Understanding and controlling for sample and platform biases in NGS assays

HORIZON DISCOVERY

Understanding and Controlling for Sample and Platform Biases in NGS Assays

2For Research Use Only

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


Clinical Application of Next Generation Sequencing

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


The Sources of Variability in the Next Generation Sequencing Workflow


Quantitative Multiplex

BRAF V600E KIT D816V EGFR ΔE746 - A750

EGFR L858R EGFR T790M

EGFR G719S KRAS G13D KRAS G12D NRAS Q61K PIK3CA H1047R

PIK3CA E545K

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AmpliSeq Panel in three laboratories


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• Personalized Medicine




RNAtranscriptomics

DNAmetagenomics

And more…


DNAepigenomics

DNAresequencing

DNAde novo assembly


RNAtranscriptomics

DNAmetagenomics

And more…


DNAepigenomics

DNAresequencing

DNAde novo assembly

We will focus on: • Biological Sample• Library Preparation• Sequencing Platform• Informatics Pipeline

o View our previous webinar for more on informatics



NGS Workflow – Reference Materials


Source of Error: Biological Sample

Potential Sources of bias/error include: • User errors

o Exogeneous DNA contaminationo Mislabelling

• Heterogenous sampleo Non-tumor cells o Mixed-cell populations (xenografts)

• Limited sample availabilityo Low Quantity

• Degradation/fragmentationo FFPEo cfDNA


Formalin Compromised DNA Reference Standards

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

• HD-C749 (Formalin-Compromised DNA) – (mild formalin treatment, low-level degraded)• Lanes 2 and 4 on right

• HD-C751 (Formalin-Compromised DNA) – (harsh formalin treatment, highly degraded)• Lanes 3 and 5 on right

The Quantitative Multiplex also comes in the following formats:

• HD701 (DNA) – high molecular weight DNA extracted directly from cells

• HD200 (FFPE) - mild-formalin fixation, embedded in paraffin once extracted shows little degradation

Genomic DNA Tapescreen assay

[bp] 1 2 3 4 5


Formalin-Compromised Multiplex Reference Standard

HD-C751 HD-C749

How does formalin treatment affect downstream analysis?

Amplification bias may not be detected without appropriate controls.


Formalin-Compromised Multiplex Reference Standard

Variant Expected Ratio

“Acceptable Range”

Determined Ratio

Batch 1

Determined Ratio

Batch 2

Determined Ratio

Batch 3

Determined Ratio

Batch 1

Determined Ratio

Batch 2

Determined Ratio

Batch 3

EGFR G719S 25% 22.1%-27% 23.4% 23.8% 23.4% 24.1% 22.7% 23.2%

PI3KCA H1047R 18% 14%-21% 19.6% 20.0% 18.8% 20.7% 20.4% 20.7%

KRAS G13D 15% 12%-18% 13.8% 14.8% 12.9% 15.3% 17.8% 14.0%

NRAS Q61K 13% 10%-15% 10.4% 10.1% 12.0% 12.8% 13.5% 13.2%

BRAF V600E 11% 8.6%-12.8% 12.4% 12.5% 11.9% 12.3% 11.6% 12.7%

PI3KCA E545K 9% 7.2%-10.8% 8.0% 8.1% 8.8% 10.7% 13.1% 13.0%

KIT D816V 10% 8%-12% 10.5% 10.2% 10.2% 10.5% 21.9% 20.1%

KRAS G12D 6% 4.8%-7.2% 5.9% 6.0% 5.3% 7.2% 5.9% 7.2%

EGFR L858R 3% 2.1%-3.9% 3.2% 3.3% 3.3% 3.4% 4.3% 3.5%

EGFR ∆E746-A750 2% 1.4%-2.6% 1.9% 2.0% 1.9% 1.9% 3.3% 3.2%

EGFR T790M 1% 0.7%-1.3% 1.3% 1.3% 1.0% 1.1% 1.6% 1.2%

HD-C749 HD-C751


Bias/Errors in Library Preparation

Robasky, K. et al. The role of replicates for error mitigation in next-generation sequencing. Nature Rev. Genet. 15, 56-62 (2014).


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.


Source of Error: Library Preparation

Errors arising from sequencing library preparation include:• Uneven sequencing coverage• Sequence changes• Length biasing/preferential amplification• Primer bias Mispriming Multiple Displacement Amplification (MDA) Incorporation of errors

From NuGEN


Variant Type Mutation Expected Fractional Abundance (%) or CNV:

SNV High GC GNA11 Q209L 5.6SNV High GC AKT1 E17K 5.6SNV Low GC KRAS G13D 5.6SNV Low GC Pi3Ka E545K 5.6Long Insertion EGFR V769 ins 5.6

Long DeletionEGFR (delE746-A750)

5.3

Fusion ROS1 translocation 5.6

Fusion RET translocation 5.6

CNV MET amplification 4.5 x amplification

CNV MYC amplification 9.5 x amplification

SNP EGFR_G719S 5.3Short Deletion MET_p.V237fs 4.8*SNV High GC NOTCH1_p.P668S 5.0Short Deletion FLT3_p.S985fs 5.6Short Deletion BRCA2_p.A1689fs 5.6Short Deletion FBXW7_p.G667fs 5.6

Structural Multiplex Reference Standard

*This product is part of our early access program. It is the responsibility of the individual laboratory to determine expected results specific to its assay.


Bias/Errors in Library Preparation



Platform Bias – Overview

3 Common Platforms: Common sources of bias/error include: • User error Sample overloading• Machine failure Laser, hard drive, software, fluidics

failures• Nucleotide malfunction Fluorophore quenching, nucleotide

damage, signal overlap• Sequence context errors High GC content, low-complexity

regions, homopolymers• Dephasing Incomplete extension, addition of

multiple nucleotides


Platform Bias – Illumina

Images from Illumina.


Platform Bias – Illumina

Images from Illumina.


Platform Bias – Ion Torrent

Illustration: James Provosthttp://spectrum.ieee.org/biomedical/devices/the-gene-machine-and-me

Erro

r Rat

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Homopolymer length


Platform Bias – PacBio

Single Molecule Real Time (SMRT) Sequencing

Image from PacBio.


Platform Bias – How can replicates help?

DNA samples from blood and saliva were sequenced on two different

platforms — Illumina and Complete Genomics — which resulted in 88.1%

concordance of single-nucleotide variants (SNVs) across replicates.

Cross Platform Replicates


Value of Replicates – Biological and Technical


R = replicates


Value of Technical Replicates – Process Noise

PlatformQX100 Droplet

Digital PCR (Internal QC)

Ampliseq Cancer HotspotPanel v2*

Gene Mutation Specification Observed mutant ratio, % COV

BRAF V600E 10.5 10.2 10.3 0.01

KIT D816V 10.0 10.4 10.1 0.01

EGFR ΔE746 - A750 2.0 2.0 Not detected -

EGFR L858R 3.0 2.7 2.4 0.07

EGFR T790M 1.0 0.9 Not detected -

EGFR G719S 24.5 24.4 24.8 0.01

KRAS G13D 15.0 16.1 15.5 0.03

KRAS G12D 6.0 5.0 5.1 0.03

NRAS Q61K 12.5 12.8 12.6 0.01

PIK3CA H1047R 17.5 18.6 17.9 0.01

PIK3CA E545K 9.0 8.9 8.8 0.01

*Average of 8 runs, average coverage 2000x

Quantitative Multiplex Reference Standard

Available as gDNA or FFPE ready for extraction


NGS Workflow


Source of Error: Bioinformatics

http://www.horizondx.com/bioinformatics-webinar.html




Next-Generation Sequencing – Wrap up

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Horizon Discovery – Your Partners in Personalized Medicine

Powering Genomic Research and Translational Medicine, from Sequence to Treatment

Horizon’s mission is to be a fully integrated life science company that provides enabling products, services and research programs to clients engaged at every stage of the healthcare continuum from sequence to treatment

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Horizon’s Range of Products/Services

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Horizon’s Range of Products/Services


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?


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

StructuralStandard

DNA

Sample Complexity

SampleFeatures QMRS

DNA andFFPE

GIABFFPE

Gene-SpecificMultiplex

DNA and FFPE

Tru-QDNA

Your Horizon Contact:

t + 44 (0)1223 655580f + 44 (0)1223 655581e [email protected] www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

Natalie LaFranzo, PhDUS Customer/Technical Support [email protected]+1-844-655-7800