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/EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/EF02-EM02-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/EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz to EF02-EM02-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 EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz (144270632 sequences in total) Writing a C -> T converted version of the input file EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz to EF02-EM02-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 EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz (144270632 sequences in total) Input files are EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-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 EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 77 * 0 0 * * 0 0 ANAAAAAAAATTTATTTTTATATTTATAAAAAAAATTTATTTTTATATATAAATTTTATTTTTTTTTTTTTTAATTTTTTTATTAAAATATTAATTTTATTAAATAATTTTTTAATATTTTTAAAAAAATAATTAATAATAAAAATTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 141 * 0 0 * * 0 0 TCATAAATCATTAAAACAAATAATAACAATAAAAACTAATAAAATTAATTTTATTAATTTTTATTATTAATTATTTTTTTAAAAATATTAAAAAATTATTTAATAAAATTAATATTTTAATAAAAAAATTAAAAAAAAAAAAAATAAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:,FFFF,FFFFF:FFFFFFF:FF:FF,FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFFFFFF:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 99 NC_035784.1_GA_converted 91080327 0 4M9I2M1I6M2I9M5I6M1I105M = 91080361 182 ANAAAAAAAATCTATCTCTATACCTACAAAAAAAATCTATCTCTATACATAAATTTTATCTCTCTCTTCTCTAACTTTTTTATTAAAATACCAACTCTACCAAATAATTTTTCAACATTTTTAAAAAAATAACCAACAATAAAAACTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, AS:i:-104 XN:i:0 XM:i:5 XO:i:5 XG:i:18 NM:i:23 MD:Z:1C5T6T1T11C103 YS:i:-80 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 147 NC_035784.1_GA_converted 91080361 0 113M1I15M1D4M1I6M1I9M = 91080327 -182 ATTTTATCTCTCTCTTCTCTAACTTTTTTATTAAAATACCAACTCTACCAAATAATTTTTCAACATTTTTAAAAAAATAACCAACAATAAAAACTAATAAAACTAATCTCATTAATTTTTATTATTATTATTTATTTTAATAATTTATAA F:FFFFFFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFF:FFFF,FFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF,FF:FF:FFFFFFF:FFFFF,FFFF,:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-80 XN:i:0 XM:i:8 XO:i:4 XG:i:4 NM:i:12 MD:Z:115C1C2A3A3^C1A0A12C0C2 YS:i:-104 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 77 * 0 0 * * 0 0 ANAAAAAAAATCTATCTCTATACCTACAAAAAAAATCTATCTCTATACATAAATTTTATCTCTCTCTTCTCTAACTTTTTTATTAAAATACCAACTCTACCAAATAATTTTTCAACATTTTTAAAAAAATAACCAACAATAAAAACTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 141 * 0 0 * * 0 0 TTATAAATTATTAAAATAAATAATAATAATAAAAATTAATGAGATTAGTTTTATTAGTTTTTATTGTTGGTTATTTTTTTAAAAATGTTGAAAAATTATTTGGTAGAGTTGGTATTTTAATAAAAAAGTTAGAGAAGAGAGAGATAAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:,FFFF,FFFFF:FFFFFFF:FF:FF,FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFFFFFF:F YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF02-EM02-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:GW210315000:4:1101:26811:1047_1:N:0:ATCCACTG+ACGCACCT/1 77 * 0 0 * * 0 0 ANAAAAAAAATTTATTTTTATATTTATAAAAAAAATTTATTTTTATATATAAATTTTATTTTTTTTTTTTTTAATTTTTTTATTAAAATATTAATTTTATTAAATAATTTTTTAATATTTTTAAAAAAATAATTAATAATAAAAATTAAT F#FFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFF:FFFF:F:FFFFFFFFFFFFFFFFFFFFFFF,FFFFFFFFFFFFFFFF:FFFFFF:FFFFFFFFFFFFFF:FFFFFFFFFFFFFFF:FFF, YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26811:1047_2:N:0:ATCCACTG+ACGCACCT/2 141 * 0 0 * * 0 0 TCATAAATCATTAAAACAAATAATAACAATAAAAACTAATAAAATTAATTTTATTAATTTTTATTATTAATTATTTTTTTAAAAATATTAAAAAATTATTTAATAAAATTAATATTTTAATAAAAAAATTAAAAAAAAAAAAAATAAAAT FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:,FFFF,FFFFF:FFFFFFF:FF:FF,FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFF,FFFF:FFFFFFFFFFFFFFFFF:F,F:FFFFFFFFFFFFFFFFFFFFF:F YT:Z:UP >>> Writing bisulfite mapping results to EF02-EM02-Zygote_pe.bam <<< Reading in the sequence files ../../data/EF02-EM02-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF02-EM02-Zygote_R2_001.fastp-trim.20220827.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1125:12147:26005_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17163 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1132:18502:12054_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17137 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1158:7907:2816_1:N:0:ATCCACTG+ACGCACCT NC_035780.1 2 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1173:20283:22905_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17155 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1213:18177:13338_1:N:0:GTCCACTG+ACGCACCT NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1213:18457:13855_1:N:0:GTCCACTG+ACGCACCT NC_007175.2 2 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far 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:GW210315000:4:1353:15528:34554_1:N:0:ATCCACTG+ACGCACCT NC_035780.1 2 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1437:19208:17816_1:N:0:ATCCACTG+ACGCACCT NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1444:23231:35023_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17124 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1445:22896:2973_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17124 Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1469:22055:30170_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 1 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1526:17960:3944_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17163 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1545:9118:10457_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 2 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1570:29107:32205_1:N:0:ATCCACTG+ACGCACCT NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1570:28339:32910_1:N:0:ATCCACTG+ACGCACCT NC_035780.1 2 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1616:13376:24001_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17163 Processed 26000000 sequence pairs so far Processed 27000000 sequence pairs so far Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1667:21784:26443_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17158 Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2138:11288:10582_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 2 Processed 32000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2155:12915:14340_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 2 Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2226:23122:2268_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17091 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2244:30843:27915_1:N:0:ATCCACTG+ACGCACCT NC_035780.1 2 Processed 37000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2254:9815:14544_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 2 Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2305:24080:23844_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 1 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2307:24017:25895_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17145 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2307:24135:30170_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17145 Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2352:14064:18176_1:N:0:ATCCACTG+GCGCACCT NC_007175.2 17159 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2352:3143:29637_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17104 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2403:11333:35243_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17137 Processed 44000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2418:23918:19210_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17183 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2443:1669:10207_1:N:0:ATCCACTG+ACGCACCT NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2454:14751:31485_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17137 Processed 47000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2461:24885:24142_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17107 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2461:26250:24940_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17107 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2464:7365:22670_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17163 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2467:7446:32299_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17163 Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2546:22083:10895_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17162 Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Processed 56000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2667:20130:30467_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17158 Processed 57000000 sequence pairs so far Processed 58000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1013:195:GW2105211192nd:2:1108:22516:13651_1:N:0:CTCCACTG+ACGCCCCT NC_007175.2 2 Processed 59000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1013:195:GW2105211192nd:2:1119:27959:24518_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17163 Chromosomal sequence could not be extracted for GWNJ-1013:195:GW2105211192nd:2:1119:27453:25770_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17163 Processed 60000000 sequence pairs so far Processed 61000000 sequence pairs so far Processed 62000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1013:195:GW2105211192nd:2:1157:9552:9643_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17164 Processed 63000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1013:195:GW2105211192nd:2:1162:31611:24142_1:N:0:ATCCACTG+ACGCACCT NC_007175.2 17141 Processed 64000000 sequence pairs so far