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/03B_1.fastq.gz /mmfs1/gscratch/coenv/sr320/cod-bs/03B_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/03B_1.fastq.gz and /mmfs1/gscratch/coenv/sr320/cod-bs/03B_2.fastq.gz Input files are in FastQ format Writing a C -> T converted version of the input file 03B_1.fastq.gz to 03B_1.fastq.gz_C_to_T.fastq Created C -> T converted version of the FastQ file 03B_1.fastq.gz (52740330 sequences in total) Writing a G -> A converted version of the input file 03B_2.fastq.gz to 03B_2.fastq.gz_G_to_A.fastq Created G -> A converted version of the FastQ file 03B_2.fastq.gz (52740330 sequences in total) Input files are 03B_1.fastq.gz_C_to_T.fastq and 03B_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 03B_1.fastq.gz_C_to_T.fastq and 03B_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: LH00160:538:22WG5VLT4:6:1101:22954:1042_1:N:0:TGAGCTAG+GANCGGTT/1 99 NC_082392.1_CT_converted 20421397 6 151M = 20421475 233 TNGATAAAATTTATATAATAGAAAATGTTTGTTTTAATATATTTGTAGATTTGTAATTGTAAATTTTATTGAAATAATTTGTAGTTTAATAAGTTTATGGTTGTTTTTGTGTTATTTGTTATTGGGTTATATAGTGTAAAATTTTATTAGG I#IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-7 XS:i:-7 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:1T50A98 YS:i:-58 YT:Z:CP LH00160:538:22WG5VLT4:6:1101:22954:1042_2:N:0:TGAGCTAG+GANCGGTT/2 147 NC_082392.1_CT_converted 20421475 6 85M6D54M2I10M = 20421397 -233 TTGTAGTTTAATAAGTTTATGGTTGTTTTTGTGTTATTTGTTATTGGGTTATATAGTGTAAAATTTTATTAGGTGATTAGTTGGTTGTTTAGTTGTTTGTTGTTGTATGAGATATTTTATTGTATAATAAAATGATTAGTTTTTTTTTTTT I-9IIIIIIIIIIIIIIIIIIIIIIIII9IIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:-58 XS:i:-58 XN:i:0 XM:i:4 XO:i:2 XG:i:8 NM:i:12 MD:Z:85^TAAAAA54G2A1A1G2 YS:i:-7 YT:Z:CP Now starting a Bowtie 2 paired-end alignment for CTread1GAread2GAgenome (reading in sequences from 03B_1.fastq.gz_C_to_T.fastq and 03B_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: LH00160:538:22WG5VLT4:6:1101:22954:1042_1:N:0:TGAGCTAG+GANCGGTT/1 83 NC_082382.1_GA_converted 23587368 6 151M = 23587292 -227 CCTAATAAAATTTTACACTATATAACCCAATAACAAATAACACAAAAACAACCATAAACTTATTAAACTACAAATTATTTCAATAAAATTTACAATTACAAATCTACAAATATATTAAAACAAACATTTTCTATTATATAAATTTTATCNA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII#I AS:i:-1 XS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:149A1 YS:i:-35 YT:Z:CP LH00160:538:22WG5VLT4:6:1101:22954:1042_2:N:0:TGAGCTAG+GANCGGTT/2 163 NC_082382.1_GA_converted 23587292 6 3M2I146M = 23587368 227 AAAAAAAAAAAACTAATCATTTTATTATACAATAAAATATCTCATACAACAACAAACAACTAAACAACCAACTAATCACCTAATAAAATTTTACACTATATAACCCAATAACAAATAACACAAAAACAACCATAAACTTATTAAACTACAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIII9IIIIIIIIIIIIIIIIIIIIIIIII9-I AS:i:-35 XS:i:-58 XN:i:0 XM:i:4 XO:i:1 XG:i:2 NM:i:6 MD:Z:2C1T1T2C139 YS:i:-1 YT:Z:CP >>> Writing bisulfite mapping results to 03B_pe.bam <<< Reading in the sequence files /mmfs1/gscratch/coenv/sr320/cod-bs/03B_1.fastq.gz and /mmfs1/gscratch/coenv/sr320/cod-bs/03B_2.fastq.gz Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1116:18034:24015_1:N:0:TGAGCTAG+GAACGGTT NC_082394.1 22312309 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1120:37043:29240_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077817 Processed 1000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1122:23027:16437_1:N:0:TGAGCTAG+GAACGGTT NC_082393.1 27586963 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1123:5240:16745_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077810 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1132:29056:6617_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 2 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1132:8696:10371_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077817 Processed 2000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1142:13729:27293_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077846 Processed 3000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1163:38977:21802_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 2 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1173:29897:16899_1:N:0:TGAGCTAG+GAACGGTT NC_082396.1 24646084 Processed 4000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1186:5507:3619_1:N:0:TGAGCTAG+GAACGGTT NC_082400.1 17993492 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1196:34429:18468_1:N:0:TGAGCTAG+GAACGGTT NC_082393.1 2 Processed 5000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1205:42546:17193_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077819 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1214:17605:28147_1:N:0:TGAGCTAG+GAACGGTT NC_082394.1 22312316 Processed 6000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1230:18285:14560_1:N:0:TGAGTTAG+GAACGGTT NC_082383.1 23805013 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1237:34745:23468_1:N:0:TGAGCTAG+GAACGGTT NC_082388.1 28186552 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1240:2157:18384_1:N:0:CGAGCTAG+GAACGGTT NC_082385.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1242:1097:17305_1:N:0:TGAGCTAG+NAACGGTT NC_082399.1 21077812 Processed 7000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1257:20219:7990_1:N:0:TGAGCTAG+GAACGGTT NC_082400.1 17993479 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1257:20235:7990_1:N:0:TGAGCTAG+GAACGGTT NC_082400.1 17993479 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1262:8105:14055_1:N:0:TGAGCTAG+GAACGGTT NC_082388.1 28186551 Processed 8000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1265:42157:20639_1:N:0:TGAGCTAG+GAACGGTT NC_082402.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1270:15178:16072_1:N:0:TGAGCTAG+GAACGGTT NC_082395.1 2 Processed 9000000 sequence pairs so far Processed 10000000 sequence pairs so far Processed 11000000 sequence pairs so far Processed 12000000 sequence pairs so far Processed 13000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1371:28125:14363_1:N:0:TGAGTTAG+GAACGGTT NC_082399.1 21077813 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1385:18795:11072_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077817 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1386:47967:4866_1:N:0:TGAGCTAG+GAACGGTT NC_082390.1 23393734 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1386:47959:4880_1:N:0:TGAGCTAG+GAACGGTT NC_082390.1 23393734 Processed 14000000 sequence pairs so far Processed 15000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1412:50007:15512_1:N:0:TGAGCTAG+GAACGGTT NC_082383.1 23804997 Processed 16000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1433:49804:27797_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077818 Processed 17000000 sequence pairs so far Processed 18000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:1471:50274:7205_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077816 Processed 19000000 sequence pairs so far Processed 20000000 sequence pairs so far Processed 21000000 sequence pairs so far Processed 22000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2176:1696:21872_1:N:0:TGAGATAG+GAACGGTT NC_082403.1 19411193 Processed 23000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2190:22792:13705_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077802 Processed 24000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2211:40061:8102_1:N:0:TGAGCTAG+GAACGGTT NC_082385.1 2 Processed 25000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2228:5507:5721_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077817 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2228:5499:5735_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077817 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2234:22420:4264_1:N:0:TGAGCTAG+GAACGGTT NC_082394.1 22312306 Processed 26000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2261:43881:16423_1:N:0:TGAGCTAG+GAACGGTT NC_036931.1 1 Processed 27000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2275:48688:10988_1:N:0:TGAGCTAG+GAACAGTT NC_082399.1 21077818 Processed 28000000 sequence pairs so far Processed 29000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2327:34202:22306_1:N:0:TGAGCTAG+GAACGGTT NC_082387.1 1 Processed 30000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2340:2036:19406_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077815 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2341:10719:27741_1:N:0:TGAGCTAG+GAACGGTT NC_082392.1 2 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 LH00160:538:22WG5VLT4:6:2415:9295:9026_1:N:0:TGAGCTAG+GAACGGTT NC_082402.1 1 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2422:44099:16353_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077818 Processed 34000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2433:30456:11646_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077818 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2437:45831:29352_1:N:0:TGAGCTAG+GAACGGTT NC_036931.1 16440 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2444:15938:12066_1:N:0:TGAGCTAG+GAACGGTT NC_082384.1 1 Processed 35000000 sequence pairs so far Processed 36000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2479:44828:2569_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077818 Chromosomal sequence could not be extracted for LH00160:538:22WG5VLT4:6:2479:9796:19728_1:N:0:TGAGCTAG+GAACGGTT NC_082382.1 26289609 Processed 37000000 sequence pairs so far Processed 38000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:1154:40916:10360_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077814 Processed 39000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:1197:21848:26068_1:N:0:TGAGCTAG+GAACGGTT NC_082386.1 1 Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:1212:6267:6479_1:N:0:TGAGCTAG+GAACGGTT NC_082386.1 22175831 Processed 40000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:1233:4228:10851_1:N:0:TGAGCTAG+GAACGGTT NC_082382.1 26289599 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 LH00586:292:22WG5WLT4:1:1447:47396:20351_1:N:0:TGAGCTAG+GAACGGTT NC_082383.1 23805027 Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:1453:19453:3172_1:N:0:TGAGCTAG+GAACGGTT NC_036931.1 1 Processed 45000000 sequence pairs so far Processed 46000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2144:33999:7404_1:N:0:TGAGCTAG+GAACGGTT NC_082390.1 23393735 Processed 47000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2222:12302:4573_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077819 Processed 48000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2260:15093:12854_1:N:0:TGAGCTAG+GAACGGTT NC_082390.1 1 Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2298:45471:13149_1:N:0:TGAGCTAG+GAACGGTT NC_082386.1 22175846 Processed 49000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2302:26022:15531_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077818 Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2309:3848:15825_1:N:0:TGAGCTAG+GAACGGTT NC_036931.1 16440 Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2312:17981:17156_1:N:0:TGAGCTAG+GAACGGTT NC_082389.1 23759925 Processed 50000000 sequence pairs so far Processed 51000000 sequence pairs so far Processed 52000000 sequence pairs so far Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2466:33044:25367_1:N:0:TGAGCTAG+GAACGGTT NC_082388.1 28186554 Chromosomal sequence could not be extracted for LH00586:292:22WG5WLT4:1:2495:38206:27469_1:N:0:TGAGCTAG+GAACGGTT NC_082399.1 21077817 52740330 reads; of these: 52740330 (100.00%) were paired; of these: 25829160 (48.97%) aligned concordantly 0 times 14841155 (28.14%) aligned concordantly exactly 1 time 12070015 (22.89%) aligned concordantly >1 times 51.03% overall alignment rate 52740330 reads; of these: 52740330 (100.00%) were paired; of these: 25822178 (48.96%) aligned concordantly 0 times 14845056 (28.15%) aligned concordantly exactly 1 time 12073096 (22.89%) aligned concordantly >1 times 51.04% overall alignment rate Processed 52740330 sequences in total Successfully deleted the temporary files 03B_1.fastq.gz_C_to_T.fastq and 03B_2.fastq.gz_G_to_A.fastq Final Alignment report ====================== Sequence pairs analysed in total: 52740330 Final Cytosine Methylation Report ================================= Total number of C's analysed: 2348843863 Total methylated C's in CpG context: 244927516 Total methylated C's in CHG context: 15312678 Total methylated C's in CHH context: 53564039 Total methylated C's in Unknown context: 4756331 Total unmethylated C's in CpG context: 87259909 Total unmethylated C's in CHG context: 466272303 Total unmethylated C's in CHH context: 1481507418 Total unmethylated C's in Unknown context: 7775025 C methylated in CpG context: 73.7% C methylated in CHG context: 3.2% C methylated in CHH context: 3.5% C methylated in Unknown context (CN or CHN): 38.0% Bismark completed in 0d 3h 53m 34s ==================== Bismark run complete ====================