Bowtie 2 seems to be working fine (tested command '/home/shared/bowtie2-2.4.4-linux-x86_64/bowtie2 --version' [2.4.4]) Output format is BAM (default) Alignments will be written out in BAM format. Samtools found here: '/usr/bin/samtools' Reference genome folder provided is ../data/ (absolute path is '/home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/)' FastQ format assumed (by default) Attention: using more than 4 cores per alignment thread has been reported to have diminishing returns. If possible try to limit -p to a value of 4 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 '/home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/code'): ../data/106M_R1.fastp-trim.fq.gz ../data/106M_R2.fastp-trim.fq.gz Library is assumed to be strand-specific (directional), alignments to strands complementary to the original top or bottom strands will be ignored (i.e. not performed!) Output will be written into the directory: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/output/01.2-bismark/ Setting parallelization to single-threaded (default) Summary of all aligner options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 Current working directory is: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/code Now reading in and storing sequence information of the genome specified in: /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are ../data/106M_R1.fastp-trim.fq.gz and ../data/106M_R2.fastp-trim.fq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 106M_R1.fastp-trim.fq.gz to 106M_R1.fastp-trim.fq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 106M_R1.fastp-trim.fq.gz (87376764 sequences in total) Writing a G -> A converted version of the input file 106M_R2.fastp-trim.fq.gz to 106M_R2.fastp-trim.fq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 106M_R2.fastp-trim.fq.gz (87376764 sequences in total) Input files are 106M_R1.fastp-trim.fq.gz_C_to_T.fastq and 106M_R2.fastp-trim.fq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /home/shared/16TB_HDD_01/sr320/github/project-mytilus-methylation/data/ with the specified options: -q --score-min L,0,-1.0 -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 106M_R1.fastp-trim.fq.gz_C_to_T.fastq and 106M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --norc)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:3533:1014_1:N:0:AGCGTGTT+CTTCGCAA/1 99 NC_086377.1_CT_converted 70497303 6 131M = 70497425 253 AATGAAGATGAATTATAATGTGTTTTTTGTAATTGTAAGTATATTATTAAATATGTTTTAATTAATTGTATTGATTTTGTTGATATTTGTAAGAAGTATTTTAATGTTAATAATATGAATGATTTATTTAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:117T13 YS:i:0 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:3533:1014_2:N:0:AGCGTGTT+CTTCGCAA/2 147 NC_086377.1_CT_converted 70497425 6 131M = 70497303 -253 TTTATTTAATAATGTTTTATTTATAAATATTGTTGTTTTTTTAAAAGAAATTGGAATTTATTATAGAATATAAAAATGTTATTATATTGAAATTGATTTTAATTTTTATTATGTTATTTTATTGTTGATTT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:-6 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:131 YS:i:-6 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 106M_R1.fastp-trim.fq.gz_C_to_T.fastq and 106M_R2.fastp-trim.fq.gz_G_to_A.fastq, with the options: -q --score-min L,0,-1.0 -p 8 --reorder --ignore-quals --no-mixed --no-discordant --dovetail --maxins 500 --nofw)) Found first alignment: LH00469:254:22HGFVLT4:2:1101:3533:1014_1:N:0:AGCGTGTT+CTTCGCAA/1 83 NW_026963295.1_GA_converted 4463939 6 131M = 4463817 -253 TTAAATAAATCATTCATATTATTAACATTAAAATACTTCTTACAAATATCAACAAAATCAATACAATTAATTAAAACATATTTAATAATATACTTACAATTACAAAAAACACATTATAATTCATCTTCATT IIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-18 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:13A117 YS:i:-6 YT:Z:CP LH00469:254:22HGFVLT4:2:1101:3533:1014_2:N:0:AGCGTGTT+CTTCGCAA/2 163 NW_026963295.1_GA_converted 4463817 6 131M = 4463939 253 AAATCAACAATAAAATAACATAATAAAAATTAAAATCAATTTCAATATAATAACATTTTTATATTCTATAATAAATTCCAATTTCTTTTAAAAAAACAACAATATTTATAAATAAAACATTATTAAATAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-6 XS:i:-6 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:42A88 YS:i:-6 YT:Z:CP >>> Writing bisulfite mapping results to 106M_pe.bam <<< Reading in the sequence files ../data/106M_R1.fastp-trim.fq.gz and ../data/106M_R2.fastp-trim.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 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1142:38160:29352_1:N:0:AGCGTGTT+CTTCGCAA NW_026963517.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1144:9796:27209_1:N:0:AGCGTGTT+CTTCGCAA NW_026963489.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1146:15048:4642_1:N:0:AGCGTGTT+CTTCGCAA NW_026963315.1 4066329 Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1160:8032:19336_1:N:0:AGCGTGTT+CTTCGCAA NW_026963598.1 2 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1176:48736:4124_1:N:0:AGCGTGTT+CTTCGCAA NW_026963303.1 249870 Processed 9000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1178:50889:9811_1:N:0:AGCGTGTT+CTTCGCAA NW_026963443.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1178:34745:11562_1:N:0:AGCGTGTT+CTTCGCAA NW_026963326.1 30746 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1179:24370:9405_1:N:0:AGCGTGTT+CTTCGCAA NW_026963377.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1182:15396:14994_1:N:0:AGCGTGTT+CTTAGCAA NW_026963415.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1184:49715:18510_1:N:0:AGCGTGTT+CTTCGCAA NW_026963308.1 2 Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1209:28862:25836_1:N:0:AGCGTGTT+CTTCGCAA NW_026963667.1 1 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 LH00469:254:22HGFVLT4:2:1242:49125:8886_1:N:0:AGCGTGTT+CTTCGCAA NW_026963491.1 1 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1261:36064:16086_1:N:0:AGCGTGTT+CTTCGCAA NW_026963309.1 1 Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1264:51447:2345_1:N:0:AGCGTGTT+CTTCGCAA NW_026963402.1 95206 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1271:34607:14013_1:N:0:AGCGTGTT+CTTCGCAA NW_026963646.1 21598 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1271:34599:14027_1:N:0:AGCGTGTT+CTTCGCAA NW_026963646.1 21598 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1287:28773:27783_1:N:0:AGCGTGTT+CTTCGCAA NW_026963542.1 23756 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1292:15428:20261_1:N:0:AGCGTGTT+CTTCGCAA NW_026963684.1 18482 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1293:1599:18706_1:N:0:AGCGTGTT+CTTCGCAA NW_026963684.1 18467 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1303:2610:7289_1:N:0:AGCGTGTT+CTTCGCAA NW_026963731.1 22032 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1312:50144:21325_1:N:0:AGCGTGTT+CTTCGCAA NW_026963677.1 25108 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1319:35473:11058_1:N:0:AGCGTGTT+CTTCGCAA NW_026963378.1 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 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1369:23796:22446_1:N:0:AGCGTGTT+CTTCGCAA NW_026963514.1 23944 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1374:47288:17893_1:N:0:AGCGTGTT+CTTCGCAA NW_026963564.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1374:47279:17907_1:N:0:AGCGTGTT+CTTCGCAA NW_026963564.1 2 Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1398:50160:3704_1:N:0:AGCGTGTT+CTTCGCAA NW_026963646.1 21626 Processed 35000000 sequence pairs so far 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 LH00469:254:22HGFVLT4:2:1435:12564:11744_1:N:0:AGCGTGTT+CTTCGCAA NW_026963415.1 3 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Processed 41000000 sequence pairs so far Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:1491:29679:22348_1:N:0:AGCGTGTT+CTTCGCAA NW_026963307.1 124870 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Processed 47000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2119:17443:20863_1:N:0:AGCGTGTT+CTTCGCAA NW_026963293.1 1 Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2127:20438:8396_1:N:0:AGCGGGTT+CTTCGCAA NW_026963443.1 56339 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2132:48550:7107_1:N:0:AGCGTGTT+CTTCGCAA NW_026963684.1 18483 Processed 49000000 sequence pairs so far Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2154:18835:3633_1:N:0:AGCGTGTT+CTTCGCAA NW_026963377.1 1 Processed 52000000 sequence pairs so far Processed 53000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2168:43606:11156_1:N:0:AGCGTGTT+CTTCGCAA NW_026963739.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2173:13454:25135_1:N:0:AGCGTGTT+CTTCGCAA NW_026963717.1 2 Processed 54000000 sequence pairs so far Processed 55000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2189:46664:8746_1:N:0:AGCGTGTT+CTTCGCAA NW_026963788.1 1 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2193:28700:18776_1:N:0:AGCGTGTT+CTTCGCAA NW_026963786.1 1 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2251:44229:3353_1:N:0:AGCGTGTT+CTTCGCAA NW_026963384.1 2 Processed 62000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2254:9618:25219_1:N:0:AGCGTGTT+CTTCGCAA NW_026963717.1 2 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2256:34664:4250_1:N:0:AGCGTGTT+CTTCGCAA NW_026963547.1 15331 Processed 63000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2268:50694:18580_1:N:0:AGCGTGTT+CTTCGCAA NW_026963303.1 1 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2332:38200:1939_1:N:0:AGCGTGTT+CTTCGCAA NW_026963443.1 2 Processed 71000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2344:12750:21872_1:N:0:AGCGTGTT+CTTCGCAA NW_026963639.1 24143 Processed 72000000 sequence pairs so far Processed 73000000 sequence pairs so far Processed 74000000 sequence pairs so far 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 Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2438:49780:6295_1:N:0:AGCGTGTT+CTTCGCAA NW_026963689.1 1 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 LH00469:254:22HGFVLT4:2:2473:19264:21830_1:N:0:AGCGTGTT+CTTCGCAA NW_026963415.1 1 Processed 85000000 sequence pairs so far Processed 86000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00469:254:22HGFVLT4:2:2490:8493:10301_1:N:0:AGCGTGTT+CTTCGCAA NW_026963377.1 1 Processed 87000000 sequence pairs so far 87376764 reads; of these: 87376764 (100.00%) were paired; of these: 38223100 (43.75%) aligned concordantly 0 times 17230147 (19.72%) aligned concordantly exactly 1 time 31923517 (36.54%) aligned concordantly >1 times 56.25% overall alignment rate 87376764 reads; of these: 87376764 (100.00%) were paired; of these: 38229042 (43.75%) aligned concordantly 0 times 17229454 (19.72%) aligned concordantly exactly 1 time 31918268 (36.53%) aligned concordantly >1 times 56.25% overall alignment rate Processed 87376764 sequences in total Failed to close filehandle AMBIG_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2641, line 349507056. Failed to close filehandle AMBIG_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2642, line 349507056. Failed to close filehandle UNMAPPED_1: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2643, line 349507056. Failed to close filehandle UNMAPPED_2: Bad file descriptor at /home/shared/Bismark-0.24.0/bismark line 2644, line 349507056. Successfully deleted the temporary files 106M_R1.fastp-trim.fq.gz_C_to_T.fastq and 106M_R2.fastp-trim.fq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 87376764 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2110434862 Total methylated C's in CpG context: 28540071 Total methylated C's in CHG context: 3093726 Total methylated C's in CHH context: 14478620 Total methylated C's in Unknown context: 352529 Total unmethylated C's in CpG context: 212795157 Total unmethylated C's in CHG context: 334591138 Total unmethylated C's in CHH context: 1516936150 Total unmethylated C's in Unknown context: 11611613 C methylated in CpG context: 11.8% C methylated in CHG context: 0.9% C methylated in CHH context: 0.9% C methylated in unknown context (CN or CHN): 2.9% Bismark completed in 0d 6h 7m 38s ==================== Bismark run complete ====================