exploring monoallelic methylation using high-throughput sequencing

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Exploring Monoallelic Methylation Using High-throughput Sequencing. Cristian Coarfa Ronald Harris Aleksandar Milosavljevic Joe Costello. Sequence-based profiling of DNA methylation: comparisons of methods and catalogue of allelic epigenetic modifications - PowerPoint PPT Presentation

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  • 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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