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/EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz ../../data/EF06-EM01-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/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/EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz to EF06-EM01-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 EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz (142739602 sequences in total) Writing a C -> T converted version of the input file EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq Writing a G -> A converted version of the input file EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz to EF06-EM01-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 EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz (142739602 sequences in total) Input files are EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-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,-0.8 -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 EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-Larvae_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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 77 * 0 0 * * 0 0 TNTAAAATAAATAATTAATTATTAAATAATAAATATTATAATTAATTTTTATTTTTTATTAAATTAAATTTTTTAAAATAAAATTTTTATAATAAAAATTTTATTAAATATTTAAAAATTAAATATATAAAATAATTAAAAAATTATAAA F#FFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFF:FF::FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 141 * 0 0 * * 0 0 AATTAATTTTTTTTTTAAAATTATTATTTATTTTTTTTATAATTTTTTAATTATTTTATATATTTAATTTTTAAATATTTAATAAAATTTTTATTATAAAAATTTTATTTTAAAAAATTTAATTTAATAAAAAATAAAAATTAATTATAA FF,FFFFFFFFFFFFFFFFFFFF,FFFFFF,FFFFFFFF:FFFFFFFFF::F,FFFFF:F:F:FF:FFFFFFFF:FFFFFFF,:FFFFFFFFFFF,FFFFFFFFFFFFF::FFFFFFFFF:FF:FFFF:FFFF,:FFF:FFFFFFFFFFF YT:Z:UP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2GAgenome (reading in sequences from EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM01-Larvae_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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 99 NC_035783.1_GA_converted 33762010 42 150M = 33762045 186 CNTAAAATAAACAATCAACCACCAAATAACAAATATTACAACCAATCTCTACCCTTCATTAAATTAAATTCTTTAAAACAAAACTTTTACAACAAAAATCTCACTAAATATCCAAAAATCAAACACACAAAACAACTAAAAAATTATAAA F#FFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFF:FF::FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:-7 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:1C101A46 YS:i:-20 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 147 NC_035783.1_GA_converted 33762045 42 134M1D16M = 33762010 -186 TTACAACCAATCTCTACCCTTCATTAAATTAAATTCTTTAAAACAAAACTTTTACAACAAAAATCTCACTAAATATCCAAAAATCAAACACACAAAACAACTAAAAAATTATAAAAAAAATAAACAACAATCCCAAAAAAAAAACTAACC FFFFFFFFFFF:FFF:,FFFF:FFFF:FF:FFFFFFFFF::FFFFFFFFFFFFF,FFFFFFFFFFF:,FFFFFFF:FFFFFFFF:FF:F:F:FFFFF,F::FFFFFFFFF:FFFFFFFF,FFFFFF,FFFFFFFFFFFFFFFFFFFF,FF AS:i:-20 XN:i:0 XM:i:2 XO:i:1 XG:i:1 NM:i:3 MD:Z:68A50C14^A16 YS:i:-7 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for GAread1CTread2CTgenome (reading in sequences from EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_G_to_A.fastq and EF06-EM01-Larvae_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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 83 NC_035783.1_CT_converted 21935408 42 150M = 21935372 -186 TTTATAATTTTTTAGTTGTTTTGTGTGTTTGATTTTTGGATATTTAGTGAGATTTTTGTTGTAAAAGTTTTGTTTTAAAGAATTTAATTTAATGAAGGGTAGAGATTGGTTGTAATATTTGTTATTTGGTGGTTGATTGTTTATTTTANG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF,FFF::FF:FFFFFFFFFFFF:FFFFF:FFFFFFFFFFFFFFF#F AS:i:-13 XN:i:0 XM:i:3 XO:i:0 XG:i:0 NM:i:3 MD:Z:48A1A97G1 YS:i:-32 YT:Z:CP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 163 NC_035783.1_CT_converted 21935372 42 6M1D144M = 21935408 186 GGTTAGTTTTTTTTTTGGGATTGTTGTTTATTTTTTTTATAATTTTTTAGTTGTTTTGTGTGTTTGATTTTTGGATATTTAGTGAGATTTTTGTTGTAAAAGTTTTGTTTTAAAGAATTTAATTTAATGAAGGGTAGAGATTGGTTGTAA FF,FFFFFFFFFFFFFFFFFFFF,FFFFFF,FFFFFFFF:FFFFFFFFF::F,FFFFF:F:F:FF:FFFFFFFF:FFFFFFF,:FFFFFFFFFFF,FFFFFFFFFFFFF::FFFFFFFFF:FF:FFFF:FFFF,:FFF:FFFFFFFFFFF AS:i:-32 XN:i:0 XM:i:4 XO:i:1 XG:i:1 NM:i:5 MD:Z:6^T16T7G52A1A64 YS:i:-13 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz_C_to_T.fastq and EF06-EM01-Larvae_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:GW210315000:4:1101:26142:1047_1:N:0:ATGAGGCC+CAATTAAC/1 77 * 0 0 * * 0 0 TNTAAAATAAATAATTAATTATTAAATAATAAATATTATAATTAATTTTTATTTTTTATTAAATTAAATTTTTTAAAATAAAATTTTTATAATAAAAATTTTATTAAATATTTAAAAATTAAATATATAAAATAATTAAAAAATTATAAA F#FFFFFFFFFFFFFFF:FFFFF:FFFFFFFFFFFF:FF::FFF,FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF YT:Z:UP GWNJ-1012:512:GW210315000:4:1101:26142:1047_2:N:0:ATGAGGCC+CAATTAAC/2 141 * 0 0 * * 0 0 AATTAATTTTTTTTTTAAAATTATTATTTATTTTTTTTATAATTTTTTAATTATTTTATATATTTAATTTTTAAATATTTAATAAAATTTTTATTATAAAAATTTTATTTTAAAAAATTTAATTTAATAAAAAATAAAAATTAATTATAA FF,FFFFFFFFFFFFFFFFFFFF,FFFFFF,FFFFFFFF:FFFFFFFFF::F,FFFFF:F:F:FF:FFFFFFFF:FFFFFFF,:FFFFFFFFFFF,FFFFFFFFFFFFF::FFFFFFFFF:FF:FFFF:FFFF,:FFF:FFFFFFFFFFF YT:Z:UP >>> Writing bisulfite mapping results to EF06-EM01-Larvae_pe.bam <<< Reading in the sequence files ../../data/EF06-EM01-Larvae_R1_001.fastp-trim.20220827.fq.gz and ../../data/EF06-EM01-Larvae_R2_001.fastp-trim.20220827.fq.gz Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1123:10176:4429_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1139:17571:23500_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1156:5367:20776_1:N:0:ATGAGGCC+CAATTAAC NC_035781.1 2 Processed 4000000 sequence pairs so far 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 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1308:16486:11162_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 11000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1326:31566:33614_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 3 Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1362:26476:32033_1:N:0:ATGAGGCC+CAATTAAC NC_035781.1 2 Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1405:3613:18615_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 1 Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1424:19217:8594_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1516:8612:34381_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1516:8621:34397_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1516:8368:35180_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1518:12463:1658_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1567:5321:13745_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1620:6406:23202_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:1632:21621:3771_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 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 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2136:29451:22592_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2139:15854:13730_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2144:7563:10551_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2148:1280:13322_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 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 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:GW210315000:4:2362:4128:17472_1:N:0:ATGAGGCC+CAATTAAC NC_035781.1 2 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2417:2917:6825_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2451:24587:30076_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 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 Chromosomal sequence could not be extracted for GWNJ-1012:512:GW210315000:4:2554:21486:11741_1:N:0:ATGAGGCC+CAATTAAC NC_035780.1 2 Processed 51000000 sequence pairs so far