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/CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/CF05-CM05-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/CF05-CM05-Zygote_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/CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF05-CM05-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 CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF05-CM05-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 CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-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,-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 CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-Zygote_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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 99 NC_035786.1_CT_converted 44505755 1 80M1D6M = 44505755 87 TNTTTTTTTTTTAGTATATATATTTATTTTTGAGTAAAGTTAAGTATAGGAAAAAAAGGAAATAGTTGTATTTTATTGAAATTTAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:F,FFFFFFF::F,FF:FFFFFF:FFFFFFFFFFFFFF:F:FFFFFFFFFFFFF AS:i:-21 XS:i:-21 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:1A0A44G32^T6 YS:i:-26 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 147 NC_035786.1_CT_converted 44505755 1 80M1D6M = 44505755 -87 TTTTTTTTTTTTAGTATATATATTTATTTTTGAGTAAAGTTAAGTATAGGAAAAAAAGGAAATAGTTGTATTTTATTGAAATTTAT FFFFFFFFF:FFFFFFF,FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-26 XS:i:-26 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:1A0A44G32^T6 YS:i:-21 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM05-Zygote_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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 77 * 0 0 * * 0 0 TNTTTTTTTTTCAATACATATATTTATTTTTAAATAAAATTAAATATAAAAAAAAAAAAAAATAATTATATTTTATTAAAATTTAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:F,FFFFFFF::F,FF:FFFFFF:FFFFFFFFFFFFFF:F:FFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 141 * 0 0 * * 0 0 GTAAATTTTAATAAAATATAATTATTTTTTTTTTTTTTTATATTTAATTTTATTTAAAAATAAATATATGTATTGAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF,FFFFFFF:FFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM05-Zygote_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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 77 * 0 0 * * 0 0 TNTTTTTTTTTCAATACATATATTTATTTTTAAATAAAATTAAATATAAAAAAAAAAAAAAATAATTATATTTTATTAAAATTTAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:F,FFFFFFF::F,FF:FFFFFF:FFFFFFFFFFFFFF:F:FFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 141 * 0 0 * * 0 0 GTAAATTTTAATAAAATATAATTATTTTTTTTTTTTTTTATATTTAATTTTATTTAAAAATAAATATATGTATTGAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF,FFFFFFF:FFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-Zygote_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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 83 NC_035780.1_GA_converted 36883175 6 6M1D80M = 36883175 -87 ATAAATTTCAATAAAATACAACTATTTCCTTTTTTTCCTATACTTAACTTTACTCAAAAATAAATATATATACTAAAAAAAAAANA FFFFFFFFFFFFF:F:FFFFFFFFFFFFFF:FFFFFF:FF,F::FFFFFFF,F:FF:FFFFFFFFFFFFFFFFFFFFFFFFFFF#F AS:i:-15 XS:i:-21 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:6^A77T0T1 YS:i:-20 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 163 NC_035780.1_GA_converted 36883175 6 6M1D80M = 36883175 87 ATAAATTTCAATAAAATACAACTATTTCCTTTTTTTCCTATACTTAACTTTACTCAAAAATAAATATATATACTAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF,FFFFFFF:FFFFFFFFF AS:i:-20 XS:i:-20 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:6^A77T0T1 YS:i:-15 YT:Z:CP >>> Writing bisulfite mapping results to CF05-CM05-Zygote_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz 1000010000 reads; of these: reads; of these: 1000010000 ( (100.00100.00%%) were paired; of these:) were paired; of these: 79537935 ( (79.5379.35%%) aligned concordantly 0 times) aligned concordantly 0 times 826823 ( (8.268.23%%) aligned concordantly exactly 1 time) aligned concordantly exactly 1 time 12211242 ( (12.2112.42%%) aligned concordantly >1 times) aligned concordantly >1 times 20.4720.65%% overall alignment rate overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8077 (80.77%) aligned concordantly 0 times 772 (7.72%) aligned concordantly exactly 1 time 1151 (11.51%) aligned concordantly >1 times 19.23% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8143 (81.43%) aligned concordantly 0 times 716 (7.16%) aligned concordantly exactly 1 time 1141 (11.41%) aligned concordantly >1 times 18.57% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-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: 120678 Total methylated C's in CpG context: 3476 Total methylated C's in CHG context: 955 Total methylated C's in CHH context: 5339 Total methylated C's in Unknown context: 400 Total unmethylated C's in CpG context: 12718 Total unmethylated C's in CHG context: 23302 Total unmethylated C's in CHH context: 74888 Total unmethylated C's in Unknown context: 1834 C methylated in CpG context: 21.5% C methylated in CHG context: 3.9% C methylated in CHH context: 6.7% C methylated in Unknown context (CN or CHN): 17.9% Bismark completed in 0d 0h 0m 14s ==================== Bismark run complete ====================