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Molecular QC: Using Reference Standards in NGS Pipelines

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HORIZON DISCOVERY Molecular QC: Using Reference Standards in NGS Pipelines 21 st May 2015 Jonathan Frampton, PhD and Natalie LaFranzo, PhD
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HORIZON DISCOVERY

Molecular QC: Using Reference Standards in NGS Pipelines21st May 2015

Jonathan Frampton, PhD and Natalie LaFranzo, PhD

2

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

3

Clinical Application of Next Generation Sequencing

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

4

The Sources of Variability in the Next Generation Sequencing Workflow

Reference Materials

External Quality Assessment

5

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)

6

For Research Use Only

Next-Generation Sequencing Introduction

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

โ€ข Has been applied to scientific questions across industriesโ€ข Pharma โ€ข Biotechโ€ข Biofuelsโ€ข Agricultureโ€ข Food Scienceโ€ข Archeologyโ€ข Medicineโ€ข โ€ฆ

vs.

7

For Research Use Only

Next-Generation Sequencing Introduction

DNA de novo

assembly

DNAresequencing

DNAepigenomics

RNAtranscriptomics

DNAmetagenomics

And moreโ€ฆ

8

For Research Use Only

Next-Generation Sequencing Introduction

DNA de novo

assembly

DNAresequencing

DNAepigenomics

RNAtranscriptomics

DNAmetagenomics

And moreโ€ฆ

9

For Research Use Only

Patient-derived Samples โ€“ DNA Analysis

Unique challenges:

โ€ข Heterogeneous

โ€ข Low quantity

โ€ข Poor quality

โ€ข Low-allelic frequency detection desired

10

For Research Use Only

NGS Workflow

Reference Materials

Analysis

11

For Research Use Only

Implementation in your own Laboratory

ACMG Recommendations

12

For Research Use Only

NGS Workflow

Reference Materials

Analysis

13

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.

14

For Research Use Only

NGS Workflow

Reference Materials

Analysis

15

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

16

For Research Use Only

NGS Workflow

Reference Materials

Analysis

17

For Research Use Only

Sequencing Library Preparation

Enrichment options:

โ€ข whole-genome (not enriched)

โ€ข whole-exome capture

โ€ข custom capture

โ€ข capture-based panels

โ€ข off-the-shelf amplicon panels

โ€ข custom amplicon panels

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

18

For Research Use Only

NGS Workflow

Reference Materials

Analysis

19

For Research Use Only

Variability

Platform: QX100โ„ข 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 ฮ”E746 - 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

20

For Research Use Only

NGS Workflow

Reference Materials

Analysis

21

For Research Use Only

Influence of Analytical Pipelines

22

For Research Use Only

NGS Workflow

Reference Materials

Analysis

23

For Research Use Only

Theoretical Limit of DetectionDependent on:

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

Reference Sequence

24

For Research Use Only

Theoretical Limit of Detection

4๐‘Ÿ๐‘’๐‘Ž๐‘‘๐‘  h๐‘ค๐‘–๐‘ก ๐‘š๐‘ข๐‘ก๐‘Ž๐‘ก๐‘–๐‘œ๐‘›๐‘œ๐‘“ ๐‘–๐‘›๐‘ก๐‘’๐‘Ÿ๐‘’๐‘ ๐‘ก3333๐‘Ÿ๐‘’๐‘Ž๐‘‘๐‘ (๐‘š๐‘œ๐‘™๐‘’๐‘๐‘ข๐‘™๐‘’๐‘ )

=0.12% ๐‘Ž๐‘™๐‘™๐‘’๐‘™๐‘–๐‘ ๐‘“๐‘Ÿ๐‘’๐‘ž๐‘ข๐‘’๐‘›๐‘๐‘ฆ

Reference Sequence

For 10 ng input amount (or 3333 molecules):

25

For Research Use Only

Theoretical Limit of Detection

Reference Sequence

For high input: 4๐‘Ÿ๐‘’๐‘Ž๐‘‘๐‘  h๐‘ค๐‘–๐‘ก ๐‘š๐‘ข๐‘ก๐‘Ž๐‘ก๐‘–๐‘œ๐‘›๐‘œ๐‘“ ๐‘–๐‘›๐‘ก๐‘’๐‘Ÿ๐‘’๐‘ ๐‘ก

ยฟ๐‘Ÿ๐‘’๐‘Ž๐‘‘๐‘ =๐ฟ๐‘–๐‘š๐‘–๐‘ก ๐‘œ๐‘“ ๐‘‘๐‘’๐‘ก๐‘’๐‘๐‘ก๐‘–๐‘œ๐‘›

4๐‘Ÿ๐‘’๐‘Ž๐‘‘๐‘  h๐‘ค๐‘–๐‘ก ๐‘š๐‘ข๐‘ก๐‘Ž๐‘ก๐‘–๐‘œ๐‘›๐‘œ๐‘“ ๐‘–๐‘›๐‘ก๐‘’๐‘Ÿ๐‘’๐‘ ๐‘กยฟ๐‘Ÿ๐‘’๐‘Ž๐‘‘๐‘ 

=0.01% ๐‘‘๐‘’๐‘ ๐‘–๐‘Ÿ๐‘’๐‘‘

# reads = 40,000x!

26

For Research Use Only

Actual Limit of Detection

Target Allelic Frequency

50% Mutant DNA (orange lid)

Wild Type DNA (white lid)

20% 4ยตl 6ยตl

10% 2ยตl 8ยตl

5% 1ยตl 9ยตl

1%1ยตl of prepared 10%

dilution 9ยตl

0% 0ยตl 10ยตl

Total Volume 7ยตl 42ยตl

27

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

28

For Research Use Only

Exciting Challenges

HD701 vs. HD751 vs. HD200

โ€ข Multiple formats for Quantitative Multiplex Reference Standardโ€ข 11 validated positive mutationsโ€ข Frequency range: 24%-1%

โ€ข HD701 โ€“high molecular weight DNA extracted directly from cells

โ€ข HD751 โ€“ formalin-compromised DNA (harsh formalin treatment, highly degraded)โ€ข Lanes 3 and 5 on right

โ€ข HD200 โ€“ mild-formalin fixation, embedded in paraffin (FFPE format), once extracted shows little degradationโ€ข Lanes 2 and 5 on right

Genomic DNA Tapescreen assay

[bp] 1 2 3 4 5

29

For Research Use Only

Exciting ChallengesHD701 vs. HD751 vs. HD200

30

For Research Use Only

Implementation in your own Laboratory

ACMG Recommendations

31

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

32

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?

www.horizondx.com


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