Dr. Robert Loewe CEO GeneWake with

MD Napier , Senior Product Manager

Recorded May 26th, 2015 @ Agilent’s Santa Clara site

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June 3, 2015

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Dr. Robert Loewe from GeneWake speaking on genotyping with HRM

Dr. Robert Loewe is the founder of GeneWake, and has been chief executive

officer since the company´s inception in 2009. Special field of interest is here

qPCR and NGS to establish practical approaches in the field of

pharmacogenomics and cancer.

Mary Dell Napier MS, MBA Cantab speaking on HRM commercialization

Mary Napier is passionate about commercializing products that relate to

genomic information. Now as senior product manager at Agilent she

directs her interest into molecular biology portfolio. She has an MBA from

the University of Chicago and studied cancer genetics at the Wellcome

Trust Centre in Cambridge UK.

WHAT TECHNOLOGY ARE YOU USING TODAY? WE WANT TO TALK ABOUT HRM AS A COMPANION ASSAY TO THE WORK YOU ARE ALREADY DOING

For Research Only. Not For Use in Diagnostic Procedures.

• Less than 0.2ºC diff in melt temp

(A/T mutation in class IV SNP)

• Large deletions

• Thermocycler calibration

• Multiplex limited to 2-3 in a

single well

Technology Limitations User Concerns Assay Details

• Oligo length: ~65-120bp

• Assay time: 8 hours to 4 days • Turn around time

• Cost

• DNA or RNA input

• Absolute quantification

• Amplicon length: ~80-300bp

• Assay time: 30-100 minutes

• Turn-around time

• Expense

• Oligo length: ~300-600bp+

• Assay time: ~2-3days.

• Annotation at hyper-variable loci is

weak

• Loci with high repeat segments is

prone to adapter mis-match or

amplification bias.

• Reliability from first

generation platforms

• De Novo deletions imputed only

• Hyper-variable loci & Loci with high

repeat segments= unreliable results

• Can only genotype what is on array,

rare alleles

HRM

Array

NGS

HRM when used appropriately can be a great alternative for difficult to genotype variants

Thermocycler calibration

Release on Demand dyes

Software design

Learning curve: Mix and Go kits

Minutes - not hours or days

Easy integration with existing workflow

Cytochrome p450

P53

HLA

CFTR

De novo

Assay reliability

Time to answer

Target

rare or hyper-variable loci

For Research Only. Not For Use in Diagnostic Procedures.

Tip #1: SPEND YOUR TIME AND MONEY WISELY

• Choose the right chemistry: EvaGreen dye is established as superior to

SYBR

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Spend your money wisely: EvaGreen Dye is better and here is why

Typical dyes used in HRM: SYBR Green, SYTO9, LC Green and EvaGreen

SYBR Green (non-saturating dye)

• Inhibits PCR thus can’t be added at saturating concentrations (At high concentrations it stabilizes the duplex DNA and inhibits the DNA polymerase)

• At lower concentrations it redistributes from the melted regions of ssDNA back to the regions of dsDNA

( dye jumping results in poor base-difference discrimination)

EvaGreen

• Non-toxic (non-mutagenic and impermeable to cell membranes)

• Non-fluorescent by itself, must be bound to dsDNA to fluoresce

• Very little PCR inhibition thus can be added at saturating concentrations (more signal, lower signal to noise ratio)

• Shorter PCR time (minimal interference of amplicon chain extension)

• Reasonably priced

• Homebrew titrating your master mix is time consuming and it is

extremely difficult to create a consistent product

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Spend your time wisely

Tip #2: ALL PROTOCOLS ARE NOT CREATED EQUAL

GOAL: correct protocol avoids costly mistakes

• Avoid batch processing errors by optimizing with methodical protocols with appropriate

melting & amplification times.

• Conquer this challenge: try small and large target amplicon.

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INTIAL DENATURATION: 3 minutes CYCLING PROTOCOLS BELOW 1. Top protocol 10s 95°C 15s 60°C 25s 72°C 2. Middle protocol 10s 95°C 30s 60°C Best for mid range amplicons 3. Bottom protocol (fast mode) 5s 95°C 10s 60° Best for shorter amplicons

OPTIMIZING PROTOCOLS, WHAT IT LOOKS LIKE OPTIMIZATION INCLUDED: TWO AMPLICONS | TESTED THREE PROTOCOLS

69 bp amplicon 112 bp amplicon

Takeaway: Match your protocol to your amplicon size.

• Fluorescence output

• Plateau reached

Our protocol (fast mode) is for short amplicons

Tip #3: THE ATCG OF TARGET AMPLICON SIZE SELECTION

Discrimination becomes difficult when base substitutions occur between A&T

and C&G.

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Importance of calibration Class IV SNPs used to be illusive to Genotype

by HRM due to thermocycler block accuracy.

Now it is part of a “base performance” solution

Tip #4: DO NOT BLINDLY TRUST AUTOMATIC GENOTYPE CALLS

Especially important for Calling rare alleles, FFPE fragmented samples and star alleles

Where annotation is challenging

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R2: 0.979; Eff: 95%

Great Curves Great R2

Bummer of

genotype

call

Tip #5: NORMALIZE AND BE CONFIDENT Important to normalize mass relative to fluorescence signal (rather than the other way around)

Perform two methods

• Normalize PCR kinetics so fragments are amplified equally

• Genetic spike in to produce heterozygote for proper normalization for genotype calling (functional assay)

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Tip #6: WATCH OUT FOR COMPOUNDS WHICH INHIBIT CHEMISTRY & ALTER FLUORESCENCE Inhibitors delay the Cq and /or may reduce fluorescence.

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What is doing this?

Salt concentration

Melting temp

Others?

Tip #7: DETECTING VERY DIFFICULT ALLELES:

SOMATIC MUTATIONS

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• Analyze both amplification and melting curve for efficiency

• Design and run a known functional assay to determine that you

have a quantitative assay

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FUNCTIONAL ASSAY Spike in experiment to simulate discrimination for lower abundant mutation 5% Detection 5% mutant | 95% WT 112 base pair allele

Tip #7: DETECTING VERY DIFFICULT ALLELES: SOMATIC MUTATIONS

INITIAL DENATURATION: 3 minutes

CYCLING PROTOCOL: 10s at 95°C & 30s at 60°C

Allele A homozygote

Normalized melting curve: green is

heterozygote at 50% on Y axis.

Temperature shift to get the 50%

intersection.

Picture perfect results for the bottom

graph: Homozygote identical,

duplicates are almost identical.

Allele B heterozygote

All three alleles

Tip #7: DETECTING VERY DIFFICULT ALLELES: SOMATIC MUTATIONS Picture perfect results, most difficult alleles

Agilent’s complete HRM solution A dramatically faster and more robust solution

Manufacturer reagents matched to manufacturer instruments

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2

3

64

54

37

105

45

29

0 50 100 150 200 Time (minutes)

Hot start

Amplification

HRM Segment

69 minutes

185 mins

Agilent

AriaMx-

HRM Ultra Fast Loci

101 minutes Competitor B-Reagent B

Competitor AB-Reagent AB

Takeaway on complete solution speed: Time savings from (1) Scan time (2) Data acquisition method (3) Tm interpolation (4) hot start (5) protocol time from

EvaGreen use For Research Only. Not For Use in Diagnostic Procedures.

Summary 7 Tips & Tricks 1. SPEND YOUR TIME AND MONEY WISELY

• Choose the right chemistry: EvaGreen dye is established as superior to SYBR

• Homebrew titrating your master mix is time consuming and it is extremely difficult to create a consistent product

2. ALL PROTOCOLS ARE NOT CREATED EQUALLY

• Avoid batch processing errors by optimizing with methodical protocols with appropriate melting & amplification times.

• Conquer this challenge: : try small and large target amplicon.

3. THE ATCG OF TARGET AMPLICON SIZE SELECTION

• Discrimination becomes difficult when base substitutions occur between A&T and C&G.

4. DO NOT BLINDLY TRUST AUTOMATIC GENOTYPE CALLS

• Calling rare alleles, FFPE fragmented samples and star alleles

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Summary 7 Tips & tricks, cont’d 5. NORMALIZE AND BE CONFIDENT

• Important to normalize mass relative to fluorescence signal (rather than the other way around)

• Normalize PCR kinetics so fragments are amplified equally

• Genetic spike in to produce heterozygote for proper normalization for genotype calling (functional assay)

6. WATCH OUT FOR COMPOUNDS WHICH INHIBIT CHEMISTRY & ALTER FLUORESCENCE

• Inhibitors delay the Cq and / or may reduce fluorescence.

• Determine if high salt concentrations is impacting melting temperature of amplicons and interfering with your

ability to detect different alleles

7. DETECTING VERY DIFFICULT ALLELES: SOMATIC MUTATIONS

• Analyze both amplification and melting curve for efficiency

• Design & run a known functional assay to determine that you have a quantitative assay

June 3, 2015

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June 3, 2015

For Research Use Only. Not for Use in Diagnostic Procedures.

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THANK YOU FOR YOUR TIME Learn more at:

www.agilent.com/genomics/ariamx

Contact us to collaborate on hyper-variable alleles with HRM

Master Mix: Part number 5190-7827