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/EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/EF01-EM01-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/EF01-EM01-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/EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF01-EM01-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 EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF01-EM01-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 EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF01-EM01-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 EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF01-EM01-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:GW210315000:4:1101:27353:1047_1:N:0:TAATACAG+GTGAATAT/1 77 * 0 0 * * 0 0 ANTAATATTTTTTTTTTAATAATTATTTTTTTTTTTTTATAATTAATATATAATATATTTTTTTTTTTTTATTTTTAATAATATTAGAA F#FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:27353:1047_2:N:0:TAATACAG+GTGAATAT/2 141 * 0 0 * * 0 0 TTCTAATATTATTAAAAATAAAAAAAAAAAAATATATTATATATTAATTATAAAAAAAAAAAAATAATTATTAAAAAAAAAATATTAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF01-EM01-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:GW210315000:4:1101:27353:1047_1:N:0:TAATACAG+GTGAATAT/1 99 NC_035784.1_GA_converted 35865154 0 57M1I31M = 35865154 88 ANCAACACTTTTTCTTTAATAATCATTTTTTTCTTTTTATAACTAACATATAATATACTTTTTTCCTCTTATTTCTAACAACATTAAAA F#FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-69 XS:i:-69 XN:i:0 XM:i:11 XO:i:1 XG:i:1 NM:i:12 MD:Z:1T3T11C1C1T2T32A4A22T0T0T0 YS:i:-68 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:27353:1047_2:N:0:TAATACAG+GTGAATAT/2 147 NC_035784.1_GA_converted 35865154 0 57M1I31M = 35865154 -88 ATCAACACTTTTTCTTTAATAATCATTTTTTTCTTTTTATAACTAACATATAATATACTTTTTTCCTCTTATTTCTAACAACATTAAAA FFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-68 XS:i:-68 XN:i:0 XM:i:10 XO:i:1 XG:i:1 NM:i:11 MD:Z:5T11C1C1T2T32A4A22T0T0T0 YS:i:-69 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF01-EM01-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:GW210315000:4:1101:27353:1047_1:N:0:TAATACAG+GTGAATAT/1 83 NC_035786.1_CT_converted 23981983 0 25M1I63M = 23981983 -88 TTTTAATGTTGTTAGAAATAAGAGGAAAAAAGTATATTATATGTTAGTTATAAAAAGAAAAAAATGATTATTAAAGAAAAAGTGTTGNT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFF#F AS:i:-69 XS:i:-69 XN:i:0 XM:i:11 XO:i:1 XG:i:1 NM:i:12 MD:Z:0A0A0A22T4T32A2A1G1G11A3A1 YS:i:-68 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:27353:1047_2:N:0:TAATACAG+GTGAATAT/2 163 NC_035786.1_CT_converted 23981983 0 25M1I63M = 23981983 88 TTTTAATGTTGTTAGAAATAAGAGGAAAAAAGTATATTATATGTTAGTTATAAAAAGAAAAAAATGATTATTAAAGAAAAAGTGTTGAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF AS:i:-68 XS:i:-68 XN:i:0 XM:i:10 XO:i:1 XG:i:1 NM:i:11 MD:Z:0A0A0A22T4T32A2A1G1G11A5 YS:i:-69 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF01-EM01-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:GW210315000:4:1101:27353:1047_1:N:0:TAATACAG+GTGAATAT/1 77 * 0 0 * * 0 0 ANTAATATTTTTTTTTTAATAATTATTTTTTTTTTTTTATAATTAATATATAATATATTTTTTTTTTTTTATTTTTAATAATATTAGAA F#FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:27353:1047_2:N:0:TAATACAG+GTGAATAT/2 141 * 0 0 * * 0 0 TTCTAATATTATTAAAAATAAAAAAAAAAAAATATATTATATATTAATTATAAAAAAAAAAAAATAATTATTAAAAAAAAAATATTAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF YT:Z:UP >>> Writing bisulfite mapping results to EF01-EM01-Zygote_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 8181 (81.81%) aligned concordantly 0 times 768 (7.68%) aligned concordantly exactly 1 time 1051 (10.51%) aligned concordantly >1 times 18.19% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8229 (82.29%) aligned concordantly 0 times 729 (7.29%) aligned concordantly exactly 1 time 1042 (10.42%) aligned concordantly >1 times 17.71% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8279 (82.79%) aligned concordantly 0 times 694 (6.94%) aligned concordantly exactly 1 time 1027 (10.27%) aligned concordantly >1 times 17.21% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8225 (82.25%) aligned concordantly 0 times 750 (7.50%) aligned concordantly exactly 1 time 1025 (10.25%) aligned concordantly >1 times 17.75% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF01-EM01-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF01-EM01-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF01-EM01-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: 101303 Total methylated C's in CpG context: 2970 Total methylated C's in CHG context: 1125 Total methylated C's in CHH context: 6153 Total methylated C's in Unknown context: 357 Total unmethylated C's in CpG context: 10515 Total unmethylated C's in CHG context: 18893 Total unmethylated C's in CHH context: 61647 Total unmethylated C's in Unknown context: 1470 C methylated in CpG context: 22.0% C methylated in CHG context: 5.6% C methylated in CHH context: 9.1% C methylated in Unknown context (CN or CHN): 19.5% Bismark completed in 0d 0h 0m 25s ==================== Bismark run complete ====================