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/CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/CF03-CM03-Zygote_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/CF03-CM03-Zygote_score_L0-0.6/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.6 -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/CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF03-CM03-Zygote_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 CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF03-CM03-Zygote_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 CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM03-Zygote_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,-0.6 -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 CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25274:1047_1:N:0:GGACTTGG+CGTCTGCG/1 77 * 0 0 * * 0 0 TNATTAAATATAATATAAAATATTTTTAAATTAAAAATAAATAAAAAATTATATATTATTTTAAAAAAAATAAAAATAAAATAATAAAAAAATATATTTTAAATTTTAAAATTTTTTTTAAAATTATTAAATTAATTTTAAATAAATT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFF:FFFF:FFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25274:1047_2:N:0:GGACTTGG+CGTCTGCG/2 141 * 0 0 * * 0 0 AATTTATTTAAAATTAATTTAATAATTTTAAAAAAAATTTTAAAATTTAAAATATATTTTTTTATTATTTTATTTTTATTTTTTTTAAAATAATATATAATTTTTTATTTATTTTTAATTTAAAAATATTTTATATTATATTTAATTA FFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFF::FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:F,FFFFFFFFF:FFFFFFFFFFFF: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25274:1047_1:N:0:GGACTTGG+CGTCTGCG/1 77 * 0 0 * * 0 0 CNACCAAACATAACACAAAACATTCTTAAATTAAAAATAAATAAAAAACCATATACTATCCCAAAAAAAATAAAAACAAAACAATAAAAAAACATATTCCAAATTTTAAAATCTTCTTCAAAACCACTAAACCAATTTCAAACAAACT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFF:FFFF:FFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25274:1047_2:N:0:GGACTTGG+CGTCTGCG/2 141 * 0 0 * * 0 0 AGTTTGTTTGAAATTGGTTTAGTGGTTTTGAAGAAGATTTTAAAATTTGGAATATGTTTTTTTATTGTTTTGTTTTTATTTTTTTTGGGATAGTATATGGTTTTTTATTTATTTTTAATTTAAGAATGTTTTGTGTTATGTTTGGTTG FFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFF::FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:F,FFFFFFFFF:FFFFFFFFFFFF: YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25274:1047_1:N:0:GGACTTGG+CGTCTGCG/1 83 NC_035784.1_CT_converted 37499203 42 78M1D70M = 37499203 -149 AGTTTGTTTGAAATTGGTTTAGTGGTTTTGAAGAAGATTTTAAAATTTGGAATATGTTTTTTTATTGTTTTGTTTTTATTTTTTTTGGGATAGTATATGGTTTTTTATTTATTTTTAATTTAAGAATGTTTTGTGTTATGTTTGGTNG FFFFFFFFFFFFFFFFFFFF:FFFF:FFFF:FFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF#F AS:i:-15 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:78^T32G35T1 YS:i:-14 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25274:1047_2:N:0:GGACTTGG+CGTCTGCG/2 163 NC_035784.1_CT_converted 37499203 42 78M1D70M = 37499203 149 AGTTTGTTTGAAATTGGTTTAGTGGTTTTGAAGAAGATTTTAAAATTTGGAATATGTTTTTTTATTGTTTTGTTTTTATTTTTTTTGGGATAGTATATGGTTTTTTATTTATTTTTAATTTAAGAATGTTTTGTGTTATGTTTGGTTG FFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFF::FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:F,FFFFFFFFF:FFFFFFFFFFFF: AS:i:-14 XN:i:0 XM:i:1 XO:i:1 XG:i:1 NM:i:2 MD:Z:78^T32G37 YS:i:-15 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.6 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:25274:1047_1:N:0:GGACTTGG+CGTCTGCG/1 77 * 0 0 * * 0 0 TNATTAAATATAATATAAAATATTTTTAAATTAAAAATAAATAAAAAATTATATATTATTTTAAAAAAAATAAAAATAAAATAATAAAAAAATATATTTTAAATTTTAAAATTTTTTTTAAAATTATTAAATTAATTTTAAATAAATT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFF:FFFF:FFFF:FFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25274:1047_2:N:0:GGACTTGG+CGTCTGCG/2 141 * 0 0 * * 0 0 AATTTATTTAAAATTAATTTAATAATTTTAAAAAAAATTTTAAAATTTAAAATATATTTTTTTATTATTTTATTTTTATTTTTTTTAAAATAATATATAATTTTTTATTTATTTTTAATTTAAAAATATTTTATATTATATTTAATTA FFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFF::FFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFF:F,FFFFFFFFF:FFFFFFFFFFFF: YT:Z:UP >>> Writing bisulfite mapping results to CF03-CM03-Zygote_L0-0.6_pe.bam <<< Reading in the sequence files ../../data/CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 8605 (86.05%) aligned concordantly 0 times 675 (6.75%) aligned concordantly exactly 1 time 720 (7.20%) aligned concordantly >1 times 13.95% overall alignment rate 10000 reads; of these: 10000 (10000100.00 reads; of these:% ) were paired; of these: 10000 (8645 (100.0086.45%%) were paired; of these:) aligned concordantly 0 times 8802602 ( (88.026.02%%) aligned concordantly 0 times) aligned concordantly exactly 1 time 581753 ( (5.817.53%%) aligned concordantly exactly 1 time) aligned concordantly >1 times 13.55617% ( overall alignment rate6.17 %) aligned concordantly >1 times 11.98% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8861 (88.61%) aligned concordantly 0 times 541 (5.41%) aligned concordantly exactly 1 time 598 (5.98%) aligned concordantly >1 times 11.39% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, CF03-CM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, CF03-CM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM03-Zygote_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: 70747 Total methylated C's in CpG context: 1698 Total methylated C's in CHG context: 487 Total methylated C's in CHH context: 2445 Total methylated C's in Unknown context: 138 Total unmethylated C's in CpG context: 7047 Total unmethylated C's in CHG context: 13683 Total unmethylated C's in CHH context: 45387 Total unmethylated C's in Unknown context: 466 C methylated in CpG context: 19.4% C methylated in CHG context: 3.4% C methylated in CHH context: 5.1% C methylated in Unknown context (CN or CHN): 22.8% Bismark completed in 0d 0h 0m 27s ==================== Bismark run complete ====================