AdamAmeur,PhDIGP,ScienceforLifeLaboratory,UppsalaUniversity

NationalGenomicsInfrastructure,Sweden

Targeted NGS in inherited disease research:

Example from Noonan syndrome

IonTorrent Symposium,ESHG,May28,2017

National Genomics Infrastructure

NGIstaff:75FTE,includingHeadoffacility,labresearchengineers,bioinformaticians,IT-experts,projectcoordinators

NGIproductionduring2016• 1000projects• 34000samples

NGIStockholm NGIUppsala

NGI Sequencing equipment

• 15SXL(Thermo Fisher) Microbialgenome,Targetedseq,Biodiversity• 5IonProton (Thermo Fisher) Largeprojects– WES,Transcriptome• 10IlluminaHiSeqX (Xten) Wholegenomesequencing&bisulfiteseq• 10IlluminaHiSeq2500/2000 Largeprojects– WGS,WES,Transcriptome• 3IlluminaMiSeq Microbialgenomes,Targetedseq,Biodiversity• 1IlluminaNextSeq Technologydevelopment• 2PacificBiosciencesRSII DeNovosequencing,longamplicon,Basemodifications• 1PacificBiosciencesSequel DeNovosequencing,longamplicon,Iso-seq• 1SangerABI3730 Variantvalidation• 1OxfordNanopore MinIon Technologydevelopment• 210XGenomicsChromium Haplotypephasing,RNA-seq• 1BioNano Irys system DeNovosequencing,structuralvariation

DNAsequencingatallscales

Ion S5XL System in Uppsala

IonS5XLsysteminstalledin2015(EarlyAccess)

WeuseitmainlyforIonAmpliSeq panelsequencing- Exome,wholetranscriptome,genepanels,…

Now we have tested theAmpliSeq On-Demand product!

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Ion AmpliSeq Designer Offers a Design Strategy for Every Need

For Research Use only. Not for use in diagnostic procedures.Proprietary and Confidential

Ready-to-Use Panels

• Pre-designed, ready-made DNA and RNA panels for multiple research applications

• Small reaction packs including 8 and 24

Made-to-Order Panels

• Flexibility to create DNA or RNA designs for any genome or choose from community pre-designed panels

• Large reaction packs typically starting from ~3,000

• Configurable DNA panels from pre-tested catalog of genes for inherited disease research

• Smaller reaction packs including 8 and 24

On-Demand Panels

New

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Ion AmpliSeq™ On-Demand Panels: a Flexible and Reliable Custom Solution

Powerful content selection engine helps to jump start experiments

Configurable panel design in small pack sizes allows much needed flexibility

Optimized genes with wet-lab performance ensures high level of confidence in the outcome of experiments

Proprietary and Confidential For Research Use only. Not for use in diagnostic procedures.

7

Less Time on Panel Development, More Time on Delivering Results

Powerful content selection engine enables optimal gene selection by disease research area

Proprietary and Confidential For Research Use only. Not for use in diagnostic procedures.

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Simple and Fast Workflow for Inherited Disease Research Using Targeted NGS

Configure an IonAmpliSeq On-Demand Panel atampliseq.com

• Building a custom panel is easy and quick using Ion AmpliSeq™ Designer

• Minimum hands-on time with the Ion Chef™ and Ion S5™ or Ion S5™ XL Systems.

• Automated data analysis with Torrent Suite™ Software and pre-configured Ion AmpliSeq™ workflows in Ion Reporter™ Software

Go from DNA to data in less than 24 hours with about 45 mins of hands-on time

For Research Use only. Not for use in diagnostic procedures.

RASopathies and Noonan Syndrome• TheRAS/mitogen-activatedproteinkinase(MAPK)pathwayis

essentialandcontrolscellproliferation,differentiationandsurvival.

• DysregulationofRAS/MAPKcausesclinicallyoverlappinggeneticdisorders,termed‘RASopathies’or‘RAS/MAPKsyndromes’

• Noonansyndromeoccursinapprox.1in1,000to2,500people

• Noonansyndrome– Congenitalheartdefects– Deformationofthechestbone

(Pectusexcavatum)– Shortstature– Learningproblems– Characteristicfacialfeatures https://en.wikipedia.org/wiki/Noonan_syndrome

Known genes for RASopathies and NS

Estimatedthat20–30%ofthecausativegenesbehindNSandNS-likedisordersareunidentified.

Importantwithaflexiblegenepaneltoeasilyincludenovelgenesandgetdatawithahighcoverage.

Ampliseq On-Demand Noonan panelwith 19genesdesigned!Incollaboration with MariaWilbe,PhD,UppsalaUniversity

Experimental setup

sample gene mutation1 KRAS c.458A>T(p.Asp153Val)2 MAP2K1 c.607G>C(p.Glu203Gln)3 PTPN11 c.1226G>C(p.Gly409Ala)4 RAF1 c.770C>T(p.Ser257Leu)5 MAP2K2 c.400T>C(p.Tyr134His)6 SOS1 c.1297G>A(p.Glu433Lys)7 NF1 c.6789delTTAC;p.Y2234Tfs8 HRAS c.35G>C(p.Gly12Ala)9 NF1 c.2991-1G>A(r.spl)10 NRAS c.179G>A(p.Gly60Glu)11 SOS1 c.806T>G(p.Met269Arg)12 RIT1 c.246T>A(Phe82Leu)13 BRAF c.736G>C(p.Ala246Pro)14 SHOC2 c.4A>G(p.Ser2Gly)15 NF1 c.1307C>A(p.Ser436Ter)16 PTPN11(amnion) c.1504T>A(p.Ser502Thr)17 SOS2 c.800T>G(p.Met267Arg)18 PTPN11 c.922A>G(p.Asn308Asp)19 SOS1 c.806T>C(p.Met269Thr)

Samplefromammiotic fluid

24Noonansamplesselectedfortestonnewpaneldesign:

NF1/PTPN11arenormallychallengingtoscreenusingNGSpanels

Results – Sequencing 24 samples on S5XL

~18millionreadsgeneratedona530chip

High coverage across all amplicons

All previously identified variants recovered

sample gene mutationDetectedwithAS On-Demand Coverage

Allelefrequency

1 KRAS c.458A>T(p.Asp153Val) yes 1763 50,20%2 MAP2K1 c.607G>C(p.Glu203Gln) yes 2940 50,60%3 PTPN11 c.1226G>C(p.Gly409Ala) yes 2713 51,10%4 RAF1 c.770C>T(p.Ser257Leu) yes 727 48,60%5 MAP2K2 c.400T>C(p.Tyr134His) yes 931 51,60%6 SOS1 c.1297G>A(p.Glu433Lys) yes 3321 48%7 NF1 c.6789delTTAC;p.Y2234Tfs yes 2299 50.5%8 HRAS c.35G>C(p.Gly12Ala) yes 204 57,80%9 NF1 c.2991-1G>A(r.spl) yes 519 53.9%10 NRAS c.179G>A(p.Gly60Glu) yes 3999 49,70%11 SOS1 c.806T>G(p.Met269Arg) yes 921 50,20%12 RIT1 c.246T>A(Phe82Leu) yes 2182 50,00%13 BRAF c.736G>C(p.Ala246Pro) yes 2581 48,70%14 SHOC2 c.4A>G(p.Ser2Gly) yes 1202 44,00%15 NF1 c.1307C>A(p.Ser436Ter) yes 2260 54,20%16 PTPN11(amnion) c.1504T>A(p.Ser502Thr) yes 2205 52,20%17 SOS2 c.800T>G(p.Met267Arg) yes 1937 51,30%18 PTPN11 c.922A>G(p.Asn308Asp) yes 2144 48,90%19 SOS1 c.806T>C(p.Met269Thr) yes 986 51,40%

Example of a variant in IGV• HeterozygousdeletioninNF1forsample7

Example of a variant in IGV (2)• HeterozygousSNPinPTPN11forsample16(amnioticfluid)

The Ion Torrent 510 chip

• Anewchip– Producessmalleramountsofdata(>500Mb)– Usefulforsmallerprojects– Smallerdatafiles,quickeranalysis

sample gene mutationDetectedwith AS

On-Demand CoverageAllele

frequency

2 MAP2K1 c.607G>C(p.Glu203Gln) yes3997(2940) 51.1%

3 PTPN11 c.1226G>C(p.Gly409Ala) yes3966(2713) 50.3%

Multiplexingoftwosamplesonone510chip:

Conclusions• TheAmpliSeq On-Demandpanelworks!

• All19mutationsinNoonansamplesrecovered

• IncludesmoregenesthancurrentpanelusedinUppsala

• Givessimilarcoveragestatisticsandperformance

• Allowsforflexibilityofaddingnewgenes

• Wearenowplanningtouseittostudyothergroupsofdiseases

Acknowledgements

SciLifeLab and Uppsala UniversityMaria WilbeInger JonassonSusana HäggqvistSara OlofssonMarie-Louise Bondeson

Thermo FisherArvind KothandaramanAndreas ToblerJussi VanhataloLinnea Nyberg