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) 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/05-bismark-align-full'): ../../data/EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/EF03-EM03-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/05-bismark-align-full/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /mmfs1/gscratch/scrubbed/sr320/github/ceasmallr/output/05-bismark-align-full 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/EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF03-EM03-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 EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz (135271836 sequences in total) Writing a C -> T converted version of the input file EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF03-EM03-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 EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz (135271836 sequences in total) Input files are EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-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,-0.8 -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 EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANATTATTATAAAATATATATATAAATAAAAATTAAATAAAAAAAATTGATTAATTAATTAATAAATTATTATAAATTATTATAATAATAAATAAATATTATTATATAAAATAAAATTATTAATAAAATTTATATTTATTAAAATAATAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTAATATTTTTATATTATTTTAATAAATATAAATTTTATTAATAATTTTATTTTATATAATAATATTTATTTATTATTATAATAATTTATAATAATTTATTAATTAATTAATCAATTTTTTTTATTTAATTTTTATTTATATATATATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANACCACTACAAAATACACATATAAATAAAAACCAAATAAAAAAAACTAACTAATTAACCAACAAATTATTATAAACCACCACAACAATAAACAAACACTACTACATAAAATAAAACCATCAATAAAACCTATACCCACCAAAATAATAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTGATATTTTTGTATTATTTTGGTGGGTATAGGTTTTATTGATGGTTTTATTTTATGTAGTAGTGTTTGTTTATTGTTGTGGTGGTTTATAATAATTTGTTGGTTAATTAGTTAGTTTTTTTTATTTGGTTTTTATTTATATGTGTATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANACCACTACAAAATACACATATAAATAAAAACCAAATAAAAAAAACTAACTAATTAACCAACAAATTATTATAAACCACCACAACAATAAACAAACACTACTACATAAAATAAAACCATCAATAAAACCTATACCCACCAAAATAATAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTGATATTTTTGTATTATTTTGGTGGGTATAGGTTTTATTGATGGTTTTATTTTATGTAGTAGTGTTTGTTTATTGTTGTGGTGGTTTATAATAATTTGTTGGTTAATTAGTTAGTTTTTTTTATTTGGTTTTTATTTATATGTGTATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-0.8 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: GWNJ-1012:512:GW210315000:4:1101:26250:1047_1:N:0:CTCTCGTC+AGGTTATA/1 77 * 0 0 * * 0 0 ANATTATTATAAAATATATATATAAATAAAAATTAAATAAAAAAAATTGATTAATTAATTAATAAATTATTATAAATTATTATAATAATAAATAAATATTATTATATAAAATAAAATTATTAATAAAATTTATATTTATTAAAATAATAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26250:1047_2:N:0:CTCTCGTC+AGGTTATA/2 141 * 0 0 * * 0 0 TTTAATATTTTTATATTATTTTAATAAATATAAATTTTATTAATAATTTTATTTTATATAATAATATTTATTTATTATTATAATAATTTATAATAATTTATTAATTAATTAATCAATTTTTTTTATTTAATTTTTATTTATATATATATT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FF,FF:FF:FFFF:FFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF03-EM03-Zygote_pe.bam <<< Reading in the sequence files ../../data/EF03-EM03-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF03-EM03-Zygote_R2_001.fastp-trim.20220827.fq.gz Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1107:6958:14450_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 2 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1113:4318:18145_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 2 Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1138:12427:30467_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 1 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1162:7934:25222_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1162:5755:26710_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 2 Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1173:2401:20400_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1173:1714:34053_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1213:6750:35477_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1233:8666:22701_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 17194 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1235:23836:23672_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1313:15564:2801_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 25000000 sequence pairs so far Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1373:7039:2284_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1450:21721:19758_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1460:29975:13949_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 2 Processed 44000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1468:27950:36714_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1468:30418:37012_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Processed 54000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1558:29884:26475_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far Processed 59000000 sequence pairs so far Processed 60000000 sequence pairs so far Processed 61000000 sequence pairs so far Processed 62000000 sequence pairs so far Processed 63000000 sequence pairs so far Processed 64000000 sequence pairs so far Processed 65000000 sequence pairs so far Processed 66000000 sequence pairs so far Processed 67000000 sequence pairs so far Processed 68000000 sequence pairs so far Processed 69000000 sequence pairs so far Processed 70000000 sequence pairs so far Processed 71000000 sequence pairs so far Processed 72000000 sequence pairs so far Processed 73000000 sequence pairs so far Processed 74000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2143:27344:21386_1:N:0:CTCTCGTC+AGGTTATA NC_007175.2 17167 Processed 75000000 sequence pairs so far Processed 76000000 sequence pairs so far Processed 77000000 sequence pairs so far Processed 78000000 sequence pairs so far Processed 79000000 sequence pairs so far Processed 80000000 sequence pairs so far Processed 81000000 sequence pairs so far Processed 82000000 sequence pairs so far Processed 83000000 sequence pairs so far Processed 84000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2235:23863:25347_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2 Processed 85000000 sequence pairs so far Processed 86000000 sequence pairs so far Processed 87000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2254:31286:6668_1:N:0:CTCTCGTC+AGGTTATA NC_035780.1 2