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/EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF08-EM04-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/EF08-EM04-Larvae_score_L0-0.6/ 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/04.2-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/EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Processing reads up to sequence no. 10000 from ../../data/EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF08-EM04-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 EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Processing reads up to sequence no. 10000 from ../../data/EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz Writing a C -> T converted version of the input file EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF08-EM04-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 EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz (10001 sequences in total) Input files are EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM04-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,-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 EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM04-Larvae_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:GW210315000:4:1101:26467:1047_1:N:0:AACGTTCC+AGTACTCC/1 77 * 0 0 * * 0 0 ANAATAATATTTATAAAAAAAATTAAAATATAATATTTAAAAAAAATAAAATATTTTTTATTTTAAAAATAATTATTATATAAATATATTATTTGTAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26467:1047_2:N:0:AACGTTCC+AGTACTCC/2 141 * 0 0 * * 0 0 TTACAAATAATATATTTATATAATAATTATTTTTAAAATAAAAAATATTTTATTTTTTTTAAATATTATATTTTAATTTTTTTTATAAATATTATTTT FFFFFFF:FFFFFF:F:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:FFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF08-EM04-Larvae_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:GW210315000:4:1101:26467:1047_1:N:0:AACGTTCC+AGTACTCC/1 77 * 0 0 * * 0 0 ANAACAACATCTACAAAAAAAACTAAAACACAATACCCAAAAAAAACAAAACACTTCCCATTCCAAAAATAACCATTATACAAATATACCACCTATAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26467:1047_2:N:0:AACGTTCC+AGTACTCC/2 141 * 0 0 * * 0 0 TTATAGGTGGTATATTTGTATAATGGTTATTTTTGGAATGGGAAGTGTTTTGTTTTTTTTGGGTATTGTGTTTTAGTTTTTTTTGTAGATGTTGTTTT FFFFFFF:FFFFFF:F:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:FFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF08-EM04-Larvae_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:GW210315000:4:1101:26467:1047_1:N:0:AACGTTCC+AGTACTCC/1 83 NC_035781.1_CT_converted 8258718 2 98M = 8258718 -98 TTATAGGTGGTATATTTGTATAATGGTTATTTTTGGAATGGGAAGTGTTTTGTTTTTTTTGGGTATTGTGTTTTAGTTTTTTTTGTAGATGTTGTTNT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF#F AS:i:-19 XS:i:-25 XN:i:0 XM:i:4 XO:i:0 XG:i:0 NM:i:4 MD:Z:0A0A1A92A1 YS:i:-24 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26467:1047_2:N:0:AACGTTCC+AGTACTCC/2 163 NC_035781.1_CT_converted 8258718 2 98M = 8258718 98 TTATAGGTGGTATATTTGTATAATGGTTATTTTTGGAATGGGAAGTGTTTTGTTTTTTTTGGGTATTGTGTTTTAGTTTTTTTTGTAGATGTTGTTTT FFFFFFF:FFFFFF:F:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:FFFFFFF AS:i:-24 XS:i:-24 XN:i:0 XM:i:4 XO:i:0 XG:i:0 NM:i:4 MD:Z:0A0A1A92A1 YS:i:-19 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM04-Larvae_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:GW210315000:4:1101:26467:1047_1:N:0:AACGTTCC+AGTACTCC/1 77 * 0 0 * * 0 0 ANAATAATATTTATAAAAAAAATTAAAATATAATATTTAAAAAAAATAAAATATTTTTTATTTTAAAAATAATTATTATATAAATATATTATTTGTAA F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26467:1047_2:N:0:AACGTTCC+AGTACTCC/2 141 * 0 0 * * 0 0 TTACAAATAATATATTTATATAATAATTATTTTTAAAATAAAAAATATTTTATTTTTTTTAAATATTATATTTTAATTTTTTTTATAAATATTATTTT FFFFFFF:FFFFFF:F:FFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:FFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF08-EM04-Larvae_L0-0.6_pe.bam <<< Reading in the sequence files ../../data/EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz 10000 reads; of these: 10000 (100.00%) were paired; of these: 10000 reads; of these:8160 10000 ( reads; of these:81.6010000 % ( ) aligned concordantly 0 times10000 100.00 ( %802) were paired; of these:100.00 ( %8.02 ) were paired; of these:%8115 ) aligned concordantly exactly 1 time ( 81.158081 % (1038) aligned concordantly 0 times80.81 ( %10.38 ) aligned concordantly 0 times%806 ) aligned concordantly >1 times ( 8.0686518.40% (%) aligned concordantly exactly 1 time8.65 overall alignment rate % ) aligned concordantly exactly 1 time1079 ( 10.791054% () aligned concordantly >1 times10.54 %18.85) aligned concordantly >1 times% overall alignment rate19.19 % overall alignment rate 10000 reads; of these: 10000 (100.00%) were paired; of these: 8200 (82.00%) aligned concordantly 0 times 794 (7.94%) aligned concordantly exactly 1 time 1006 (10.06%) aligned concordantly >1 times 18.00% overall alignment rate Processed 10000 sequences in total Successfully deleted the temporary files EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, EF08-EM04-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, EF08-EM04-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF08-EM04-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: 128257 Total methylated C's in CpG context: 4008 Total methylated C's in CHG context: 778 Total methylated C's in CHH context: 2743 Total methylated C's in Unknown context: 223 Total unmethylated C's in CpG context: 15634 Total unmethylated C's in CHG context: 27621 Total unmethylated C's in CHH context: 77473 Total unmethylated C's in Unknown context: 634 C methylated in CpG context: 20.4% C methylated in CHG context: 2.7% C methylated in CHH context: 3.4% C methylated in Unknown context (CN or CHN): 26.0% Bismark completed in 0d 0h 0m 25s ==================== Bismark run complete ====================