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_L0-0.8/ 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/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,-0.8 -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 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,-0.8 -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,-0.8 -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,-0.8 -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,-0.8 -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_L0-0.8_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: 8270 (82.70%) aligned concordantly 0 times 777 (7.77%) aligned concordantly exactly 1 time 953 (9.53%) aligned concordantly >1 times 17.30% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8307 (83.07%) aligned concordantly 0 times 720 (7.20%) aligned concordantly exactly 1 time 973 (9.73%10000) aligned concordantly >1 times reads; of these: 16.93 %10000 overall alignment rate ( 100.00%) were paired; of these: 8500 (85.00%) aligned concordantly 0 times 672 (6.72%) aligned concordantly exactly 1 time 828 (8.28%) aligned concordantly >1 times 15.00% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8562 (85.62%) aligned concordantly 0 times 633 (6.33%) aligned concordantly exactly 1 time 805 (8.05%) aligned concordantly >1 times 14.38% 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: 84505 Total methylated C's in CpG context: 1907 Total methylated C's in CHG context: 618 Total methylated C's in CHH context: 3421 Total methylated C's in Unknown context: 237 Total unmethylated C's in CpG context: 8440 Total unmethylated C's in CHG context: 16145 Total unmethylated C's in CHH context: 53974 Total unmethylated C's in Unknown context: 829 C methylated in CpG context: 18.4% C methylated in CHG context: 3.7% C methylated in CHH context: 6.0% C methylated in Unknown context (CN or CHN): 22.2% Bismark completed in 0d 0h 0m 27s ==================== Bismark run complete ====================