Director, Global Marketing Programs...1. Beyond the Exome : resolving structural complexities and variation OneSeq CNV Backbone + Custom Panel • Computational methods that identify

Agilent NGS Solutions : Addressing Today’s Challenges

Charmian Cher, Ph.D

Director, Global Marketing Programs

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

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10 years of Next-Gen Sequencing

2003 Completion of the Human

Genome Project 2004

Pyrosequencing hits the

market 2006 Launch of Genome

Analyzer and SOLiD

2010 High-throughput

sequencing enabled 2011

Benchtop and single-

molecule sequencing

2015 Promise of the $1000

genome

What’s Next?

NGS tools to probe the genome and transcriptome in greater depth

to enhance understanding of phenotype and disease

Innovative technologies to make NGS faster, cheaper, more accurate

Precision medicine

Entering an Exciting Era in Advancing Human Health

Technologies and disease

understanding are moving through

the research-clinical continuum

Precision medicine enable physicians to

identify and treat maladies more effectively

at a lower cost

Building on a strong legacy and expertise

within bio-reagents, automation solutions

and informatics capabilities

For Research Use Only. Not for use in diagnostic

procedures.

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Focus on Constitutional and Cancer Research Addressing today’s challenges

Prenatal

Postnatal Pre-implantation

screening

Cancer

Sample Prep

Challenging samples : FFPE, single cells, cfDNA

Sample heterogeneity

Hard to capture/sequence regions

Structurally complex regions

Cost efficiencies

Shorter time from sample to data

Streamlined workflows

Data analysis

Cumbersome and difficult bottleneck


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8 years of Agilent NGS Innovation along the research-to-clinical continuum

2009 Launch of SureSelect

Human All Exon kit, the

first target enrichment

system

2011 Launch of first model

organism exome, the

SureSelect Mouse All Exon

2012 Launch of HaloPlex, an

amplicon-based target

enrichment system

Agilent acquires Dako, a

leading cancer

diagnostics company

2014 Most comprehensive

Clinical Research Exome

and SureSelectQXT

launched

>2000 Agilent NGS

publications

2016 OneSight for cfDNA analysis

$80M investment in Lasergen

2015 Agilent acquires

Cartagenia enabling

clinical analysis of

genomics data

Introduced HaloPlexHS for

low allele frequency

variants & OneSeq for

CNV detection

SureSelect Human All

Exon V6 and ClearSeq

Comprehensive Cancer


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Agilent’s Core Capability : High-Fidelity Oligo Synthesis

(1)

Leading

platform for

generating

long, high-

fidelity oligos

The foundation

of Agilent’s

target

enrichment

solutions

Production of

custom

microarrays and

NGS target

enrichment

High volume

& low cost

production

capability


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Advantage of Ultra-Long RNA baits


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What’s New?

1. Beyond the Exome : resolving structural complexities and variation

OneSeq CNV Backbone + Custom Panel

Collaboration with 10X Genomics on a “Premium” Exome

2. High sensitivity detection of rare variants (<1% VAF)

HaloPlexHS Target Enrichment System incorporating molecular barcodes

3. Quality control of DNA from single cells for NGS

Agilent D5000 ScreenTape Assay

4. Visualization of chromosomal aneuploidies in cell-free DNA sequencing data

OneSight software platform

5. Investment in NGS technology for clinical sequencing

Lasergen


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1. Beyond the Exome : resolving structural complexities and variation OneSeq CNV Backbone + Custom Panel

• Computational methods that identify CNVs from WES are based on analysis of read depth

• These methods assume that read depth is proportional to the number of copies of that region

• Differences in bait efficiencies and sequencing biases result in highly variable read depth

Asymmetric read depth makes

CNV calling from WES very

difficult

Same WES dataset analyzed with

different CNV-callers produce

significantly different results

(Tan et al., 2014)

WGS can identify all variants in one

assay but remains cost-prohibitive

How do we study all variants in one cost-effective NGS assay?

Distribution of read depth in exome


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1. Beyond the Exome : resolving structural complexities and variation OneSeq CNV Backbone + Custom Panel SureSelectXT Library Prep and

OneSeq Target Enrichment

Probes covering coding regions

Probes selected for calling copy number

High minor allele frequency SNPs targeted

Exon Exon Exon

Backbone

Select CNV backbone

High

resolution

backbone (300Kb

genome-

wide + 25Kb

in ClinGen

regions)

Low

resolution

backbone

(1Mb)

Select content for SNVs & indels

Catalog

ClearSeq gene panels

SureSelect Exomes,

Clinical Research Exome,

Focused Exome

SureSelect

Custom

OneSeq Target Enrichment


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1. Beyond the Exome : resolving structural complexities and variation OneSeq CNV Backbone + Custom Panel

Detection of Trisomy 13 in NA02948

300kb OneSeq backbone

1Mb OneSeq backbone

Detection of 5Mb LOH in NA20409

Copy number

[Log2Ratio]

LOH data

[B allele freq]

Microarray

OneSeq

Detection of 364kb duplication upstream of SOX9


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1. Beyond the Exome : resolving structural complexities and variation Collaboration with 10X Genomics on a “Premium” Exome

Launching in October 2016!

Standard Exome Baits

Linked-Reads from Standard Exome Baits

SureSelect Optimized Exome Baits

Linked-Reads from Optimized Baits

Agilent Target Enrichment Chromium Controller Sequencer 10x Long Ranger 10x Loupe

1. Reach beyond the exome while maintaining

high quality SNP/indel performance

2. Phase thousands of genes and assign cis

or trans to heterozygous variants with

megabase-scale phase blocks

3. Detect translocations, gene fusions and

enable CNV detection with precise

insertions, deletions events.


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2. High Sensitivity Detection of Rare Variants HaloPlexHS Target Enrichment System Incorporating Molecular Barcodes

How molecular barcoding works

Step 1 Align reads

Step 2 Group read pairs to designed probes based

on read start-stop position

Step 3 Group reads with an identical molecular

barcode sequence for each probe

Step 4 Consolidate read information to one read

per molecule (remove PCR duplicates)

HaloPlexHS Workflow

Complete library prep-free target enrichment in <6hr

Detects SNVs down to <1% allele frequency

Analysis using Agilent SureCall software


HaloPlexHS performance with FFPE samples Detection down to 0.5% allele frequency in HapMap dilutions


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HaloPlexHS makes the cover of the

Journal of Medical Genetics!

Mosaic DICER1 RNaseIIIb

missense mutations important

genetic cause of DICER1

syndrome

High Sensitivity Sequencing Reveals Multi-Organ Somatic

Mosaicism Causing DICER-1 Syndrome

Leanne de Kock et al (2015)

Dept of Human Genetics, McGill University

Journal of Medical Genetics

Tumors associated

with DICER1

Syndrome

Deep sequencing of

gDNA from blood,

tumor and normal

tissue

Detected RNaseIIIb

mutations at 0.2 – 13%

frequency in

constitutional DNA


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3. Quality Control of DNA from Single Cells for NGS Agilent Sample QC Systems

Agilent 2100 Bioanalyzer System

Widely accepted QC standard

Integrated in many protocols & extensively cited

Widest range of applications

Includes protein

Small footprint

Agilent 4200 TapeStation System

96 sample walkaway operation

Scalable throughput at a constant cost

Easy to use

Ready-to-use consumables

NEW


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3. Quality Control of DNA from Single Cells for NGS Agilent D5000 ScreenTape Assay

Agilent Application Note (5991-5259EN): Morris & Eberwine, Penn University

A B


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Compatible

with most

common

NGS library

prep kits

Compatible with

the most

common NGS

sequencing

platforms

Select analysis

pipeline and

reference set

Visually inspect

chromosome

plots

OneSight

Upload NGS

data

OneSight offers genetics laboratories a cost-effective way to perform high-quality

cell-free DNA analysis in-house



Normal Segmental aberration

Trisomy Complex aberrations


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5. $80M Investment in NGS Technology for Clinical Sequencing Lasergen

Automation

Library Prep /

Capture QC Sequencing Analysis

Interpretation /

Reporting

Lasergen

Houston, TX

Focused on advancement and

commercialization of unique

nucleotide chemistries

Proprietary photocleavable

nucleotide chemistry (“lightning

terminators”) fast reaction

times, robust cleavage

Potential to be faster, cheaper

and more accurate than current

NGS technologies

Ultimate goal:

Provide a complete workflow for routine clinical

testing

Intended to support applications within

Human & Reproductive Genetics and Cancer

Exomes and panels

Integrated solution, including automation and

analysis / reporting


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Building a Complete Agilent NGS Solution

Research Mix & match

Clinical Integrated workflow

Automated

Sample prep

Automation

(q)PCR

Sample QC

HaloPlex Target

Enrichment

Alignment

& Variant Calling

Target Enrichment

Next Generation

Sequencing

Interpretation

support and

reporting

OneSeq Target

Enrichment

SureSelect Target

Enrichment

Data Analysis

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Learn More @ Festival of Genomics Boston Stop by Agilent Booth #322!

Documents

Director, Global Marketing Programs...1. Beyond the Exome : resolving structural complexities and variation OneSeq CNV Backbone + Custom Panel • Computational methods that identify