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/CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz ../../data/CF05-CM05-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/CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz to CF05-CM05-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 CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz (146024492 sequences in total) Writing a C -> T converted version of the input file CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz to CF05-CM05-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 CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz (146024492 sequences in total) Input files are CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-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 CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 99 NC_035786.1_CT_converted 44505755 0 80M1D6M = 44505755 87 TNTTTTTTTTTTAGTATATATATTTATTTTTGAGTAAAGTTAAGTATAGGAAAAAAAGGAAATAGTTGTATTTTATTGAAATTTAT F#FFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:F,FFFFFFF::F,FF:FFFFFF:FFFFFFFFFFFFFF:F:FFFFFFFFFFFFF AS:i:-21 XS:i:-21 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:1A0A44G32^T6 YS:i:-26 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 147 NC_035786.1_CT_converted 44505755 0 80M1D6M = 44505755 -87 TTTTTTTTTTTTAGTATATATATTTATTTTTGAGTAAAGTTAAGTATAGGAAAAAAAGGAAATAGTTGTATTTTATTGAAATTTAT FFFFFFFFF:FFFFFFF,FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-26 XS:i:-26 XN:i:0 XM:i:3 XO:i:1 XG:i:1 NM:i:4 MD:Z:1A0A44G32^T6 YS:i:-21 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM05-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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 77 * 0 0 * * 0 0 TNTTTTTTTTTCAATACATATATTTATTTTTAAATAAAATTAAATATAAAAAAAAAAAAAAATAATTATATTTTATTAAAATTTAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:F,FFFFFFF::F,FF:FFFFFF:FFFFFFFFFFFFFF:F:FFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 141 * 0 0 * * 0 0 GTAAATTTTAATAAAATATAATTATTTTTTTTTTTTTTTATATTTAATTTTATTTAAAAATAAATATATGTATTGAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF,FFFFFFF:FFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and CF05-CM05-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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 77 * 0 0 * * 0 0 TNTTTTTTTTTCAATACATATATTTATTTTTAAATAAAATTAAATATAAAAAAAAAAAAAAATAATTATATTTTATTAAAATTTAC F#FFFFFFFFFFFFFFFFFFFFFFFFFFF:FF:F,FFFFFFF::F,FF:FFFFFF:FFFFFFFFFFFFFF:F:FFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 141 * 0 0 * * 0 0 GTAAATTTTAATAAAATATAATTATTTTTTTTTTTTTTTATATTTAATTTTATTTAAAAATAAATATATGTATTGAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF,FFFFFFF:FFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and CF05-CM05-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:GW210315000:4:1101:25509:1047_1:N:0:AGTTCAGG+TCTGTTGG/1 83 NC_035780.1_GA_converted 36883175 6 6M1D80M = 36883175 -87 ATAAATTTCAATAAAATACAACTATTTCCTTTTTTTCCTATACTTAACTTTACTCAAAAATAAATATATATACTAAAAAAAAAANA FFFFFFFFFFFFF:F:FFFFFFFFFFFFFF:FFFFFF:FF,F::FFFFFFF,F:FF:FFFFFFFFFFFFFFFFFFFFFFFFFFF#F AS:i:-15 XS:i:-21 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:6^A77T0T1 YS:i:-20 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:25509:1047_2:N:0:AGTTCAGG+TCTGTTGG/2 163 NC_035780.1_GA_converted 36883175 6 6M1D80M = 36883175 87 ATAAATTTCAATAAAATACAACTATTTCCTTTTTTTCCTATACTTAACTTTACTCAAAAATAAATATATATACTAAAAAAAAAAAA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF,FFFFFFF:FFFFFFFFF AS:i:-20 XS:i:-20 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:6^A77T0T1 YS:i:-15 YT:Z:CP >>> Writing bisulfite mapping results to CF05-CM05-Zygote_pe.bam <<< Reading in the sequence files ../../data/CF05-CM05-Zygote_R1_001.fastp-trim.20220827.fq.gz and ../../data/CF05-CM05-Zygote_R2_001.fastp-trim.20220827.fq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1150:29695:23109_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 5000000 sequence pairs so far 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 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1269:8856:7905_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17111 Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1274:16324:6308_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17161 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1274:16107:6370_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17161 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1274:16161:6370_1:N:0:AGTTCAGG+TCAGTTGG NC_007175.2 17161 Processed 13000000 sequence pairs so far 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:GW210315000:4:1349:28655:12759_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 1 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1377:19587:36667_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1408:7039:3474_1:N:0:AGTTCAGG+TCTGTTGG NC_035781.1 2 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1418:10610:20494_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17124 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1428:30418:24862_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 2 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1432:32027:19946_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1457:2808:9518_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 2 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1469:31738:29277_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 3 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1474:16586:31407_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17157 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1474:17128:33191_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17157 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 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1557:26413:12790_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17097 Processed 30000000 sequence pairs so far Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1607:22028:2002_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 32000000 sequence pairs so far 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:GW210315000:4:1651:19027:29590_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 1 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1652:25943:13135_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17166 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1654:32099:1407_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1662:16260:5102_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17165 Processed 36000000 sequence pairs so far Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2120:7292:27806_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17163 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2131:22535:24205_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2137:18991:10864_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 2 Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2159:10239:9893_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2225:24017:34538_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2225:22309:34867_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2225:25129:35524_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2263:20220:32878_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2267:14570:13980_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 49000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2306:14624:4272_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 50000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2311:26747:31438_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2313:5918:22106_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 1 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2319:7211:24846_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 17100 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 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2377:19054:28103_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 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 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2460:2709:5713_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2468:7048:8312_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2468:7880:8907_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 61000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2504:18285:5572_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 3 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2505:8947:35994_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2505:8223:36151_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2506:9326:2957_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2506:8513:19179_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2 Processed 62000000 sequence pairs so far Processed 63000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2526:18692:9972_1:N:0:AGTTCAGG+TCTGTTGG NC_035781.1 2 Processed 64000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2537:13476:14591_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2537:13331:22169_1:N:0:AGTTCAGG+TCTGTTGG NC_035780.1 2 Processed 65000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2550:29170:13150_1:N:0:AGTTCAGG+TCTGTTGG NC_007175.2 2