haloplex disease research panels - agilent · 2015-09-04 · sensitive variant detection . research...

Research Use Only

Not for Use in Diagnostic Procedures

The Next Generation of Panels has Arrived…

HaloPlex Disease Research Panels

Premium Performance with Content you can Trust!

Research Use Only


Data Analysis with SureCall

Agenda – HaloPlex

HaloPlex Introduction

How it Works

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Research Use Only


Next generation sequencing

Disruptive technology – transforming clinical research

Research Use Only


Why targeted resequencing?

Leverage throughput and accuracy to sequence at higher coverage

while multiplexing samples

NGS

Transcriptome Genome

Gene

expression

Small

RNA Whole

genome

Epigenome

De novo Whole

transcriptome

ChIP-Seq Methylation

Targeted

resequencing

Research Use Only


Targeted resequencing vs. whole genome

Required throughput = genome size x average coverage

v

Target = 50Mb x 100 = 5Gb

Target = 5Mb x 100 = 500Mb

Target = 0.5Mb x 100 = 50Mb

Target =50Kb x 100 = 5Mb

Human Genome

3Gb x 30 = 90Gb

~1 Sample/Run

$$$$$$

Research Use Only


DISCOVERY FOLLOW-UP CLINICAL

RESEARCH

From Discovery to Clinical Research

Whole Exome

Whole Genome

GWAS

Follow-up Exome

Follow-Up WGAS

Follow-up GWAS

Clinical Research

Panels

Research Use Only


FOLLOW-UP CLINICAL RESEACH

From discovery to clinical research

All Solutions

currently available

are based on

Multiplex PCR and

complex data

analysis Challenges:

Fast turnaround Time

Flexibility in panel size

Simple workflow

High Coverage and Specificity

High Throughput

Data Analysis Solution

Research Use Only


Advantages of traditional PCR

High Coverage

High Specificity

Fast

Cost Effective

Simple

Well established

Research Use Only


The problems with multiplex PCR

Primer Cross reactivity

Limited in # target regions

Long optimization

Hard to change

Dropouts

Artifacts

Research Use Only


HaloPlex – Premium performance

Low DNA Input (200ng)

High Coverage

Uniform Amplification

High Specificity

Sensitive variant detection

Research Use Only



Agenda – HaloPlex


How it Works

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Research Use Only


SureDesign - Create custom designs in <10 mins

1. Input gene ID/name/coordinate

2. Define regions of interest (eg.

Exons, UTRs, etc)

3. Click “Start Design”

4. Design report in 10 minutes

www.agilent.com/genomics/suredesign

Research Use Only


SureDesign - Create custom designs in <10 mins

www.agilent.com/genomics/suredesign

Report with target coverage

metrics

link to UCSC browser to review

target regions and amplicons

Design options available for

fragmented samples

Improved design coverage of

pseudogenes

Research Use Only


HaloPlex workflow Simple & fast 4-step protocol

Probe/Fragment

hybrids are retrieved

with magnetic beads.

Circular molecules are

then ligated.

Only circular DNA

targets are amplified.

Sample barcodes are

introduced.

3

4

Sample is

fragmented using

restriction enzymes

Probe library is added

& hybridized to the

target fragments

resulting in a Halo

shape.

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2

Regions of interest are ready for sequencing

Research Use Only


Improves design coverage

Redundancy reduces risk of

loosing completeness if a

probe fails; protects against

primer site mutations

Specificity of the restriction

enzymes add specificity to

the capture

Amplicon tiling

HaloPlex workflow

Each DNA sample is fragmented in

eight double-digest reactions

Digest and

denature DNA

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Research Use Only


Same hybridization requirement

as regular PCR

• High specificity

Both primers incorporated on

the probe

• No cross-reactivity

Integrated index or sample

barcode

• 96 sample format available

HaloPlex workflow

Hybridize probes Fragments are mixed with custom

HaloPlex probes and a primer cassette.

Hybridization overnight.

2

Correctly hybridized fragments will form a nicked

dsDNA structure with HaloPlex probe

Research Use Only


Purify and ligate

targets

Probe/fragment hybrids are retrieved with

streptavidin magnetic beads.

Circular molecules are ligated.

HaloPlex workflow

3

Level of enrichment is

increased by removal of

non-targeted fragments

Only perfectly

hybridized fragments

will be ligated

Research Use Only


HaloPlex workflow

Precise target is ready for sequencing

Amplify enriched

fragments

Only circular DNA targets are amplified. 4

PCR is primed

from the primer

cassette

Thousands of

amplicons

One primer pair

Research Use Only


HaloPlex technology Eight different fragments cover each target position.

Genomic region Target

Amplicons

are tiled over

the region

Research Use Only


HaloPlex technology

Genomic region Target

A subset covering

the target region is

selected using

HaloPlex probes Multiple

fragments

cover the

target region

Research Use Only


HaloPlex technology

Genomic region Target exon

HaloPlex baits guide circularization

Research Use Only


HaloPlex technology The selected subset is equipped with sequencing primer

sequences by the selector probes.


Research Use Only


HaloPlex technology The sequencing primers are used to sequence in from the

fragment ends (paired-end sequencing).


Research Use Only


HaloPlex technology


0001111122322222333455422221111222111110000 Read coverage

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HaloPlex technology


0001111122322222333455422221111222111110000

By tiling

amplicons, high

target coverage is

achieved

Research Use Only


Pileup in integrative genome viewer

Outstanding

coverage of

targeted bases

Highly

specific

Research Use Only


HaloPlex Others

TARGET TARGET

With HaloPlex each target base is

covered by multiple amplicons

(different start and stop sites)!

With other multiplex PCR based

technologies, each target base is

covered by only one amplicon

(same start and stop sites)

If a variant occurs – it can be

checked by multiple amplicons with

HaloPlex

If a variant occurs, it is hard to

know if it is a real mutations and

not a PCR artifact

DNA variant DNA variant

If an unknown mutation appears in

a restriction site, it may affect one

or two fragments but all others will

be present

If an unknown mutation appears in

a primer site it causes a complete

dropout in the target region

What makes HaloPlex better than other PCR methods?

Research Use Only


Complete clinical research solution

Design your

gene panel

using

SureDesign

Prepare

samples with

HaloPlex Kit

Sequence on

NGS

sequencer

~10 mins <1 day <1 day

1 2 3 4

Mutation

Analysis and

report on

SureCall

~1 hour

Research Use Only



Agenda – HaloPlex


How it Works

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HaloPlex Disease Research Panels A new family of premium performance gene panels developed with the help of

clinical research thought leaders.

Available in Catalog or Pre-Designed formats

Compatible with Illumina and Ion Torrent platforms

Developed in collaboration with Clinical research thought

leaders, for key disease research areas;

High Performance, already tested;

Allow comprehensive analysis of several genes at the same

time;

Save money and time, compared to Sanger Sequencing single

gene assays;

Research Use Only


Benefits of HaloPlex disease research panels

1. Confidence in Genetic Content

– Our Panels have been developed

with key thought leaders in the

Clinical Research field

– Why is that important?

Best set of gene targets selected for

each disease

No need to design

Pre-tested

Prof. Paul Waring

Dr. Berivan Baskin

ICCG (ISCA) Consortium

leaders

Research Use Only


Anyone can do it!

• Library-free, <1 day protocol

• Automatable protocol

• All you need is the kit!

• SureCall for intuitive variant

calling

Benefits of HaloPlex disease research panels

2. Ease of use from sample to analysis

Research Use Only


Positioning HaloPlex Disease Research Panels

Multiple amplicon tiling of targets provides:

• Fewer PCR artifacts

• Fewer lost targets

• Reliable mutation/variant calling

3. Results you can trust

Research Use Only


HaloPlex disease research panels

Two Panels available as catalog products:

Catalog panels are available off the shelf, for fast delivery

Cancer

(already launched)

“A 47 gene panel

targeting COSMIC

mutations”

NEW Cardiomyopathy

“A 34 gene panel for analysis

of inherited forms of

cardiomyopathies”

Research Use Only


HaloPlex Cancer Research Panel

http://www.sanger.ac.uk/genetics/CGP/cosmic

Catalog kit

10Kb design

47 genes targeting COSMIC mutations

o Designed with relevant cancer targets

Designed for challenging, degraded samples

o Includes shorter fragments

o Design with both polarities

Analyze data in SureCall




Research Use Only


HaloPlex Cardiomyopathy Research Panel

Content: 34 genes known to be associated with:

Hypertrophic cardiomyopathy,

Dilated cardiomyopathy,

Arrythmogenic right ventricular cardiomyopathy

Developed in

collaboration with

Dr. Berivan Baskin

“Save time and money by analyzing all 34 genes in the same assay, and being able to

evaluate many different forms of the disease in a single panel”

Designed specifically for inherited forms

of cardiomyopathy.

Cardiomyopathy Research Panel – Gene Content

Research Use Only



Five Panels available as PRE-DESIGNED products:

Pre-Designed panels are available as DESIGN IDs in SureDesign. Each design

ID can be ordered as a CUSTOM product

ICCG (ISCA)

Connective Tissue

Disorder

X Chromosome

Arrhythmia

Noonan

Syndrome

Research Use Only



Any panel can be CUSTOMIZED in SureDesign

Any Panel

(Catalog or Pre-

Designed)

Custom Targets

The Best

research panel

for your lab!

Research Use Only



CATALOG PANEL: Order off the shelf

PRE-DESIGNED PANEL: Choose the panel DESIGN ID

available in SureDesign and order as a CUSTOM kit

OR

Integrated

Workflow

Research Use Only


Reports 1-page mutation report

QC report

Make your own report

Reports

Research Use Only



0%

20%

40%

60%

80%

100%

Enrichment Performance – HaloPlex Catalog Panels

HaloPlex Cancer

HaloPlex

Cardiomyopathy

Premium Performance Results you

can trust!

Research Use Only



0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

100%

HaloPlex Pre-Designed Panels

HaloPlex Arrhythmia

HaloPlex Noonan

Syndrome

HaloPlex Connective

Tissue Disorder

HaloPlex ICCG

HaloPlex X Chromosome

Premium Performance Results you

can trust!

Research Use Only



Agenda – HaloPlex


How it Works

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Research Use Only


Thank You!

haloplex disease research panels - agilent · 2015-09-04 · sensitive variant detection . research...

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