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-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-bismark-align/ 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-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,-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 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,-0.6 -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,-0.6 -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 ANCAACACTTTTTCTTTAATAATCATTTTTTTCTTTTTATAACTAACATATAATATACTTTTTTCCTCTTATTTCTAACAACATTAAAA 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 TTTTAATGTTGTTAGAAATAAGAGGAAAAAAGTATATTATATGTTAGTTATAAAAAGAAAAAAATGATTATTAAAGAAAAAGTGTTGAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF YT:Z:UP 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,-0.6 -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 ANCAACACTTTTTCTTTAATAATCATTTTTTTCTTTTTATAACTAACATATAATATACTTTTTTCCTCTTATTTCTAACAACATTAAAA 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 TTTTAATGTTGTTAGAAATAAGAGGAAAAAAGTATATTATATGTTAGTTATAAAAAGAAAAAAATGATTATTAAAGAAAAAGTGTTGAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF YT:Z:UP 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,-0.6 -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_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 (10000100.00 reads; of these:% ) were paired; of these: 10000 (8908 (89.08100.00%%) aligned concordantly 0 times) were paired; of these: 5288918 ( (5.2889.18%%) aligned concordantly exactly 1 time) aligned concordantly 0 times 564528 ( (5.645.28%%) aligned concordantly >1 times) aligned concordantly exactly 1 time 10.92 %554 overall alignment rate ( 5.54%) aligned concordantly >1 times 10.82% overall alignment rate 1000010000 reads; of these: reads; of these: 1000010000 ( (100.00100.00%%) were paired; of these:) were paired; of these: 89198904 ( (89.1989.04%%) aligned concordantly 0 times) aligned concordantly 0 times 520524 ( (5.205.24%%) aligned concordantly exactly 1 time) aligned concordantly exactly 1 time 561572 ( (5.615.72%%) aligned concordantly >1 times) aligned concordantly >1 times 10.8110.96%% overall alignment rate 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: 70163 Total methylated C's in CpG context: 2191 Total methylated C's in CHG context: 740 Total methylated C's in CHH context: 3057 Total methylated C's in Unknown context: 128 Total unmethylated C's in CpG context: 7136 Total unmethylated C's in CHG context: 13593 Total unmethylated C's in CHH context: 43446 Total unmethylated C's in Unknown context: 481 C methylated in CpG context: 23.5% C methylated in CHG context: 5.2% C methylated in CHH context: 6.6% C methylated in Unknown context (CN or CHN): 21.0% Bismark completed in 0d 0h 0m 35s ==================== Bismark run complete ====================