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/EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF04-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/EF04-EM05-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/EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF04-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 EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF04-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 EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF04-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,-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 EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF04-EM05-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:GW210315000:4:1101:25979:1047_1:N:0:CTTGGTAT+GGACTTGG/1 77 * 0 0 * * 0 0 ANTAATGGGGAGTTATAGATTTTTTATGTGATAGTATAAATAGTGGTTTGATTATAGTTTGATTAAATTTTATTTAAAGATTTTTTTGTTAAGTTTGGTTGTAATTGGTTTAGTTATTTTGGTAAAGGAAATGAAAATGTGAAAAAATTT F#FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFF:FFFF:FFFFFFFFF:FFFF:FFFFFFF,FFFFFF:FFFFFF:FFFFFFFFFF:FFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25979:1047_2:N:0:CTTGGTAT+GGACTTGG/2 141 * 0 0 * * 0 0 ATCAAAATTTAAATATAATCAAATAAAAAACCACTCTCCCTTTTAAATAATAAATTATTTAAATTAACAAAAATATAAAAATAATATCATAAATAAATTTTCTCCTAAAAATCAAACTTTTTCACTAAATCTCATATTTAAACTCATCCA FF:FFFFFFFFFFFF:FFFFFFFFFFFFFFF:F:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFF,FF,FFFFF:FFFF,:F:FF:FFF:FFFF,FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF04-EM05-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:GW210315000:4:1101:25979:1047_1:N:0:CTTGGTAT+GGACTTGG/1 77 * 0 0 * * 0 0 ANTAATAAAAAATTATAAATTTTTTATATAATAATATAAATAATAATTTAATTATAATTTAATTAAATTTTATTTAAAAATTTTTTTATTAAATTTAATTATAATTAATTTAATTATTTTAATAAAAAAAATAAAAATATAAAAAAATTT F#FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFF:FFFF:FFFFFFFFF:FFFF:FFFFFFF,FFFFFF:FFFFFF:FFFFFFFFFF:FFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25979:1047_2:N:0:CTTGGTAT+GGACTTGG/2 141 * 0 0 * * 0 0 ATTAAAATTTAAATATAATTAAATAAAAAATTATTTTTTTTTTTAAATAATAAATTATTTAAATTAATAAAAATATAAAAATAATATTATAAATAAATTTTTTTTTAAAAATTGAATTTTTTTATTAAATTTTATATTTAAATTTATTTG FF:FFFFFFFFFFFF:FFFFFFFFFFFFFFF:F:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFF,FF,FFFFF:FFFF,:F:FF:FFF:FFFF,FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF04-EM05-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:GW210315000:4:1101:25979:1047_1:N:0:CTTGGTAT+GGACTTGG/1 77 * 0 0 * * 0 0 ANTAATAAAAAATTATAAATTTTTTATATAATAATATAAATAATAATTTAATTATAATTTAATTAAATTTTATTTAAAAATTTTTTTATTAAATTTAATTATAATTAATTTAATTATTTTAATAAAAAAAATAAAAATATAAAAAAATTT F#FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFF:FFFF:FFFFFFFFF:FFFF:FFFFFFF,FFFFFF:FFFFFF:FFFFFFFFFF:FFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25979:1047_2:N:0:CTTGGTAT+GGACTTGG/2 141 * 0 0 * * 0 0 ATTAAAATTTAAATATAATTAAATAAAAAATTATTTTTTTTTTTAAATAATAAATTATTTAAATTAATAAAAATATAAAAATAATATTATAAATAAATTTTTTTTTAAAAATTGAATTTTTTTATTAAATTTTATATTTAAATTTATTTG FF:FFFFFFFFFFFF:FFFFFFFFFFFFFFF:F:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFF,FF,FFFFF:FFFF,:F:FF:FFF:FFFF,FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF04-EM05-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:GW210315000:4:1101:25979:1047_1:N:0:CTTGGTAT+GGACTTGG/1 83 NC_035785.1_GA_converted 37740619 42 134M1D16M = 37740383 -387 AAATTTTTTCACATTTTCATTTCCTTTACCAAAATAACTAAACCAATTACAACCAAACTTAACAAAAAAATCTTTAAATAAAATTTAATCAAACTATAATCAAACCACTATTTATACTATCACATAAAAAATCTATAACTCCCCATTANT FFFF:FFFFFFFFFF:FFFFFF:FFFFFF,FFFFFFF:FFFF:FFFFFFFFF:FFFF:FFFFFFFFF:FFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF#F AS:i:-33 XN:i:0 XM:i:5 XO:i:1 XG:i:1 NM:i:6 MD:Z:2T61T3T27A37^A14C1 YS:i:-26 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25979:1047_2:N:0:CTTGGTAT+GGACTTGG/2 163 NC_035785.1_GA_converted 37740383 42 44M1D106M = 37740619 387 ATCAAAATTTAAATATAATCAAATAAAAAACCACTCTCCCTTTTAAATAATAAATTATTTAAATTAACAAAAATATAAAAATAATATCATAAATAAATTTTCTCCTAAAAATCAAACTTTTTCACTAAATCTCATATTTAAACTCATCCA FF:FFFFFFFFFFFF:FFFFFFFFFFFFFFF:F:F:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF:FFFFFFF,FF,FFFFF:FFFF,:F:FF:FFF:FFFF,FFFFFFFFFFF AS:i:-26 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:1A0A41^A101T4 YS:i:-33 YT:Z:CP >>> Writing bisulfite mapping results to EF04-EM05-Larvae_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz 1000010000 reads; of these: reads; of these: 10000 10000 reads; of these:10000 ( ( 100.0010000100.00% (%) were paired; of these:) were paired; of these:100.00 % ) were paired; of these:77007612 ( ( 77.0076.127263%) aligned concordantly 0 times% ( ) aligned concordantly 0 times72.63 %905 ) aligned concordantly 0 times (952 9.05 ( %9.521080) aligned concordantly exactly 1 time% ( ) aligned concordantly exactly 1 time10.80 %1395 ) aligned concordantly exactly 1 time (1436 13.95 ( %14.361657) aligned concordantly >1 times% ( ) aligned concordantly >1 times16.5723.00 %%23.88) aligned concordantly >1 times overall alignment rate% overall alignment rate27.37 % overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7310 (73.10%) aligned concordantly 0 times 1077 (10.77%) aligned concordantly exactly 1 time 1613 (16.13%) aligned concordantly >1 times 26.90% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF04-EM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF04-EM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF04-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: 195843 Total methylated C's in CpG context: 4712 Total methylated C's in CHG context: 677 Total methylated C's in CHH context: 3878 Total methylated C's in Unknown context: 365 Total unmethylated C's in CpG context: 23970 Total unmethylated C's in CHG context: 40555 Total unmethylated C's in CHH context: 122051 Total unmethylated C's in Unknown context: 2150 C methylated in CpG context: 16.4% C methylated in CHG context: 1.6% C methylated in CHH context: 3.1% C methylated in Unknown context (CN or CHN): 14.5% Bismark completed in 0d 0h 0m 15s ==================== Bismark run complete ====================