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:GW210315000:4:1101:26178:1047_1:N:0:CCAAGTCT+TCATCCTT/1 77 * 0 0 * * 0 0 ANATTATTAATATAATTTTTTAATTATTTAATTTTTTATTATTTATAATTATATATATTTTTTTTTAATTAAATATATAAAAAAAAGTTGGATTAATTAGAAAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26178:1047_2:N:0:CCAAGTCT+TCATCCTT/2 141 * 0 0 * * 0 0 TTTTCTAATTAATCCAACTTTTTTTTATATATTTAATTAAAAAAAAATATATATAATTATAAATAATAAAAAATTTAATAATTATAAAATTATATTTATTATTT FFFFFFFFFFFFFF:FF:FFFFFFFFFFFF,:FFFF:FFFF:,FFF,FFF:FFFFF:FF:,FF:FF,,FFFF,FF,FFF,,FFF,FFF,FFFFFFF,FF,,FFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM06-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:GW210315000:4:1101:26178:1047_1:N:0:CCAAGTCT+TCATCCTT/1 99 NC_035782.1_GA_converted 53013450 3 86M11I7M = 53013450 93 ANACCATCAATATAACTTTCTAACCATTCAACTTTTCATTATTCATAATTATACATACTTTCCTTTAATTAAACATATAAAAAAAAATTAAATTAATTAAAAAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF AS:i:-69 XN:i:0 XM:i:6 XO:i:1 XG:i:11 NM:i:17 MD:Z:1A5A20T11T45C4T1 YS:i:-74 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26178:1047_2:N:0:CCAAGTCT+TCATCCTT/2 147 NC_035782.1_GA_converted 53013450 3 86M11I7M = 53013450 -93 AAACAATAAATATAACTTTATAACCATTAAACTTTTCATTATTCATAATTATACATACTTTCCTTTAATTAAACATATAAAAAAAAATTAAATTAATTAAAAAA FFF,,FF,FFFFFFF,FFF,FFF,,FFF,FF,FFFF,,FF:FF,:FF:FFFFF:FFF,FFF,:FFFF:FFFF:,FFFFFFFFFFFF:FF:FFFFFFFFFFFFFF AS:i:-74 XN:i:0 XM:i:6 XO:i:1 XG:i:11 NM:i:17 MD:Z:4C14C8T11T45C4T1 YS:i:-69 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM06-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:GW210315000:4:1101:26178:1047_1:N:0:CCAAGTCT+TCATCCTT/1 77 * 0 0 * * 0 0 ANACCATCAATATAACTTTCTAACCATTCAACTTTTCATTATTCATAATTATACATACTTTCCTTTAATTAAACATATAAAAAAAAATTAAATTAATTAAAAAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26178:1047_2:N:0:CCAAGTCT+TCATCCTT/2 141 * 0 0 * * 0 0 TTTTTTAATTAATTTAATTTTTTTTTATATGTTTAATTAAAGGAAAGTATGTATAATTATGAATAATGAAAAGTTTAATGGTTATAAAGTTATATTTATTGTTT FFFFFFFFFFFFFF:FF:FFFFFFFFFFFF,:FFFF:FFFF:,FFF,FFF:FFFFF:FF:,FF:FF,,FFFF,FF,FFF,,FFF,FFF,FFFFFFF,FF,,FFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM06-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:GW210315000:4:1101:26178:1047_1:N:0:CCAAGTCT+TCATCCTT/1 77 * 0 0 * * 0 0 ANATTATTAATATAATTTTTTAATTATTTAATTTTTTATTATTTATAATTATATATATTTTTTTTTAATTAAATATATAAAAAAAAGTTGGATTAATTAGAAAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26178:1047_2:N:0:CCAAGTCT+TCATCCTT/2 141 * 0 0 * * 0 0 TTTTCTAATTAATCCAACTTTTTTTTATATATTTAATTAAAAAAAAATATATATAATTATAAATAATAAAAAATTTAATAATTATAAAATTATATTTATTATTT FFFFFFFFFFFFFF:FF:FFFFFFFFFFFF,:FFFF:FFFF:,FFF,FFF:FFFFF:FF:,FF:FF,,FFFF,FF,FFF,,FFF,FFF,FFFFFFF,FF,,FFF YT:Z:UP >>> Writing bisulfite mapping results to EF06-EM06-Larvae_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz 1000010000 reads; of these: reads; of these: 10000 reads; of these:1000010000 ( ( 10000100.00 (100.00%%) were paired; of these:100.00) were paired; of these: % ) were paired; of these: 7957 8004 ( (79.57795380.04% (%) aligned concordantly 0 times79.53) aligned concordantly 0 times % ) aligned concordantly 0 times 876 879 ( (8.768368.79% (%) aligned concordantly exactly 1 time8.36) aligned concordantly exactly 1 time % ) aligned concordantly exactly 1 time 1167 1117 ( (11.67121111.17% (%) aligned concordantly >1 times12.11) aligned concordantly >1 times % 20.43) aligned concordantly >1 times19.96% % overall alignment rate20.47 overall alignment rate % overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8048 (80.48%) aligned concordantly 0 times 820 (8.20%) aligned concordantly exactly 1 time 1132 (11.32%) aligned concordantly >1 times 19.52% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF06-EM06-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF06-EM06-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM06-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: 117904 Total methylated C's in CpG context: 2690 Total methylated C's in CHG context: 1078 Total methylated C's in CHH context: 6063 Total methylated C's in Unknown context: 405 Total unmethylated C's in CpG context: 12570 Total unmethylated C's in CHG context: 21437 Total unmethylated C's in CHH context: 74066 Total unmethylated C's in Unknown context: 2097 C methylated in CpG context: 17.6% C methylated in CHG context: 4.8% C methylated in CHH context: 7.6% C methylated in Unknown context (CN or CHN): 16.2% Bismark completed in 0d 0h 0m 26s ==================== Bismark run complete ====================