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ExecutiveSummaryThemovementtowardpersonalizeddiagnosticsandsyndromictestingislargelyresponsiblefortheincreaseddemandformultiplexmoleculardiagnostic technologies. While the current multiplex molecularsolutions havemade a significant impact in the clinical and researchworlds,theglobalpenetrationofthesetechnologieshasbeengreatlylimitedbythehighinstrumentationandpertestcost,andlimitedtestthroughput to meet the needs of high volume laboratories.ChromaCodehas recentlydevelopedanovelmultiplexing technologycalled High-Definition PCR (HDPCR™) that enhances themultiplexingcapabilityofreal-timePCR(qPCR)anddigitalPCR(dPCR) instrumentswithout requiring instrument hardware alterations. To demonstratehowHDPCR™ can increasemultiplexing levelwithout sacrificing therobustness of single-plex qPCR assays, ChromaCode developed theHDPCR™ RVPDemoPlatformDemonstratorAssay, a respiratory viralpanelthatsimultaneouslydetectsninerespiratoryvirusesinthreecolorchannels inasingle reaction. InaseriesofstudiesperformedontheApplied BioSystems® 7500 Fast (Thermo Fisher Scientific, Waltham,MA) and Roche LightCycler® 480 (Roche, Basel, Switzerland) usingsynthetic DNA samples and previously characterized clinicalnasopharyngeal swab (NPS) specimens, HDPCR™ RVPDemo had asensitivityandspecificityof99.1%and99.8%onsyntheticsamplesand95.8% and 99.5% on NPS samples. The HDPCR™ multiplexingtechnologyhasthepotentialtoaddressthelimitationsofthecurrentlyavailablemultiplexmoleculardiagnosticoptionsandgreatlyscalethemarketthatcanaffordtoperformmultiplexingmoleculartesting.

IntroductionOver the past decade, the demand for multiplexing moleculartechnologies has increased greatly as more and more clinical andresearch applications require testing of 5-50 biomarkers in a singlereaction.Thereareavarietyofmultiplexmoleculartechnologiesthatcurrentlyservicethismarket,includingtheBioFireFilmArray™System(BioMerieux, Marcy l’Etoile, France), GenMark eSensor® XT-8 andePlex® Systems (GenMark Diagnostics, Carlsbad, CA), and LuminexVerigene® System (Luminex, Austin, TX), to name a few. Thesetechnologies have cemented multiplex molecular testing as thestandard of care for a variety of clinical applicationswith syndromictestingoptions forupper respiratory tract, gastrointestinal tract, andbloodstream infection testing, as well as countless researchapplications.However, thepenetrationof thesetechnologiesbeyondlarge,well-resourcedlaboratoriesbothintheU.S.andgloballyhasbeenlimitedbythehigh instrumentationandpertestcostandthe limitedtestthroughputofthesetechnologies.Newsolutionsareneededthat

canovercomecostand throughput limitations tobetterdemocratizemolecularmultiplex testingandspread thevalue this testingbeyondresourcerichclinicalandresearchsettings.Based on California Institute of Technology intellectual property,ChromaCode has developed a novel multiplexing technology calledHigh Definition PCR (HDPCR™), which can enhance multiplexcapabilitiesonreal-timePCR(qPCR)anddigitalPCR(dPCR)instrumentscurrentlydeployed inclinicalandresearch labsaroundtheworld.Byleveragingthegreaterthan80,000qPCRanddPCRinstrumentsalreadyin use worldwide, HDPCR™ can greatly expand access to multiplexmolecular testing on high-throughput instrumentation withoutburdening laboratorieswith the added cost of new instrumentation.HDPCR™isdesignedtobeflexiblesothatitcanintegrateseamlesslyintoopenqPCRanddPCRsystems,aswellasmostclosed,sampletoanswer qPCR platforms, giving this technology significant ability toscale.ChromaCodedevelopedaninetarget,threecolorchannelRespiratoryViral Panel Assay (HDPCR™ RVPDemo)with an internal control as aplatformdemonstratorassaytohighlightthemultiplexingcapabilitiesofHDPCR™,showthatHDPCR™multiplexqPCRassaysmaintaintherobust performanceof traditional single-plex qPCR assays, and showHDPCR™ technology can seamlessly be deployed onto open qPCRinstruments. This white paper details the performance of HDPCR™RVPDemoontheAppliedBioSystems®7500FastandRocheLightCyler®480acrossaseriesofanalyticalstudiesperformedusingsyntheticDNAsamples and previously characterized, de-identified nasopharyngealswab(NPS)specimensinviraloruniversaltransport.

Methods&MaterialsHDPCR™ Overview: HDPCR™ is a highly engineered and tunablemultiplexing technology that couples traditional TaqMan® (ThermoFisher Scientific, Waltham, MA) chemistry with a proprietary signalprocessing algorithm. While traditional qPCR and dPCR multiplexingrely on differentiation of targets by color, HDPCR™ uses probeconcentrationasthelimitingreagentinreactions,whichallowsmultipletargetsinthesamecolorchanneltobedistinguishedbysignalintensityratherthancolor.Sincefluorescentintensityscaleslinearlywithprobeconcentration, each qPCR curve has a distinct and predictable signalintensity at reaction completion for each individual target orcombinationoftargetsdetectedwithinasinglecolorchannel.Byusing

High-DefinitionPCR(HDPCR™):TransformingDiagnosticsThroughDataScience

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signal intensitytodeterminewhattargetorcombinationoftargetsispresent,HDPCR™ effectively improves themultiplexing capability ofexisting qPCR and dPCR instruments without requiring hardwarechanges(Figure1).

Figure1:Currently,HDPCR™qPCRassayscansimultaneouslydetectthreedifferenttargetsinasingleqPCRcolorchannel.Withineachcolorchannelutilized,thereareeightpotentialresults,rangingfromnotargetsdetectedtoallthreetargetsdetected.TheproprietarysignalprocessingalgorithmofHDPCR™thatresidesinChromaCode’s cloud-based software along with the probe-limitedchemistry together drive reproducible inter- and intra-instrumentperformance.HDPCR™isfarlesspronetoissuesthatplaguetraditionalqPCRasaresultofsample,thermaland/oropticalvariance.Thiswaspreviouslydemonstratedacrossaseriesofstudiesthat lookedatthetechnology’simpactonreductionincoefficientofvariationinendpointfluorescence,removalofcrosstalkresultsfrombleedthroughbetweenfluorescencechannels,abilitytodetectmultipletargetssimultaneouslyacrossa titration serieswith consistent fluorescenceendpoints, andsensitivecopynumberdetection(Figure2).1,2,3

Figure2:(A)Asingletargettested288timesbetweentwooperatorsoveroneweek.AfterprocessingwiththeHDPCR™ signalprocessingalgorithm,theCV=1.5%. (B)RemovalofFAMchannelbleedthroughintoVICchannel.TherawVICsignal is inthefirstpanel.Themiddleshowscrosstalksignalremovingusingtheinstrumentmulti-componentalgorithm.ThelastpanelshowscrosstalksignalremovalusingtheHDPCR™algorithm, illustratingasignificant improvement over existingmethods. (C) Influenza A and Influenza A/H3 dualpresencetargettitrationfrom10copyinput(red)to100,000copyinput(orange).(D)RSVAdilutionseriesinthepresenceofaverylowcopynumberInfluenzaB.TheblackdottedlinereflectswherethefluorescenceofRSVAalonewouldbe.ThegreencurveontopofthedottedlineshowstheadditivesignalintensityfromtheamplificationofinfluenzaB.HDPCR™providesbettercorrectionforthermalandopticalvariancesinherenttoqPCRinstrumentationthanthecorrectionsprovidedbytheinstrumentmanufacturer’s software. The HDPCR™ signal processingalgorithm also ensures consistent performance of an assay acrossmultiple instruments. Figure 3 shows the high intra-instrumentreproducibilityfordetectionoftargetsacrossmultiplecolorchannelsonmultipleinstruments.

Figure3: Intra-instrument reproducibilityofHDPCR™RVPDemoacrossaQuantStudio7,twoViiA7s,andanABI7500Fast.RVPDemo Design: ChromaCode’s HDPCR™ RVPDemo assay is aqualitative, qPCR assay for the detection and identification of ninerespiratoryviralpathogentargetsacrossthreecolorchannels,aswellasaninternalcontrol(Table1).HDPCR™RVPDemoisforresearchuseonly(RUO)andisnotforuseindiagnosticprocedures.

Level Channel1 Channel2 Channel4 Channel5Level1 InfluenzaB InfluenzaA

InternalControl

Parainfluenza2Level2 RSVA InfluenzaA/H3 Parainfluenza3Level4 RSVB InfluenzaA/H1 Parainfluenza1

Table1:DesignofChromaCode’sHDPCR™RVPDemoAssay.InstrumentDyeCalibrationandEqualization:HDPCR™RVPDemousesHDPCR™-specificdyes.TheABI7500FastandRocheLightCycler®480instrumentsusedinthisstudywerefirstcalibratedwithHDPCR™dyes,followingtheinstrumentmanufacturer’sinstructionsforcalibrationofcustom dyes. Next, a brief instrument equalization study wasperformedoneachinstrument.Thestudyconsistedoftestingplatesofsynthetic DNA in each channel utilized in by HDPCR™ RVPDemo togenerate an equalizationmatrix that corrects for instrument specificopticalandthermalvariances.Analytical Studies with Synthetic DNA Samples: Limit of detection,singleanddualpositive,andtitrationstudieswereperformedonboththeABI7500FastandLightCycler®480 tocharacterize theanalyticalperformanceofHDPCR™RVPDemo.Synthetictargetsforeachofthemultiplexed primer and probe sequences included in HDPCR™RVPDemo and were manufactured by Integrated DNA Technologies(Danaher,Washington,D.C.).ThesesampleswerereconstitutedinTEbufferpriortouse.AnalyticalStudieswithClinicalNPSSpecimens:Clinicalnasopharyngealswab (NPS) samples previously tested with BioFire FilmArrayRespiratory Panel (BioMerieux, Marcy l’Etoile, France), Cepheid(Danaher, Washington, D.C.), or GenMark eSensor RVP (GenMarkDiagnostics, Carlsbad, CA), were tested in duplicate with HDPCR™RVPDemoontheABI7500FastandRocheLightCycler®480.Discrepantresolutionwasperformedby testing the sample in a singleplexPCRreactionorbysequencingtodetermineifthevirusstrainwasinclusivetotheHDPCR™RVPDemotargetdesign.ThosesamplesnotinclusivetoanHDPCR™RVPDemotargetwereexcludedfromthefinalanalysis.

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ResultsSynthetic DNA Studies: In total, 344 synthetic samples were tested on the ABI 7500 Fast and Roche LightCycler® 480 (172 samples on eachinstrument).TheperformanceofHDPCR™RVPDemoonboththeABI7500FastandRocheLightCycler®480aresummarizedinTable2.TheoverallsensitivityandspecificityofHDPCR™RVPDemoacrossbothinstrumentswas99.1%and99.8%,respectively.ThegraphsofeachHDPCR™curvefromallofthesyntheticDNAstudiesontheABI7500FastandRocheLightCycler®480aredisplayedinFigure4.Theconsistencyinsignalintensityatcurvetruncationacrossthelimitofdetection,singleanddualpositive,andtitrationseriesstudiesonboththeABI7500FastandRocheLightCycler®480speaktotheintra-andinter-instrumentreproducibilityofHDPCR™.

ABI7500Fast RocheLightCycler®480 Target TP TN FP FN Sensitivity Specificity TP TN FP FN Sensitivity Specificity FluB 24 148 0 0 100%(82.8%-100%) 100%(96.9%-100%) 24 148 0 0 100%(82.8%-100%) 100%(96.9%-100%)

Ch.1 RSVA 24 148 0 0 100%(82.8%-100%) 100%(96.9%-100%) 24 148 0 0 100%(82.8%-100%) 100%(96.9%-100%)

RSVB 51 121 0 0 100%(91.3%-100%) 100%(96.2%-100%) 51 121 0 0 100%(91.3%-100%) 100%(96.2%-100%)

Ch.2FluA 39 132 1 0 100%(88.8%-100%) 99.3%(95.3%-99.9%) 38 131 2 1 97.4%(84.9%-99.9%) 98.5%(94.1%-99.7%)FluA/H3 19 151 0 2 90.5%(68.2%-98.3%) 100%(96.9%-100%) 20 150 1 1 95.2%(74.1%-99.8%) 99.3%(95.8%-99.9%)FluA/H1 21 151 0 0 100%(80.8%-100%) 100%(96.9%-100%) 21 151 0 0 100%(80.8%-100%) 100%(96.9%-100%)

Ch.5Para2 12 160 0 0 100%(69.9%-100%) 100%(97.1%-100%) 12 160 0 0 100%(69.9%-100%) 100%(97.1%-100%)Para3 12 160 0 0 100%(69.9%-100%) 100%(97.1%-100%) 12 160 0 0 100%(69.9%-100%) 100%(97.1%-100%)Para1 12 160 0 0 100%(69.9%-100%) 100%(97.1%-100%) 12 160 0 0 100%(69.9%-100%) 100%(97.1%-100%)

Total 214 1,331 1 2 99.1%(96.3%-99.8%) 99.9%(99.5%-99.9%) 214 1,329 3 2 99.1%(96.3%-99.8%) 99.8%(99.3%-99.9%)

Table2.ResultsfromsyntheticDNAsamplestudiesontheABI7500FastandRocheLightCycler®480.

Channel1 Channel2 Channel5

Figure 4. Compilation of results from the 172 synthetic DNA studies on both an ABI 7500 Fast and Roche LightCycler® 480. These results demonstrate the inter- and intra-instrumentreproducibilityofHDPCR™RVPDemoacrosslimitofdetection,singleanddualpositive,andtitrationstudies.ClinicalNPSStudies:Intotal,274uniqueclinicalNPSsamplesweretestedinduplicateonboththeABI7500FastandRocheLightCycler®480foratotalof548samplesoneachinstrumentand1,096samplestotal.Twenty-twoNPSsampleswereexcludedfromthefinalanalysisfollowingsingleplexPCRtestingthatshowedthepresenceofstrainsnotinclusivetoanHDPCR™RVPDemotarget.TwelveadditionalNPSsampleswereexcludedaftersequencingresolutiondeterminedstrainstobenon-inclusivetoanHDPCR™RVPDemotarget.Followingdiscrepantresolution,240uniqueNPSsampleswere included in the finalanalysis.TheperformanceofHDPCR™RVPDemoonboththeABI7500FastandRocheLightCycler®480aresummarizedinTable3.TheoverallsensitivityandspecificityofHDPCR™RVPDemoacrossbothinstrumentswas95.8%and99.5%,respectively.ThegraphsofHDPCR™curvesforalloftheRSVA(channel1;n=56),influenzaA/H3(channel2;n=96)andparainfluenza2(channel5;n=14)clinicalNPSsamplestestedontheABI7500FastaredisplayedinFigure5.ThesensitivityandspecificityofHDPCR™RVPDemowithclinicalNPSsamplescompares favorablywith the performancemarket leadingmultiplexmolecular respiratory panels and shows to the intra- and inter-instrumentreproducibilityofHDPCR™withclinicalsamples.

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ABI7500Fast RocheLightCycler®480 Target TP TN FP FN Sensitivity Specificity TP TN FP FN Sensitivity Specificity FluB 95 383 1 1 99.0%(93.5%-99.9%) 99.7%(98.3%-100%) 93 382 2 3 96.9%(90.5%-99.2%) 99.5%(97.9%-99.9%)Ch.1 RSVA 55 424 0 1 98.2%(89.2%-99.9%) 100%(98.9%-100%) 56 421 3 0 100%(92.0%-100%) 99.3%(97.8%-99.8%) RSVB 38 442 0 0 100%(88.6%-100%) 100%(98.9%-100%) 38 442 0 0 100%(88.6%-100%) 100%(98.9%-100%)

Ch.2FluA 153 320 0 7 95.6%(90.8%-98.1%) 100%(98.5%-100%) 149 318 2 11 93.1%(87.7%-96.3%) 99.4%(97.5%-99.9%)FluA/H3 96 380 4 0 100%(95.2%-100%) 99.0%(97.2%-99.7%) 96 381 3 0 100%(95.2%-100%) 99.2%(97.5%-99.8%)FluA/H1 61 416 0 3 95.3%(86.0%-98.8%) 100%(98.9%-100%) 60 415 1 4 93.8%(84.0%-98.0%) 99.8%(98.5%-100%)

Ch.5Para2 14 462 4 0 100%(73.2%-100%) 99.1%(97.7%-99.7%) 13 452 14 1 92.9%(64.2%-99.6%) 97.0%(94.9%-98.3%)Para3 57 418 0 5 91.9%(81.5%-97.0%) 100%(98.9%-100%) 54 412 6 8 87.1%(75.6%-93.9%) 98.6%(96.7%-99.4%)Para1 55 422 0 3 94.8%(84.7%-98.7%) 100%(98.9%-100%) 51 422 0 7 87.9%(76.1%-94.6%) 100%(98.9%-100%)

Total 624 3,667 9 20 96.9%(95.2%-98.0%) 99.8%(99.5%-99.9%) 610 3,645 31 34 94.7%(92.6%-96.3%) 99.2%(98.8%-99.4%)

Table3.ResultsfromtheclinicalNPSstudiesontheABI7500FastandRocheLightCycler®480.

Channel1 Channel2 Channel5

Figure5.ThegraphsofHDPCR™curvestheRSVA(channel1;n=56),influenzaA/H3(channel2;n=96)andparainfluenza2(channel5;n=14)NPSsamplestestedontheABI7500Fast.

DiscussionThecurrentmultiplexmoleculartechnologieshavemadeasignificantimpactacrossmanyclinicalandresearchapplications.Tofurtherscalethese multiplex molecular technologies, manufacturers must maketechnologyfarmorecost-effectivewithmuchgreatertestthroughput,which is a significant undertaking. The HDPCR™ multiplexingtechnologyprovidesanopportunity toaddress thekey limitationsofexistingmultiplexmoleculartechnologies.Byleveragingthemorethan80,000 high-throughput qPCR instruments in use in laboratoriesglobally,HDPCR™providesa cost-effectivealternative thatdoesnotrequirecapitalpurchaseandserviceofexpensiveinstrumentationthatonly supports a narrowmenu of test applications. Furthermore, theHDPCR™testworkflowisidenticaltotraditionalqPCRandseamlesslyintegratesintolabworkflow.AdditionalfeaturesarebeingdevelopedtoexpandthepotentialclinicalandresearchapplicationsforHDPCR™andincreasetheoverallutilityofthistechnology.

HDPCR™ Multiplex Quantitation: The HDPCR™ signal processingalgorithm analyzes the entirety of each qPCR curve and defines keycharacteristic points by extracting kinetics information from the firstandsecondderivatives.Thesoftwareusesthisinformationtosegmenteach curve into individual amplification events. If a single target ispresent, the algorithm extracts characteristic inflection pointscorresponding to the exponential, linear, and plateau phases. Ifmultiple targetsarepresent inasingleHDPCR™curve, thealgorithmseparates the amplification events to extract the exponential, linear,andplateauphasesforeachtargetpresent,essentiallyconstructinganew qPCR curve for each individual target. For both single targetdetectionsandmultiplextargetdetectionsinasinglecolorchannel,theHDPCR™signalprocessingalgorithmwillbeabletotracebackthecyclethreshold (Ct) tied to the key characteristic points for each targetpresentinordertoquantifyrelativeconcentration(Figure6).

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Figure6:HDPCR™algorithmanalyzeseachqPCRcurvebyperformingfirstandsecondderivativesalongthedurationofthecurvetosegmenteachcurveintoindividualeventsthatdefineeachtargetorcombinationoftargets.AutomatedCurveClassificationwithMachineLearning:TheHDPCR™signalprocessingalgorithmalsoemploysprinciplesofmachinelearningtohelp laboratorians flagatypicalcurves.Thesoftwareautomaticallyanalyzes each qPCR curve and automatically flags atypical curves toprovideslaboratorieswithasafetynetagainstreportingresultsfromcurveswithtoomuchvariability,toomuchnoise,insufficientsignal,lateamplification,andlinearamplification.Redundant Coding for Greater Multiplexing and ReducedDevelopmentTimes:Multiplexing level and testperformance canbepotentially enhanced through alternative coding methods. TheHDPCR™datadisplayedinthisstudywasgatheredwithabinarycodingmethod,inwhichthreetargetsarecodedintoasinglecolorchannelbydoublingprobeconcentrationacrosseachofthethreetargets(e.g.2X,4X, 8X probe concentration by target). There are additional codingmethodsthatstillrelyontitteringprobeconcentrationamongsttargetsand having probe concentration being the limiting reagent of thereaction.Onesuchcodingmethodreliesonredundantlycodingeachindividual target across multiple color channels. With a redundantcodingmethod, targets are coded intomultiplex color channels andredundantmeasurementsaretakenacrossmultiplecolorchannelstodetectagiventargetorcombinationoftargetspresent inareaction.With redundant coding, there is the potential to increase qPCRmultiplexcapabilitiesby5x–10xanddPCRmultiplexingcapabilitiesbyupto200x.Itisalsohypothesizedthatbecauseredundantcodingislessprone to errors that result from the inherent variance of qPCR anddPCR,itcanreducetheamountoftimenecessarytoperfectthesystemofinteractionsbetweenprimersandprobesinbinarycodedHDPCR™multiplexing and greatly reduce multiplex assay development time(Figure7).

Figure7:DesignofaredundantlycodedHDCPR™3-plexassayacrosstwoopticalchannels.

HDPCR™ Multiplexing of Digital PCR: Similar to qPCR, dPCRmultiplexinghasbeencriticallylimitedtomeasuringasingletargetpercolor channel, with most dPCR instruments having only 2-3 colorchannels.ThequantitationcapabilitiesofdPCRareunrivaled,butthelack ofmultiplexing has greatly restricted the use of dPCR to a verynarrow set of clinical and research applications. Both the HDPCR™binary and redundant coding strategies can be applied to dPCR toincreasemultiplexinglevelsby3x–200x.PreliminarystudieswiththeHDPCR™ binary coding method using a six target, two channelrespiratory viral panel have shown great peak separation fordelineationofeachof thesix targets,highlightingapath forward foreffectively multiplexing dPCR (Figure 8). With greater multiplexing,dPCRcouldbeaveryvaluabletoolforcellfreeDNA(cfDNA)applicationsfor oncology and non-invasive prenatal testing (NIPT) and provide amuch more cost-effective, easier to use, faster, and possibly moresensitivealternativetonextgenerationsequencing(NGS),thecurrentgoldstandardfortheseapplications.

Figure8:HDCPR™integratedintoadPCRtoincreasemultiplexinglevelsby3xwithaproofofprinciplesixtarget,twocolorchannelrespiratoryviralpanel.

ConclusionsThe HDPCR™ multiplexing technology, with its ability to enhancemultiplexing capabilities on existing qPCR and dPCR instrumentationwithnohardwarechanges required,providesacost-effective,higherthroughput, and scalable alternative to the commercially availablemultiplex molecular technologies. The results from this studydemonstrate the robust performance of the HDPCR™ RVPDemomultiplexassay,theeaseofintegrationofHDPCR™ontotheABI7500FastandRocheLightCycler®480,and the inter-and intra-instrumentreproducibility of the technology. As ChromaCode continues toenhancethecapabilitiesoftheHDPCR™multiplexingtechnology,thetechnology will have the potential to make a significant impact inclinicalandresearchdiagnosticsgloballyandwillhelptodemocratizemultiplextesting.References:1. Rajagopal et al. Multiplexing Method for Significantly Increasing the

Bandwidth of qPCR Instruments. Poster TT06, 2017 Association forMolecularPathologyAnnualMeeting,SaltLakeCity,UT.

2. Jacky et al. High-Definition PCR (HDPCR™): A Novel and EconomicMultiplexingTechnologyAppliedtoRespiratoryPathogenPanelTestingasaProofofPrinciple.Poster#33.2018ClinicalVirologySymposium,WestPalmBeach,FL.

3. Rajagopaletal.SupercolorCodingMethodsforLarge-ScaleMultiplexingofBiochemicalAssays.Anal.Chem.2013;85:7629-763.