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Om niPlex Am plification forGenetic,Epigenetic,and Expression Analysis— Applications in Genom ics,Pharm acogenom ics,Diagnostics,Biosurveillance,and Forensics Em m anuelKam berov,Eric Bruening Takao Kurihara,Alice Lu,Joseph M’Mwirichia,Jon Pinter, Irina Sleptsova,JenniferSun,Tim Tesmer,VladimirMakarov,andJohn Langmore langm ore@ rubicongenom ics.com Rubicon Genom ics,Inc.,Ann Arbor,MI,USA Abstract M any genetic studies and diagnostic tests are lim ited by the am ountofDNA orRNA thatis available forstudy. Com m on factors thatlead to inadequate am ounts ofnucleic acid are sm allinitialsam ple size,degradation atthe source orin handling/storage,cost,depletion ofexisting sam ples by unanticipated num bers oftests orcollaborators,and difficulties ofrecontacting subjects. Celltransform ation and growth is nota viable solution to these problem s. W e have developed a sim ple m ethod forwhole genom e am plification,called Om niPlex W GA,thatcan accurately and robustly am plify sub-nanogram am ounts oftotalhum an DNA using com m on reagents. The process is a random ,non- enzym atic fragm entation ofgenom ic DNA followed by addition ofadaptorsequences to both ends to form an in vitro m olecularlibrary thatis 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 ofDNA. Om niPlex W GA data willbe shown from whole blood,blood spots,buccalswabs,serum , fixed orfrozen tissue,hairfollicles,degraded archived DNA, single cells,and single sorted chrom osom es. Om niPlex has been used in academ ic,governm ent,and com m ercial projects forSNP and STR genotyping,form utation discovery by sequencing and heteroduplex analysis,forchromosome painting and CGH,and form ethylation and expression analysis. Genotype concordance between the gDNA and the W GA DNA is >99.7% on high throughputsingle base extension,ligation,and exonuclease assays. By enabling large-scale genotyping orresequencing studies to be done with blood spots,hair,orbuccalswabs,W GA allows genetic resources forlarge-scale population studies to be collected, archived,and shared m ore rapidly and econom ically than by otherm ethods. The very low background,insensitivity to DNA breakage,and high sequence accuracy ofthe am plification process m ake W GA an attractive m ethod form ore accurate and sensitive genetic and epigenetic diagnostics,and human and pathogen identification O m niPlex™ Solutions O m niPlex™ Solutions Rubicon genom ics Om niPlex™ presents an integrated solution to discovery and diagnostics ofgenetic,epigenetic,and expression m arkers. Sensitivity,speed,and accuracy ofOm niPlex DNA and RNA am plification are unsurpassed,w ith sam ples ready foranalysis in less than 3 hours. Om niPlex W hole Genom e Am plification has been validated in com m ercialgenotyping,sequencing,CGH,and cytogenetics projects. Am plified Om niPlex DNA and RNA can be analyzed on a variety of widely-available instrum entation. Non-invasive cancerdetection and m onitoring becom e feasible using Om niPlex W GA,W TA,and W MA from degraded DNA/RNA in serum . O m niPlex enables retrospective studies from fixed tissue. Om niPlex facilitates the study ofgenetic,epigenetic,and expression m arkers from a unified platform . (www.rubicongenom ics.com ) ChromosomalDNA O m niPlex Library Creation ofO 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 WGA W TA WMA Correlation Coefficient= 0.907 Array painting ofthe derivative chrom oseom es from a cellline carrying a t11;12)q21;p13.33)translocation. Probes were generated from Om niPlex™ libraries constructed from single copies ofthe derivative chrom osom es. (Data provided courtesy ofNigelCarter,W ellcom e TrustSangerInstitute) 0 200 400 600 800 1000 1200 1400 1357 9 11 13 15 17 19 21 23 25 27 29 31 one cell no cell Single CellAm plification W TA Library Am plification and Representation 0 10 20 30 40 50 60 70 80 90 100 110 1 3 57 9 11 13 15 17 19 21 23 25 27 29 10 10 2 10 3 10 4 10 5 10 6 Negative Control Dilution Factor W G A from dilutions ofa single hum an hairfollicle Normal 1 :1000 1 :100 1 :10 1 :1 Cancer p15 Prom oterM ethylation Detection In the Presence ofNorm alCells Rubicon W TA technology yields robustam plification overa wide range ofRNA inputtem plate (A). W TA can be applied to m RNA ordirectly to totalRNA. Libraries show uniform am plification ofRNA as dem onstrated by STS analysis across a setof differentially expressed genes in controltissue sam ples (B). Prostate cancerm arkerdiscovery dem onstrates a powerful application ofW TA.Norm alpooled prostate vs.prostate cancer RNA were W TA processed from a range ofinputam ounts from both polyA+ selected and totalRNA. Differentially labeled normal and cancerW TA sam ples were applied to a 18.5K cDNA m icroarray hybridization and com pared to unam plified. The correlation forthe top 509 (3 fold)overand underexpressed genes show a 91% correlation with respectto unam plified RNA (fig.C). The PCA signature genes with the m ostfrequently altered gene expression in prostate cancerare shown as a median of theirratios (fig.D). (Prostate array data provided courtesy ofArulChinnaiyan M .D., Ph.D.,Departm entofPathology,University ofMichigan Medical School) B.) C.) D.) Sensitivity ofP16 Prom oterM ethylation Detection in CancerCells Tissue Sam ple Nucleic Acid Extraction O m niPlex™ Library Prep WGA W TA WMA Genotyping G enetic M arkers Gene Copy Analysis Expression Profiling M ethylation Profiling Alternative Splicing Epigenetic M arkers Gene Im printing Expression M arkers M olecular Diagnostics & Discovery C N C N C N 10ng 1ng 0.1ng } Contro l Cancer 10ng Norm al 10ng Cancer 1ng Norm al 1ng } } } Input DNA Screening forCpG Methylation in Prom oterRegions Norm alvs CancerCells X C X C X C Norm alCells } } } } } } } } } } } } Methylated DNA KG1-A CancerCells Norm alCells Methylated DNA KG1-A CancerCells Norm alCells Methylated DNA KG1-A CancerCells Norm alCells Methylated DNA KG1-A CancerCells X C X C X C X C X C X C X C X C X C GSTP1 E-cadherin p16 p15 X C X C X C 0 20 40 60 80 100 120 1 3 5 7 9 11 13 15 17 19 21 23 25 27 Single Chrom osom e Am plification Single Chrom osom es No DNA Log2(ratio) Log2(ratio) Distance along chrom osom e (Mbp) Distance along chrom osom e (Mbp) Chrom osom e 11 Chrom osom e 12 W GA from a single hum an leukocyte W TA InputRNA 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Cycle %Max RFU 100ng 10ng A.) W GA from ~10ng free circulating DNA W GA on Serum and plasm a 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Cycle # % Max RFU Serum Plasm a M W Serum M W Plasm a 500 200 1000 2000 200 500 1000 2000 M W distribution ofW GA am plim ers from circulating DNA
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Feb 14, 2022

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Page 1: WGA WTA WMA

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)

0

200

400

600

800

1000

1200

1400

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

one cell

no cell

Single Cell Am plification

W TA Library Am plification and Representation

0

10

20

30

40

50

60

70

80

90

100

110

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

10102

103104

105106

Negative Control

Dilution Factor

W G A from dilutions of a single hum an hair follicle

No

rm

al

1 :10

00

1 :10

0

1 :

10

1 :

1

Ca

nce

r

p15 Prom oter M ethylationDetection In the Presence

of Norm al Cells

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)

B.)

C.)D.)

Sensitivity of P16 Prom oter M ethylation Detection in Cancer Cells

TissueSam ple

Nucleic AcidExtraction

O m niPlex™Library Prep

W GA

W TA

W M A

GenotypingG eneticM arkers

G ene CopyAnalysis

ExpressionProfiling

M ethylationProfiling

AlternativeSplicing

EpigeneticM arkers

G eneIm printing

ExpressionM arkers

M olecularDiagnostics& Discovery

C N C N C N

10ng 1ng 0.1ng

}

Contro

l

Cancer10ng

Norm al10ng

Cancer1ng Norm al

1ng

} } }InputDNA

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

X C X C X C

Norm

al Cells

} } }

} } }} } }

} } }

Methylated DNA

KG1-A Cancer Cells

Norm

al Cells

Methylated DNA

KG1-A Cancer Cells

Norm

al Cells

Methylated DNA

KG1-A Cancer Cells

Norm

al Cells

Methylated DNA

KG1-A Cancer Cells

X C X C X C X C X C X C X C X C X C

G STP1E-cadherinp16p15X C X C X C

0

20

40

60

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100

120

1 3 5 7 9 11 13 15 17 19 21 23 25 27

Single Chrom osom e Am plification

Single Chrom osom es

No DNA

Log2(ratio)

Log2(ratio)

Distance along chrom osom e (M bp) Distance along chrom osom e (M bp)

Chrom osom e 11 Chrom osom e 12

W G A from a single hum an leukocyte

W TA Input RNA

0

20

40

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120

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Cycle

%Max RFU

100ng 10ng

A.)

W G A from ~10ng free circulating DNA

W GA on Serum and plasm a

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14Cycle #

% Max RFU

Serum

Plasm a

M W Serum M W Plasm a

500

200

1000

2000

200

500

1000

2000

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