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 /mmfs1/gscratch/scrubbed/sr320/github/project-cod-temperature/data/genome/ (absolute path is '/mmfs1/gscratch/scrubbed/sr320/github/project-cod-temperature/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/project-cod-temperature/output/16-bismark'): /mmfs1/gscratch/coenv/sr320/cod-bs/08B_1.fastq.gz /mmfs1/gscratch/coenv/sr320/cod-bs/08B_2.fastq.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: /mmfs1/gscratch/scrubbed/sr320/github/project-cod-temperature/output/16-bismark/ 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/project-cod-temperature/output/16-bismark Now reading in and storing sequence information of the genome specified in: /mmfs1/gscratch/scrubbed/sr320/github/project-cod-temperature/data/genome/ Single-core mode: setting pid to 1 Paired-end alignments will be performed ======================================= The provided filenames for paired-end alignments are /mmfs1/gscratch/coenv/sr320/cod-bs/08B_1.fastq.gz and /mmfs1/gscratch/coenv/sr320/cod-bs/08B_2.fastq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 08B_1.fastq.gz to 08B_1.fastq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 08B_1.fastq.gz (51504358 sequences in total) Writing a G -> A converted version of the input file 08B_2.fastq.gz to 08B_2.fastq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 08B_2.fastq.gz (51504358 sequences in total) Input files are 08B_1.fastq.gz_C_to_T.fastq and 08B_2.fastq.gz_G_to_A.fastq (FastQ) Now running 2 instances of Bowtie 2 against the bisulfite genome of /mmfs1/gscratch/scrubbed/sr320/github/project-cod-temperature/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 08B_1.fastq.gz_C_to_T.fastq and 08B_2.fastq.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: LH00160:538:22WG5VLT4:6:1101:24152:1042_1:N:0:CTTGTCGA+CGNTGTTC/1 77 * 0 0 * * 0 0 GNTTGGGATGTTGATTATTTTTTTGTTGATGATTTTTTGGGTGAGTTTGAGATAAGATAAGGGTTTGTGTGTGTTGTGAGGGGTGAGGTTAATATGTTAGGTTGGTGATGGTTGGGTTTTGGGATAAGGTATATAGTATTTTATTTTTTTT I#IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII YT:Z:UP LH00160:538:22WG5VLT4:6:1101:24152:1042_2:N:0:CTTGTCGA+CGNTGTTC/2 141 * 0 0 * * 0 0 AAAAAAAAAAAATAAAATACTATATACCTTATCCCAAAACCCAACCATCACCAACCTAACATATTAACCTCACCCCTCACAACACACACAAACCCTTATCTTATCTCAAACTCACCCAAAAAATCATCAACAAAAAAATAATCAACATCCC 9IIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 08B_1.fastq.gz_C_to_T.fastq and 08B_2.fastq.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: LH00160:538:22WG5VLT4:6:1101:24152:1042_1:N:0:CTTGTCGA+CGNTGTTC/1 83 NC_082402.1_GA_converted 15689096 42 1M2I148M = 15689088 -157 AAAAAAAATAAAATACTATATACCTTATCCCAAAACCCAACCATCACCAACCTAACATATTAACCTCACCCCTCACAACACACACAAACCCTTATCTTATCTCAAACTCACCCAAAAAATCATCAACAAAAAAATAATCAACATCCCAANC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII#I AS:i:-12 XN:i:0 XM:i:1 XO:i:1 XG:i:2 NM:i:3 MD:Z:147A1 YS:i:-17 YT:Z:CP LH00160:538:22WG5VLT4:6:1101:24152:1042_2:N:0:CTTGTCGA+CGNTGTTC/2 163 NC_082402.1_GA_converted 15689088 42 6M2D145M = 15689096 157 AAAAAAAAAAAATAAAATACTATATACCTTATCCCAAAACCCAACCATCACCAACCTAACATATTAACCTCACCCCTCACAACACACACAAACCCTTATCTTATCTCAAACTCACCCAAAAAATCATCAACAAAAAAATAATCAACATCCC 9IIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIII AS:i:-17 XN:i:0 XM:i:1 XO:i:1 XG:i:2 NM:i:3 MD:Z:2T3^TT145 YS:i:-12 YT:Z:CP >>> Writing bisulfite mapping results to 08B_pe.bam <<< Reading in the sequence files /mmfs1/gscratch/coenv/sr320/cod-bs/08B_1.fastq.gz and /mmfs1/gscratch/coenv/sr320/cod-bs/08B_2.fastq.gz Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1107:20332:17039_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077819 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1114:18196:25780_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077804 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 LH00160:538:22WG5VLT4:6:1163:4026:26690_1:N:0:CTTGTCGA+CGATGTTC NC_082387.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1173:37747:4964_1:N:0:CTTGTCGA+CGATGTTC NC_082387.1 2 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1173:37763:4964_1:N:0:CTTGTCGA+CGATGTTC NC_082387.1 2 Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1213:4941:7065_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077818 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1224:23877:8130_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077817 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1229:49893:29464_1:N:0:CTTGTCGA+CGATGTTC NC_082402.1 1 Processed 9000000 sequence pairs so far 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 LH00160:538:22WG5VLT4:6:1282:23666:2219_1:N:0:CTTGTCGA+CGATGTTC NC_082382.1 26289602 Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1328:45871:6645_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077815 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1335:29250:1462_1:N:0:CTTGTCGA+CGATGTTC NC_082398.1 16295360 Processed 16000000 sequence pairs so far Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1352:40442:13859_1:N:0:CTTGTCGA+CGATGTTC NC_082383.1 23805027 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1353:17136:21395_1:N:0:CTTGTCGA+CGATGTTC NC_082397.1 2 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1357:40790:24827_1:N:0:CTTGTCGA+CGATGTTC NC_082400.1 17993499 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1358:39050:18426_1:N:0:CTTGTCGA+CGATGTTC NC_082392.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1363:40069:6099_1:N:0:CTTGTCGA+CGATGTTC NC_082396.1 24646073 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1364:11617:8704_1:N:0:CTTGTCGA+CGATGTTC NC_082382.1 26289617 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1364:23626:23076_1:N:0:CTTGTCGA+CGATGTTC NC_082388.1 28186519 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1364:23618:23090_1:N:0:CTTGTCGA+CGATGTTC NC_082388.1 28186519 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 LH00160:538:22WG5VLT4:6:1397:35934:4180_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077812 Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1427:43403:16437_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077817 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1427:43395:16451_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077817 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1444:19466:24113_1:N:0:CTTGTCGA+CGATGTTC NC_082382.1 26289601 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1454:2424:3914_1:N:0:CTTGTCGA+CGATGTTC NC_082397.1 2 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far 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 LH00160:538:22WG5VLT4:6:2123:27486:20793_1:N:0:CTTGTCGA+CGATGTTC NC_082387.1 2 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2128:35999:3479_1:N:0:CTTGTCGA+CGATGTTC NC_082387.1 3 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 LH00160:538:22WG5VLT4:6:2203:32098:6141_1:N:0:CTTGTCGA+CGATGTTC NC_082393.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2211:29533:24981_1:N:0:CTTGTCGA+CGATGTTC NC_082382.1 26289611 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2221:28740:21760_1:N:0:CTTGTCGA+CGATGTTC NC_082398.1 16295396 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2239:31208:18832_1:N:0:CTTGTCGA+CGATGTTC NC_082382.1 26289605 Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2256:35602:16521_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077816 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2256:35594:16535_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077816 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2256:35610:16535_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077816 Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2301:23699:16030_1:N:0:CTTGTCGA+CGATGTTC NC_082382.1 26289616 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2324:47199:18047_1:N:0:CTTGTCGA+CGATGTTC NC_082388.1 28186546 Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2333:27826:18160_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077815 Processed 42000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2358:18876:10651_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077811 Processed 43000000 sequence pairs so far Processed 44000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2387:2173:16226_1:N:0:CTTGTCGA+CGATGTTC NC_082399.1 21077818 Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2397:51382:14644_1:N:0:CTTGTCGA+CGATGTTC NC_082390.1 23393708 Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2413:2716:22936_1:N:0:CTTGTCGA+CGATGTTC NC_082387.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2423:10751:24015_1:N:0:CTTGTCGA+CGATGTTC NC_082387.1 1 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 LH00160:538:22WG5VLT4:6:2475:42189:29716_1:N:0:NTTGTCGA+NGATGTTC NC_082391.1 23727868 Processed 51000000 sequence pairs so far 51504358 reads; of these: 51504358 (100.00%) were paired; of these: 25638450 (49.78%) aligned concordantly 0 times 14736763 (28.61%) aligned concordantly exactly 1 time 11129145 (21.61%) aligned concordantly >1 times 50.22% overall alignment rate 51504358 reads; of these: 51504358 (100.00%) were paired; of these: 25630664 (49.76%) aligned concordantly 0 times 14742764 (28.62%) aligned concordantly exactly 1 time 11130930 (21.61%) aligned concordantly >1 times 50.24% overall alignment rate Processed 51504358 sequences in total Successfully deleted the temporary files 08B_1.fastq.gz_C_to_T.fastq and 08B_2.fastq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 51504358 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2337731160 Total methylated C's in CpG context: 252136240 Total methylated C's in CHG context: 15611825 Total methylated C's in CHH context: 54523729 Total methylated C's in Unknown context: 5035517 Total unmethylated C's in CpG context: 82838688 Total unmethylated C's in CHG context: 464625733 Total unmethylated C's in CHH context: 1467994945 Total unmethylated C's in Unknown context: 7924345 C methylated in CpG context: 75.3% C methylated in CHG context: 3.3% C methylated in CHH context: 3.6% C methylated in Unknown context (CN or CHN): 38.9% Bismark completed in 0d 4h 9m 30s ==================== Bismark run complete ====================