exploring monoallelic methylation using high-throughput sequencing

Exploring Monoallelic Methylation Using High-throughput Sequencing Cristian CoarfaRonald HarrisAleksandar MilosavljevicJoe Costello

Sequence-based profiling of DNA methylation: comparisons of methods and catalogue of allelic epigenetic modifications

Harris RA, Wang T, Coarfa C, Zhou X, Xi Y, Nagarajan RP, Hong C, Downey S, Johnson BE, Delaney A, Zhao Y, Olshen A, Ballinger T, Schillebeeckx M, Echipare L, OGeen H, Lister R, Pelizzola M, Epstein C, Bernstein BE, Hawkins RD, Ren B, Chung WY, Gu H, Bock C, Gnirke A, Zhang MQ, Haussler D, Ecker J, Li W, Farnham PJ, Waterland RA, Meissner A, Marra MA, Hirst M, Milosavljevic A, Costello JF.

In press, Nature Biotechnology

Imprinting

Non-imprinted monoallelic methylation

Cell type-specific methylation

Sites of inter-individual variation in methylation level?Biological importance of intermediate methylation levels

Methylated CpGsUnmethylated CpGsmethyl DNA immunoprecipitation(MeDIP)methylation-sensitive restriction digestion (MRE)~20 million reads/sample~100 million reads/sampleIGAII sequencingdata visualizationIllumina library constructioncombine parallel digests,ligate adapters,size-select 100-300 bpIP sonicated, adapter-ligatedDNA, size-select 100-300 bp

Unmethylated and Methylated patches within a CpG island

high MeDIP, no or low MREhigh MRE, no or low MeDIP12

Intermediate methylation levels at imprinted genes

Start Stop MRE MeDIP nearest gene GeneChr1. . . . . . . . . . . . . . . .... chr22 . . . . . . . . . . . . . . . .Initial catalogue of Intermediate methylation sites

Ting Wang, Washington University

Chr11153328115366671.034291.9069-205410HCCA2chr11194647519487870.776958.5443-18939LOC100133545chr11197514119774391.284587.55160H19chr11224568022505082.345199.4044-29211C11orf21chr11242074724232241.656529.51610KCNQ1

Using Genetic Variation to Detect Monoallelic Epigenomic and Transcription StatesMonoallelic DNA methylation (MRE and MeDIP)

Monoallelic expression (MethylC-seq and RNA-seq)

Monoallelic Histone H3K4me3 (MethylC-seq and Chip-seq)

MethylC-seq +ChIP-seqMethylC-seq + RNA-seqMRE-seq +MeDIP-seqMonoallelic Epigenomic Marks and Expression

Intermediate methylation levels in POTEB

Validation of monoallelic DNA methylation in POTEB

Searching for Monoallelic Methlylation Using Shotgun Bisulfite SequencingWe expect streaks of 50%+/-delta methylation ratiosUse 500bp windows tiling CpG IslandsCompute average CpG methylationCpG Islands1000 lociInfer distribution of methylation in 1000 lociSubselect 500bp windows tiling CpG IslandsIn the selected windows, look for allele specific methylation

Questions

How many of the 1000 loci can we rediscover ? How many of the 1000 loci show allele-specific methylation ? How many additional 500bp sites do we discover ?

Average methylation over 500 bp window in CpG Islands and 1000 loci

Chart1

0.04381694260.0055714741

0.04690808490.0039796243

0.04726356630.0049347342

0.04214772570.0071633238

0.03806741780.0084368036

0.03361617290.0079592486

0.03038592910.0074816937

0.02834577520.0101878383

0.02556374710.0062082139

0.02534736710.0087551735

0.02550192420.0105062082

0.02142161640.0084368036

0.01965966520.0081184336

0.01826865120.0093919134

0.0157030030.0066857689

0.0161357630.0078000637

0.01404924190.0078000637

0.01265822780.0073225088

0.01165360660.0074816937

0.01046351680.0070041388

0.01165360660.0079592486

0.00850064140.006049029

0.00877884420.0062082139

0.00710962740.0071633238

0.00571861330.0062082139

0.00737237450.0082776186

0.00633684180.0073225088

0.00625956320.0070041388

0.0056258790.0074816937

0.00499219490.0052531041

0.00644503180.0073225088

0.00468308060.0065265839

0.0048221820.0062082139

0.00451306780.0063673989

0.00443578920.0062082139

0.0042348650.0049347342

0.0041266750.0054122891

0.00403394070.005730659

0.00352390230.0054122891

0.00335388940.0049347342

0.00457489070.0078000637

0.00381756080.0054122891

0.00335388940.0070041388

0.00372482650.0055714741

0.00326115520.0068449538

0.00338480090.0074816937

0.00316842090.0071633238

0.00316842090.0065265839

0.00323024370.0062082139

0.00298295240.0052531041

0.0040184850.0071633238

0.00279748380.005889844

0.00361663650.0081184336

0.00315296520.0074816937

0.00346207940.0081184336

0.00333843370.0078000637

0.00306023090.0079592486

0.00316842090.0079592486

0.00289021810.006049029

0.00323024370.0066857689

0.00374028220.0093919134

0.00289021810.0101878383

0.00313750950.0098694683

0.00323024370.0095510984

0.00318387660.0133715377

0.00312205380.0100286533

0.00366300370.0085959885

0.00347753510.0120980579

0.00338480090.0113021331

0.00344662370.0114613181

0.00409576360.0128939828

0.00392575080.011620503

0.00449761210.0135307227

0.00462125780.0148042025

0.00386392790.0170327921

0.00547132190.017510347

0.00517766340.0167144222

0.0056258790.017510347

0.00590408190.0195797517

0.00649139890.0192613817

0.00874793280.0224450812

0.00834608430.0245144858

0.00913432560.0272206304

0.01103537810.0238777459

0.01163815090.0262655205

0.0142037990.0316778096

0.01540934450.0272206304

0.016939460.0289716651

0.01905689250.0248328558

0.01863958830.0245144858

0.01969057670.0194205667

0.01664580150.0122572429

0.01357011480.0093919134

0.00908795850.0049347342

0.00598136040.0033428844

0.00344662370.0028653295

0.00187014110.0007959249

0.00089643130.0003183699

0.00038639280.000159185

0.00142192550.0009551098

% of CpG Islands windows

% windows in 1000 loci

Percent methylation

% of windows

Average Methylation Scores over 500bp windows in CpG Islands and 1000 putative intermediate methylation loci

Methylation CpG Islands

Meth levelNumber of windows in CpG Islands% of CpG Islands windowsNumber of windows in 1000 loci% windows in 1000 loci

028354.38%350.56%

130354.69%250.40%

230584.73%310.49%

327274.21%450.72%

424633.81%530.84%

521753.36%500.80%

619663.04%470.75%

718342.83%641.02%

816542.56%390.62%

916402.53%550.88%

1016502.55%661.05%

1113862.14%530.84%

1212721.97%510.81%

1311821.83%590.94%

1410161.57%420.67%

1510441.61%490.78%

169091.40%490.78%

178191.27%460.73%

187541.17%470.75%

196771.05%440.70%

207541.17%500.80%

215500.85%380.60%

225680.88%390.62%

234600.71%450.72%

243700.57%390.62%

254770.74%520.83%

264100.63%460.73%

274050.63%440.70%

283640.56%470.75%

293230.50%330.53%

304170.64%460.73%

313030.47%410.65%

323120.48%390.62%

332920.45%400.64%Looking at 500 bp windows with 30-80% methylation

342870.44%390.62%Rediscover 950 of the 1000 loci

352740.42%310.49%

362670.41%340.54%

372610.40%360.57%

382280.35%340.54%

392170.34%310.49%

402960.46%490.78%

412470.38%340.54%

422170.34%440.70%

432410.37%350.56%

442110.33%430.68%

452190.34%470.75%

462050.32%450.72%

472050.32%410.65%

482090.32%390.62%

491930.30%330.53%

502600.40%450.72%

511810.28%370.59%

522340.36%510.81%

532040.32%470.75%

542240.35%510.81%

552160.33%490.78%

561980.31%500.80%

572050.32%500.80%

581870.29%380.60%

592090.32%420.67%

602420.37%590.94%

611870.29%641.02%

622030.31%620.99%

632090.32%600.96%

642060.32%841.34%

652020.31%631.00%

662370.37%540.86%

672250.35%761.21%

682190.34%711.13%

692230.34%721.15%

702650.41%811.29%

712540.39%731.16%

722910.45%851.35%

732990.46%931.48%

742500.39%1071.70%

753540.55%1101.75%

763350.52%1051.67%

773640.56%1101.75%

783820.59%1231.96%

794200.65%1211.93%

805660.87%1412.24%

815400.83%1542.45%

825910.91%1712.72%

837141.10%1502.39%

847531.16%1652.63%

859191.42%1993.17%

869971.54%1712.72%

8710961.69%1822.90%

8812331.91%1562.48%

8912061.86%1542.45%

9012741.97%1221.94%

9110771.66%771.23%

928781.36%590.94%

935880.91%310.49%

943870.60%210.33%

952230.34%180.29%

961210.19%50.08%

97580.09%20.03%

98250.04%10.02%

100920.14%60.10%

647016282

&C&A

&CPage &P

Methylation CpG Islands

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