HORIZON DISCOVERY

Molecular QC: Using Reference Standards in NGS Pipelines21st May 2015

Jonathan Frampton, PhD and Natalie LaFranzo, PhD

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What is the impact of assay failure in your laboratory and how do you monitor for it?

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Clinical Application of Next Generation Sequencing

Using just one sample, one workflow can test for mutation status across multiple genes

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The Sources of Variability in the Next Generation Sequencing Workflow

Reference Materials

External Quality Assessment

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T790M &

L858R

E746_A

750del

Wild

type

Wild

type

E746_A

750del

T790M &

L858R

E746_A

750del

Wild

type

T790M &

L858R

T790M &

L858R

E746_A

750del

T790M &

L858R

E746_A

750del

Wild

type

Wild

type

Wild

type

Wild

type

G719S

T790M &

L858R

G719S0

5

10

15

20

25

30

35

40

EGFR Genotyping ErrorsExternal Quality Assessment 2014

EGFR Sample Tested

Perc

enta

ge o

f Inc

orre

ct R

esul

ts

European Molecular Quality Network (EMQN)

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For Research Use Only

Next-Generation Sequencing Introduction

Also known as high-throughput or massively-parallel sequencing• Allows us to address questions that require a lot of data

• Has been applied to scientific questions across industries• Pharma • Biotech• Biofuels• Agriculture• Food Science• Archeology• Medicine• …

vs.

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For Research Use Only

Next-Generation Sequencing Introduction

DNA de novo

assembly

DNAresequencing

DNAepigenomics

RNAtranscriptomics

DNAmetagenomics

And more…

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For Research Use Only

Next-Generation Sequencing Introduction

DNA de novo

assembly

DNAresequencing

DNAepigenomics

RNAtranscriptomics

DNAmetagenomics

And more…

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For Research Use Only

Patient-derived Samples – DNA Analysis

Unique challenges:

• Heterogeneous

• Low quantity

• Poor quality

• Low-allelic frequency detection desired

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For Research Use Only

NGS Workflow

Reference Materials

Analysis

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For Research Use Only

Implementation in your own Laboratory

ACMG Recommendations

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For Research Use Only

NGS Workflow

Reference Materials

Analysis

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For Research Use Only

DNA Extraction Variability from FFPE

Promega

Max

well (n=12)

Promega

Mag

nesil (n

=6)

Promega

ReliaPrep (n

=6)

Qiagen Dneasy

(n=6)

Roche Cobas

(n=6)0%

10%20%30%40%50%60%70%

DNA Recovery from Total Theoretical Yield

Extraction Kit

Perc

enta

ge D

NA

Rec

over

ed

In house generated data set.

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For Research Use Only

NGS Workflow

Reference Materials

Analysis

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For Research Use Only

DNA Quantification

Lab A

Lab B

Lab C

Lab D

Lab E

Lab F

Lab G

Lab H

Lab I

Lab J

Lab K

Lab L

Lab M

Averag

e

Median

0

5

10

15

20

25

30

35

40Nanodrop:Qubit (N/Q Ratio)

Participant Laboratory

N/Q

Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644

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For Research Use Only

NGS Workflow

Reference Materials

Analysis

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For Research Use Only

Sequencing Library Preparation

Enrichment options:

• whole-genome (not enriched)

• whole-exome capture

• custom capture

• capture-based panels

• off-the-shelf amplicon panels

• custom amplicon panels

Goal: Use a reference standard that reflects your actual sample.

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For Research Use Only

NGS Workflow

Reference Materials

Analysis

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For Research Use Only

Variability

Platform: QX100™ Droplet Digital PCR

Ampliseq Cancer Hotspot Panel v2

(Average of 8 runs)Ampliseq Cancer Hotspot Panel v2

Ampliseq Cancer Hotspot Panel v2

Sequencing Depth: N/A N/A 2000x 3000-4000x Average 5000x

Gene Variant Specification In house validation Partner A Partner B Partner C

BRAF V600E 10.5 10.2 10.3 9.9 9.1

KIT D816V 10.0 10.4 10.1 10.0 11.0

EGFR ΔE746 - A750 2.0 2.0 Not detected 2.3 Not detected

EGFR L858R 3.0 2.7 2.4 2.7 2.1

EGFR T790M 1.0 0.9 Not detected 0.8 Not detected

EGFR G719S 24.5 24.4 24.8 23.7 23.1

KRAS G13D 15.0 16.1 15.5 16.3 12.4

KRAS G12D 6.0 5.0 5.1 5.2 Not detected

NRAS Q61K 12.5 12.8 12.6 9.0 12.7

PIK3CA H1047R 17.5 18.6 17.9 16.7 16.8

PIK3CA E545K 9.0 8.9 8.8 3.2 8.4

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For Research Use Only

NGS Workflow

Reference Materials

Analysis

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For Research Use Only

Influence of Analytical Pipelines

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For Research Use Only

NGS Workflow

Reference Materials

Analysis

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For Research Use Only

Theoretical Limit of DetectionDependent on:

(1) molecular uniqueness/deduplication, (2) input, and (3) coverage

Reference Sequence

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For Research Use Only

Theoretical Limit of Detection

4𝑟𝑒𝑎𝑑𝑠 h𝑤𝑖𝑡 𝑚𝑢𝑡𝑎𝑡𝑖𝑜𝑛𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡3333𝑟𝑒𝑎𝑑𝑠(𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠)

=0.12% 𝑎𝑙𝑙𝑒𝑙𝑖𝑐 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦

Reference Sequence

For 10 ng input amount (or 3333 molecules):

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For Research Use Only

Theoretical Limit of Detection

Reference Sequence

For high input: 4𝑟𝑒𝑎𝑑𝑠 h𝑤𝑖𝑡 𝑚𝑢𝑡𝑎𝑡𝑖𝑜𝑛𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡

¿𝑟𝑒𝑎𝑑𝑠=𝐿𝑖𝑚𝑖𝑡 𝑜𝑓 𝑑𝑒𝑡𝑒𝑐𝑡𝑖𝑜𝑛

4𝑟𝑒𝑎𝑑𝑠 h𝑤𝑖𝑡 𝑚𝑢𝑡𝑎𝑡𝑖𝑜𝑛𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡¿𝑟𝑒𝑎𝑑𝑠

=0.01% 𝑑𝑒𝑠𝑖𝑟𝑒𝑑

# reads = 40,000x!

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For Research Use Only

Actual Limit of Detection

Target Allelic Frequency

50% Mutant DNA (orange lid)

Wild Type DNA (white lid)

20% 4µl 6µl

10% 2µl 8µl

5% 1µl 9µl

1%1µl of prepared 10%

dilution 9µl

0% 0µl 10µl

Total Volume 7µl 42µl

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For Research Use Only

Actual Limit of Detection

0% 5% 10% 15% 20% 25%0%

5%

10%

15%

20%

25%Limit of Detection Results

Target Allelic Frequency

Aver

age

of A

ctua

l Alle

lic

Freq

uenc

y D

etec

ted

Alternative: Tru-Q Multiplex standards available at 5%, 2.5% and 1.25% allelic frequencies covering multiple mutants.

1% not detected

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For Research Use Only

Exciting Challenges

HD701 vs. HD751 vs. HD200

• Multiple formats for Quantitative Multiplex Reference Standard• 11 validated positive mutations• Frequency range: 24%-1%

• HD701 –high molecular weight DNA extracted directly from cells

• HD751 – formalin-compromised DNA (harsh formalin treatment, highly degraded)• Lanes 3 and 5 on right

• HD200 – mild-formalin fixation, embedded in paraffin (FFPE format), once extracted shows little degradation• Lanes 2 and 5 on right

Genomic DNA Tapescreen assay

[bp] 1 2 3 4 5

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For Research Use Only

Exciting ChallengesHD701 vs. HD751 vs. HD200

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For Research Use Only

Implementation in your own Laboratory

ACMG Recommendations

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For Research Use Only

How to Test the Robustness and Sensitivity of your Workflow and Assay

Sensitivity of your Assay

HD701

Formalin Intensity

HD200

Robustness and Sensitivity of your Workflow

HD-C751

FFPE

DNA

Robustness of your Assay

HD-C749

GIAB FFPE Samples

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Routinely monitor the performance of your workflows and assays with independent external controls

What extraction and quantification methods are you

using?

What is the limit of detection of your

workflow?

Is the impact of formalin treatment interesting to you?

What is the impact of assay failure in your laboratory and how do you monitor for it?

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