Om niPlex Am plification for Genetic, Epigenetic, and Expression Analysis— Applications in Genom ics, Pharm acogenom ics, Diagnostics, Biosurveillance, and ForensicsEm m anuel Kam berov, Eric Bruening Takao Kurihara, Alice Lu, Joseph M ’M w irichia, Jon Pinter,

Irina Sleptsova, Jennifer Sun, Tim Tesm er, Vladim ir M akarov, and John Langm orelangm ore@ rubicongenom ics.com

Rubicon G enom ics, Inc., Ann Arbor, M I, USA

Abstract

M any genetic studies and diagnostic tests are lim ited by the am ount of DNA or RNA that is available for study. Com m on factors that lead to inadequate am ounts of nucleic acid are sm all initial sam ple size, degradation at the source or in handling/storage, cost, depletion of existing sam ples by unanticipated num bers of tests or collaborators, and difficulties of recontacting subjects. Cell transform ation and growth is not a viable solution to these problem s.

W e have developed a sim ple m ethod for w hole genom e am plification, called Om niPlex W GA, that can accurately and robustly am plify sub-nanogram am ounts of total hum an DNA using com m on reagents. The process is a random , non-enzym atic fragm entation of genom ic DNA followed by addition of adaptor sequences to both ends to form an in vitro m olecular library that is am plified using PCR. Library preparation and am plification takes less than three hours, is autom atable, and can be repeated m ultiple tim es to produce m illigram am ounts of DNA. Om niPlex W GA data will be shown from whole blood, blood spots, buccal swabs, serum , fixed or frozen tissue, hair follicles, degraded archived DNA, single cells, and single sorted chrom osom es. O m niPlex has been used in academ ic, governm ent, and com m ercial projects for SNP and STR genotyping, for m utation discovery by sequencing and heteroduplex analysis, for chrom osom e painting and CG H, and for m ethylation and expression analysis. Genotype concordance between the gDNA and the W G A DNA is >99.7% on high throughput single base extension, ligation, and exonuclease assays. By enabling large-scale genotyping or resequencing studies to be done with blood spots, hair, or buccal swabs, W GA allows genetic resources for large-scale population studies to be collected, archived, and shared m ore rapidly and econom ically than by other m ethods.

The very low background, insensitivity to DNA breakage, and high sequence accuracy of the am plification process m ake W G A an attractive m ethod for m ore accurate and sensitive genetic and epigenetic diagnostics, and hum an and pathogen identification

O m niPlex™ SolutionsO m niPlex™ Solutions

Rubicon genom ics Om niPlex™ presents an integrated solution to discovery and diagnostics of genetic, epigenetic, and expressionm arkers.

Sensitivity, speed, and accuracy of Om niPlex DNA and RNA am plification are unsurpassed, w ith sam ples ready for analysis inless than 3 hours.

Om niPlex W hole Genom e Am plification has been validated in com m ercial genotyping, sequencing, CGH, and cytogenetics projects.

Am plified O m niPlex DNA and RNA can be analyzed on a variety of widely-available instrum entation.

Non-invasive cancer detection and m onitoring becom e feasible using Om niPlex W GA, W TA, and W M A from degraded DNA/RNA in serum .

O m niPlex enables retrospective studies from fixed tissue.

Om niPlex facilitates the study of genetic, epigenetic, and expressionm arkers from a unified platform .(www.rubicongenom ics.com )

Chrom osom al DNA

O m niPlex Library

Creation of O m niPlex Libraries

Random fragm entation

O m niPlex™ W G A Library Preparation

W hole G enom e Am plification

Am plified O m niPlex Library

W G A W TA W M A

Correlation Coefficient = 0.907

Array painting of the derivative chrom oseom es from a cell line carrying a t11;12)q21;p13.33) translocation.Probes were generated from Om niPlex™ libraries constructed from single copiesof the derivative chrom osom es. (Data provided courtesy of Nigel Carter, W ellcom e Trust Sanger Institute)

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p15 Prom oter M ethylationDetection In the Presence

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Rubicon W TA technology yields robust am plification over a w iderange of RNA input tem plate (A). W TA can be applied to m RNAor directly to total RNA. Libraries show uniform am plificationof RNA as dem onstrated by STS analysis across a set of differentiallyexpressed genes in control tissue sam ples (B).

Prostate cancer m arker discovery dem onstrates a pow erful application of W TA. Norm al pooled prostate vs. prostate cancer RNA w ere W TA processed from a range of input am ounts from both polyA+ selected and total RNA. Differentially labeled normaland cancer W TA sam ples w ere applied to a 18.5K cDNA m icroarray hybridization and com pared to unam plified. The correlation for the top 509 (3 fold) over and under expressed genes show a 91% correlation w ith respect to unam plified RNA(fig. C). The PCA signature genes with the m ost frequently alteredgene expression in prostate cancer are show n as a m edian of their ratios (fig. D).(Prostate array data provided courtesy of Arul Chinnaiyan M .D., Ph.D., Departm ent of Pathology, University of M ichigan M edical School)

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Sensitivity of P16 Prom oter M ethylation Detection in Cancer Cells

TissueSam ple

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Screening forCpG M ethylation in Prom oter RegionsNorm al vs Cancer Cells

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G STP1E-cadherinp16p15X C X C X C

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M W distribution of W GA am plim ers from circulating DNA