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-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/CF03-CM05-Larvae_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-CM05-Larvae_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/CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz to CF03-CM05-Larvae_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-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz to CF03-CM05-Larvae_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-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM05-Larvae_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 CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM05-Larvae_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:GW210315000:4:1101:25310:1047_1:N:0:CGGCGTGA+ACAGGCGC/1 77 * 0 0 * * 0 0 ANAGTATTAATTTATAAGAAATATTAAAATATTATTATATATTAAATTATTAATTTAAATTTTAATTTTATTATTATAATATTATTAATTTTATTAAAAATTATATTTTATATTTTTTTTAAAATT F#F:FFFFFFFFFFFFFFF:F,FFFFFFFF:F:FFFFFFFF:FFFF::FF:FFFF:FFFFFFF:FFFFFF::FFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25310:1047_2:N:0:CGGCGTGA+ACAGGCGC/2 141 * 0 0 * * 0 0 AATTTTAAAAAAAATATAAAATATAATTTTTAATAAAATTAATAATATTATAATAATAAAATTAAAATTTAAATTAATAATTTAATATATAATAATATTTTAATATTTCTTATAAATTAATACTAT ,FFFFFFFFFFFFF:FFFFFFF:FF:FFF:FF::FFFFFF,FFFFFF:F,FFFFFFFF:FFFFFFF,FFFFFFFFFF:FFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:FF,:FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF03-CM05-Larvae_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:GW210315000:4:1101:25310:1047_1:N:0:CGGCGTGA+ACAGGCGC/1 77 * 0 0 * * 0 0 ANAACACCAATCCATAAAAAATATCAAAACACCACTACACATTAAATCATTAATCCAAATCTTAATCCCATCACCACAACATTACCAACCTTACTAAAAACTATACCCTACATCCCCCCCAAAACC F#F:FFFFFFFFFFFFFFF:F,FFFFFFFF:F:FFFFFFFF:FFFF::FF:FFFF:FFFFFFF:FFFFFF::FFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25310:1047_2:N:0:CGGCGTGA+ACAGGCGC/2 141 * 0 0 * * 0 0 GGTTTTGGGGGGGATGTAGGGTATAGTTTTTAGTAAGGTTGGTAATGTTGTGGTGATGGGATTAAGATTTGGATTAATGATTTAATGTGTAGTGGTGTTTTGATATTTTTTATGGATTGGTGTTAT ,FFFFFFFFFFFFF:FFFFFFF:FF:FFF:FF::FFFFFF,FFFFFF:F,FFFFFFFF:FFFFFFF,FFFFFFFFFF:FFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:FF,:FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF03-CM05-Larvae_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:GW210315000:4:1101:25310:1047_1:N:0:CGGCGTGA+ACAGGCGC/1 77 * 0 0 * * 0 0 ANAACACCAATCCATAAAAAATATCAAAACACCACTACACATTAAATCATTAATCCAAATCTTAATCCCATCACCACAACATTACCAACCTTACTAAAAACTATACCCTACATCCCCCCCAAAACC F#F:FFFFFFFFFFFFFFF:F,FFFFFFFF:F:FFFFFFFF:FFFF::FF:FFFF:FFFFFFF:FFFFFF::FFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25310:1047_2:N:0:CGGCGTGA+ACAGGCGC/2 141 * 0 0 * * 0 0 GGTTTTGGGGGGGATGTAGGGTATAGTTTTTAGTAAGGTTGGTAATGTTGTGGTGATGGGATTAAGATTTGGATTAATGATTTAATGTGTAGTGGTGTTTTGATATTTTTTATGGATTGGTGTTAT ,FFFFFFFFFFFFF:FFFFFFF:FF:FFF:FF::FFFFFF,FFFFFF:F,FFFFFFFF:FFFFFFF,FFFFFFFFFF:FFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:FF,:FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM05-Larvae_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:GW210315000:4:1101:25310:1047_1:N:0:CGGCGTGA+ACAGGCGC/1 77 * 0 0 * * 0 0 ANAGTATTAATTTATAAGAAATATTAAAATATTATTATATATTAAATTATTAATTTAAATTTTAATTTTATTATTATAATATTATTAATTTTATTAAAAATTATATTTTATATTTTTTTTAAAATT F#F:FFFFFFFFFFFFFFF:F,FFFFFFFF:F:FFFFFFFF:FFFF::FF:FFFF:FFFFFFF:FFFFFF::FFFFFFFFFFFFFFFF:F::FFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25310:1047_2:N:0:CGGCGTGA+ACAGGCGC/2 141 * 0 0 * * 0 0 AATTTTAAAAAAAATATAAAATATAATTTTTAATAAAATTAATAATATTATAATAATAAAATTAAAATTTAAATTAATAATTTAATATATAATAATATTTTAATATTTCTTATAAATTAATACTAT ,FFFFFFFFFFFFF:FFFFFFF:FF:FFF:FF::FFFFFF,FFFFFF:F,FFFFFFFF:FFFFFFF,FFFFFFFFFF:FFFFFF:FFFFFFFFF:FFFFFFFFFFFFFFF:FF,:FFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to CF03-CM05-Larvae_L0-1.0_pe.bam <<< Reading in the sequence files ../../data/CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 7471 (74.71%) aligned concordantly 0 times 1035 (10.35%) aligned concordantly exactly 1 time 1494 (14.94%) aligned concordantly >1 times 25.29% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7508 (75.08%) aligned concordantly 0 times 1004 (10.04%) aligned concordantly exactly 1 time 1488 (14.88%) aligned concordantly >1 times 24.92% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7598 (75.98%) aligned concordantly 0 times 943 (9.43%) aligned concordantly exactly 1 time 1459 (14.59%) aligned concordantly >1 times 24.02% overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 7606 (76.06%) aligned concordantly 0 times 943 (9.43%) aligned concordantly exactly 1 time 1451 (14.51%) aligned concordantly >1 times 23.94% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, CF03-CM05-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, CF03-CM05-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF03-CM05-Larvae_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: 184148 Total methylated C's in CpG context: 4870 Total methylated C's in CHG context: 1412 Total methylated C's in CHH context: 5955 Total methylated C's in Unknown context: 551 Total unmethylated C's in CpG context: 23335 Total unmethylated C's in CHG context: 38705 Total unmethylated C's in CHH context: 109871 Total unmethylated C's in Unknown context: 2066 C methylated in CpG context: 17.3% C methylated in CHG context: 3.5% C methylated in CHH context: 5.1% C methylated in Unknown context (CN or CHN): 21.1% Bismark completed in 0d 0h 0m 24s ==================== Bismark run complete ====================