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/06B_1.fastq.gz /mmfs1/gscratch/coenv/sr320/cod-bs/06B_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/06B_1.fastq.gz and /mmfs1/gscratch/coenv/sr320/cod-bs/06B_2.fastq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 06B_1.fastq.gz to 06B_1.fastq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 06B_1.fastq.gz (51326454 sequences in total) Writing a G -> A converted version of the input file 06B_2.fastq.gz to 06B_2.fastq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 06B_2.fastq.gz (51326454 sequences in total) Input files are 06B_1.fastq.gz_C_to_T.fastq and 06B_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 06B_1.fastq.gz_C_to_T.fastq and 06B_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: LH00160:538:22WG5VLT4:6:1101:25269:1042_1:N:0:GAGACGAT+ACNGGTTA/1 77 * 0 0 * * 0 0 ANTATTGTGGTTATATGGGTTAGTATATATGGATTAGAATATAATGGTTTGTAGTTAGTTTTGTGTTTTAGATTGGAAGAGTATATGTTTGAATTTTAGTTATGAGATGATATTTAGTATGTTGTTTTTTGTTTGAAAAAAGGGGGGGGGG I#IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIII9I-II--I-I----I-I---I--9I99--999999999 YT:Z:UP LH00160:538:22WG5VLT4:6:1101:25269:1042_2:N:0:GAGACGAT+ACNGGTTA/2 141 * 0 0 * * 0 0 AAAACACAAAACTAACTACAAACCATTATATTCTAATCCATATATACTAACCCATATAACCACAATACTAAATCAAAAAAACATCATATAAAAAAAAAATATACCAATTCATATAAATCTCAATAATCACCATATCATTAAAAAAAAAAAA IIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III9III9IIIIIIIIIIII9IIIIIIII9I-IIII-III-II9II9I9II999IIIIIII--III-IIIIII YT:Z:UP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 06B_1.fastq.gz_C_to_T.fastq and 06B_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: LH00160:538:22WG5VLT4:6:1101:25269:1042_1:N:0:GAGACGAT+ACNGGTTA/1 77 * 0 0 * * 0 0 ANTATTGTGGTTATATGGGTTAGTATATATGGATTAGAATATAATGGTTTGTAGTTAGTTTTGTGTTTTAGATTGGAAGAGTATATGTTTGAATTTTAGTTATGAGATGATATTTAGTATGTTGTTTTTTGTTTGAAAAAAGGGGGGGGGG I#IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIII9I-II--I-I----I-I---I--9I99--999999999 YT:Z:UP LH00160:538:22WG5VLT4:6:1101:25269:1042_2:N:0:GAGACGAT+ACNGGTTA/2 141 * 0 0 * * 0 0 AAAACACAAAACTAACTACAAACCATTATATTCTAATCCATATATACTAACCCATATAACCACAATACTAAATCAAAAAAACATCATATAAAAAAAAAATATACCAATTCATATAAATCTCAATAATCACCATATCATTAAAAAAAAAAAA IIII9IIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9III9III9IIIIIIIIIIII9IIIIIIII9I-IIII-III-II9II9I9II999IIIIIII--III-IIIIII YT:Z:UP >>> Writing bisulfite mapping results to 06B_pe.bam <<< Reading in the sequence files /mmfs1/gscratch/coenv/sr320/cod-bs/06B_1.fastq.gz and /mmfs1/gscratch/coenv/sr320/cod-bs/06B_2.fastq.gz Processed 1000000 sequence pairs so far Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1133:16294:18790_1:N:0:GAGACGAT+ACCGGTTA NC_082387.1 1 Processed 3000000 sequence pairs so far Processed 4000000 sequence pairs so far Processed 5000000 sequence pairs so far Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1196:15461:24351_1:N:0:GAGACGAT+ACCGGTTA NC_082387.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1204:35659:17039_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077804 Processed 7000000 sequence pairs so far Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1235:28109:12458_1:N:0:GAGACGAT+ACCGGTTA NC_036931.1 1 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1259:23278:22726_1:N:0:GAGACGAT+ACCGGTTA NC_082392.1 2 Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1279:28781:14125_1:N:0:GAGACGAT+AACGGTTA NC_036931.1 16418 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1285:17613:5328_1:N:0:GAGACGAT+ACCGGTTA NC_036931.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1292:27130:7962_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077814 Processed 13000000 sequence pairs so far Processed 14000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1313:33239:3718_1:N:0:GAGACGAT+ACCGGTTA NC_082391.1 23727853 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1317:28506:21605_1:N:0:GAGACGAT+ACCGGTTA NC_082400.1 17993472 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1317:28514:21619_1:N:0:GAGACGAT+ACCGGTTA NC_082400.1 17993472 Processed 15000000 sequence pairs so far Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1343:34550:23468_1:N:0:GAGACGAT+ACCGGTTA NC_082389.1 23759926 Processed 17000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1361:26952:4572_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077818 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1364:30941:14027_1:N:0:GAGACGAT+ACCGGTTA NC_082385.1 2 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1364:36088:28063_1:N:0:GAGAGGAT+ACCGGTTA NC_082394.1 22312318 Processed 18000000 sequence pairs so far Processed 19000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1387:41170:2737_1:N:0:GAGACGAT+ACCGGTTA NC_082395.1 3 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1395:32131:6197_1:N:0:GAGACGAT+ACCGGTTA NC_082391.1 23727853 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1395:15857:18706_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077813 Processed 20000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1411:25948:2107_1:N:0:GAGACGAT+ACCGGTTA NC_082403.1 3 Processed 21000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1423:24767:22894_1:N:0:GAGACGAT+ACCGGTTA NC_082392.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1427:15380:4040_1:N:0:GAGACGAT+ACCGGTTA NC_082397.1 2 Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1438:21457:3269_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077814 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1438:21449:3283_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077814 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1440:44666:10133_1:N:0:GAGACGAT+ACCGGTTA NC_082396.1 2 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1451:18698:8578_1:N:0:GAGACGAT+ACCGGTTA NC_082391.1 23727853 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1453:8129:10371_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077815 Processed 24000000 sequence pairs so far Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1482:45095:20933_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077819 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1487:12790:10035_1:N:0:GAGACGAT+ACCGGTTA NC_036931.1 1 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1489:26199:4530_1:N:0:GAGACGAT+ACCGGTTA NC_036931.1 16424 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1489:32309:28329_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077815 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1489:32301:28343_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077815 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2103:20858:4782_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077820 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2118:36905:15358_1:N:0:GAGACGAT+ACCGGTTA NC_082382.1 26289593 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2118:36913:15372_1:N:0:GAGACGAT+ACCGGTTA NC_082382.1 26289593 Processed 28000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2125:41396:28875_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077817 Processed 29000000 sequence pairs so far Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2161:20211:25626_1:N:0:GAGACGAT+ACCGGTTA NC_082394.1 22312311 Processed 31000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2181:19685:13705_1:N:0:GAGACGAT+ACCGGTTA NC_082382.1 26289587 Processed 32000000 sequence pairs so far Processed 33000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2215:44763:28763_1:N:0:GAGACGAT+ACCGGTTA NC_082389.1 23759913 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2230:36404:9755_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 2 Processed 35000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2246:14037:6393_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077819 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 LH00160:538:22WG5VLT4:6:2287:23116:4684_1:N:0:GAGACGAT+ACCGGTTA NC_082402.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2296:34858:15288_1:N:0:GAGACGAT+ACCGGTTA NC_082390.1 23393738 Processed 39000000 sequence pairs so far Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2325:42133:14237_1:N:0:GAGACGAT+ACCGGTTA NC_082384.1 1 Processed 41000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2339:50427:16941_1:N:0:GAGACGAT+ACCGGTTA NC_082387.1 1 Processed 42000000 sequence pairs so far Processed 43000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2374:10800:10539_1:N:0:GAGACGAT+ACCGGTTA NC_082398.1 16295378 Processed 44000000 sequence pairs so far Processed 45000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2407:25819:24379_1:N:0:GAGACGAT+ACCGGTTA NC_082384.1 1 Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2426:45046:15694_1:N:0:GAGACGAT+ACCGGTTA NC_082382.1 26289594 Processed 47000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2443:21279:21928_1:N:0:GAGACGAT+ACCGGTTA NC_082399.1 21077817 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2446:31742:26312_1:N:0:GAGACGAT+ACCGGTTA NC_082396.1 24646067 Processed 48000000 sequence pairs so far Processed 49000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2471:12232:3718_1:N:0:GAGACGAT+ACCGGTTA NC_082390.1 23393714 Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far 51326454 reads; of these: 51326454 (100.00%) were paired; of these: 25109236 (48.92%) aligned concordantly 0 times 14442393 (28.14%) aligned concordantly exactly 1 time 11774825 (22.94%) aligned concordantly >1 times 51.08% overall alignment rate 51326454 reads; of these: 51326454 (100.00%) were paired; of these: 25110406 (48.92%) aligned concordantly 0 times 14438216 (28.13%) aligned concordantly exactly 1 time 11777832 (22.95%) aligned concordantly >1 times 51.08% overall alignment rate Processed 51326454 sequences in total Successfully deleted the temporary files 06B_1.fastq.gz_C_to_T.fastq and 06B_2.fastq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 51326454 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2278727614 Total methylated C's in CpG context: 242827929 Total methylated C's in CHG context: 15034207 Total methylated C's in CHH context: 52630375 Total methylated C's in Unknown context: 4809378 Total unmethylated C's in CpG context: 81108635 Total unmethylated C's in CHG context: 452073792 Total unmethylated C's in CHH context: 1435052676 Total unmethylated C's in Unknown context: 7627131 C methylated in CpG context: 75.0% C methylated in CHG context: 3.2% C methylated in CHH context: 3.5% C methylated in Unknown context (CN or CHN): 38.7% Bismark completed in 0d 4h 8m 9s ==================== Bismark run complete ====================