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 /mmfs1/gscratch/scrubbed/sr320/github/project-chilean-mussel/data/Mchi/ (absolute path is '/mmfs1/gscratch/scrubbed/sr320/github/project-chilean-mussel/data/Mchi/)' FastQ format assumed (by default) 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/project-chilean-mussel/output/06.1-bismark'): /mmfs1/gscratch/scrubbed/strigg/analyses/20250731_methylseq/raw-reads/LCo_BSr2_R1.fastq.gz /mmfs1/gscratch/scrubbed/strigg/analyses/20250731_methylseq/raw-reads/LCo_BSr2_R2.fastq.gz Library is assumed to be strand-specific (directional), alignments to strands complementary to the original top or bottom strands will be ignored (i.e. not performed!) Output will be written into the directory: /mmfs1/gscratch/scrubbed/sr320/github/project-chilean-mussel/output/06.1-bismark/ 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/project-chilean-mussel/output/06.1-bismark Now reading in and storing sequence information of the genome specified in: /mmfs1/gscratch/scrubbed/sr320/github/project-chilean-mussel/data/Mchi/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are /mmfs1/gscratch/scrubbed/strigg/analyses/20250731_methylseq/raw-reads/LCo_BSr2_R1.fastq.gz and /mmfs1/gscratch/scrubbed/strigg/analyses/20250731_methylseq/raw-reads/LCo_BSr2_R2.fastq.gz Input files are in FastQ format Writing a C -> T converted version of the input file LCo_BSr2_R1.fastq.gz to LCo_BSr2_R1.fastq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file LCo_BSr2_R1.fastq.gz (100000000 sequences in total) Writing a G -> A converted version of the input file LCo_BSr2_R2.fastq.gz to LCo_BSr2_R2.fastq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file LCo_BSr2_R2.fastq.gz (100000000 sequences in total) Input files are LCo_BSr2_R1.fastq.gz_C_to_T.fastq and LCo_BSr2_R2.fastq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /mmfs1/gscratch/scrubbed/sr320/github/project-chilean-mussel/data/Mchi/ 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 LCo_BSr2_R1.fastq.gz_C_to_T.fastq and LCo_BSr2_R2.fastq.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: A01057:543:H2JCGDSXF:1:1101:1832:1000_1:N:0:NCGGAGTT+GGATGTGA/1 99 Chromosome_6_CT_converted 104447923 42 151M = 104447956 186 NTTTTGTGGTTATAAAAATTGTGTTAAAATTTTATTGATTTTTATTTATTTATATTAAAGTTATTATTTAAAAATTATTTGATTTTGGAAATTTTTTTGAGGTTGTATAAAAATAATATTTTGTAATTTGTAAGATATTAAAAATAAAAAT #FFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFF:FFFFF,FFFFFFFF,FFFFFFFFFFFFFFFFF,,F:F,FFFFFFFFFFFFFFFFF,,FFFF:F:FFF,FFFF:FFF::FFFFFFF AS:i:-13 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:0T98T21G29 YS:i:-19 YT:Z:CP A01057:543:H2JCGDSXF:1:1101:1832:1000_2:N:0:NCGGAGTT+GGATGTGA/2 147 Chromosome_6_CT_converted 104447956 42 147M2D4M = 104447923 -186 ATTGATTTTTATTTATTTATATTAAAGTTATTATTTAAAAATTATTTGATTTTGGAAATTTTTTTGTGGTTGTATAAAAATNATATTTGGTAATTTGTAAGATATTAAAAATAAAAATTATAGTTTTAGTAAAAGTAATGTAGTTTTTTTN :FF,FFF,FFFFFFFFFFFFFFFFFF,FFFFFFF,FFFFFFFFFFF:FFFFFFF:FFFFFFF:FFFFFFFFFFFFFFFFFF#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF,FFFFFFFFFFFFFFFFFFFF:FF:F# AS:i:-19 XN:i:0 XM:i:3 XO:i:1 XG:i:2 NM:i:5 MD:Z:81A62G2^GA3T0 YS:i:-13 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from LCo_BSr2_R1.fastq.gz_C_to_T.fastq and LCo_BSr2_R2.fastq.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: A01057:543:H2JCGDSXF:1:1101:1832:1000_1:N:0:NCGGAGTT+GGATGTGA/1 77 * 0 0 * * 0 0 NTTTTGTGGTTATAAAAATTGTGTTAAAATTTTATTGATTTTTATTTATTTATATTAAAGTTATTATTTAAAAATTATTTGATTTTGGAAATTTTTTTGAGGTTGTATAAAAATAATATTTTGTAATTTGTAAGATATTAAAAATAAAAAT #FFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF:FFFFF:FFFFF,FFFFFFFF,FFFFFFFFFFFFFFFFF,,F:F,FFFFFFFFFFFFFFFFF,,FFFF:F:FFF,FFFF:FFF::FFFFFFF YT:Z:UP A01057:543:H2JCGDSXF:1:1101:1832:1000_2:N:0:NCGGAGTT+GGATGTGA/2 141 * 0 0 * * 0 0 NAAAAAAACTACATTACTTTTACTAAAACTATAATTTTTATTTTTAATATCTTACAAATTACCAAATATNATTTTTATACAACCACAAAAAAATTTCCAAAATCAAATAATTTTTAAATAATAACTTTAATATAAATAAATAAAAATCAAT #F:FF:FFFFFFFFFFFFFFFFFFFF,FFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF#FFFFFFFFFFFFFFFFFF:FFFFFFF:FFFFFFF:FFFFFFFFFFF,FFFFFFF,FFFFFFFFFFFFFFFFFF,FFF,FF: YT:Z:UP >>> Writing bisulfite mapping results to LCo_BSr2_pe.bam <<< Reading in the sequence files /mmfs1/gscratch/scrubbed/strigg/analyses/20250731_methylseq/raw-reads/LCo_BSr2_R1.fastq.gz and /mmfs1/gscratch/scrubbed/strigg/analyses/20250731_methylseq/raw-reads/LCo_BSr2_R2.fastq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for A01057:543:H2JCGDSXF:1:1420:11641:3458_1:N:0:CCGGAGTT+GGATGTGA Chromosome_12 2 Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for A01057:543:H2JCGDSXF:1:1476:31132:29105_1:N:0:CCGGAGTT+GGATGTGA Chromosome_1 1 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for A01057:543:H2JCGDSXF:1:1535:4309:20008_1:N:0:CCGGAGTT+GGATGTGA Chromosome_2 140585419 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far Chromosomal sequence could not be extracted for A01057:543:H2JCGDSXF:1:2164:7907:7952_1:N:0:CCGGAGTT+GGATGTGA Chromosome_9 133209698 Processed 59000000 sequence pairs so far Processed 60000000 sequence pairs so far Processed 61000000 sequence pairs so far Processed 62000000 sequence pairs so far Processed 63000000 sequence pairs so far Processed 64000000 sequence pairs so far Processed 65000000 sequence pairs so far Chromosomal sequence could not be extracted for A01057:543:H2JCGDSXF:1:2251:24433:5008_1:N:0:CCGGAGTT+GGATGTGA Chromosome_13 113386513 Processed 66000000 sequence pairs so far Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Processed 69000000 sequence pairs so far Processed 70000000 sequence pairs so far Processed 71000000 sequence pairs so far Processed 72000000 sequence pairs so far Processed 73000000 sequence pairs so far Processed 74000000 sequence pairs so far Processed 75000000 sequence pairs so far Processed 76000000 sequence pairs so far Processed 77000000 sequence pairs so far Processed 78000000 sequence pairs so far Processed 79000000 sequence pairs so far Chromosomal sequence could not be extracted for A01057:543:H2JCGDSXF:1:2426:8630:7670_1:N:0:CCGGAGTT+GGATGTGA Chromosome_12 2 Processed 80000000 sequence pairs so far Processed 81000000 sequence pairs so far Processed 82000000 sequence pairs so far Processed 83000000 sequence pairs so far Processed 84000000 sequence pairs so far Chromosomal sequence could not be extracted for A01057:543:H2JCGDSXF:1:2475:27227:12477_1:N:0:CCGGAGTT+GGATGTGA Chromosome_12 2 Processed 85000000 sequence pairs so far Processed 86000000 sequence pairs so far Processed 87000000 sequence pairs so far Processed 88000000 sequence pairs so far Processed 89000000 sequence pairs so far Processed 90000000 sequence pairs so far Processed 91000000 sequence pairs so far Processed 92000000 sequence pairs so far Processed 93000000 sequence pairs so far Processed 94000000 sequence pairs so far Processed 95000000 sequence pairs so far Processed 96000000 sequence pairs so far Processed 97000000 sequence pairs so far Processed 98000000 sequence pairs so far Processed 99000000 sequence pairs so far 100000000 reads; of these: 100000000 (100.00%) were paired; of these: 43497982 (43.50%) aligned concordantly 0 times 22028679 (22.03%) aligned concordantly exactly 1 time 34473339 (34.47%) aligned concordantly >1 times 56.50% overall alignment rate 100000000 reads; of these: 100000000 (100.00%) were paired; of these: 43426702 (43.43%) aligned concordantly 0 times 22059134 (22.06%) aligned concordantly exactly 1 time 34514164 (34.51%) aligned concordantly >1 times 56.57% overall alignment rate Processed 100000000 sequence pairs so far Processed 100000000 sequences in total Could not delete temporary files LCo_BSr2_R1.fastq.gz_C_to_T.fastq and LCo_BSr2_R2.fastq.gz_G_to_A.fastq properly: No such file or directory Final Alignment report ====================== Sequence pairs analysed in total: 100000000 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2661442491 Total methylated C's in CpG context: 50458999 Total methylated C's in CHG context: 9841039 Total methylated C's in CHH context: 48270635 Total methylated C's in Unknown context: 1856194 Total unmethylated C's in CpG context: 248851060 Total unmethylated C's in CHG context: 402932871 Total unmethylated C's in CHH context: 1901087887 Total unmethylated C's in Unknown context: 9879125 C methylated in CpG context: 16.9% C methylated in CHG context: 2.4% C methylated in CHH context: 2.5% C methylated in Unknown context (CN or CHN): 15.8% Bismark completed in 0d 4h 47m 58s ==================== Bismark run complete ====================