HaloPlex

For research use only 1

eSeminar:

Diseño de un nuevo test

NGS para el análisis

rutinario de cáncer

hereditario

5 marzo 2014

Ponente Invitado

Dr. Alberto Acedo

Co-fundador y Dr. Técnico

AC-Gen Reading Life S.L.

Amparo Dávila

Especialista Producto Genómica para Iberia

Agilent Technologies

HaloPlex

For research use only

DISCOVERY FOLLOW-UP CLINICAL

The NGS Applications Workflow

Whole Exome

Whole Genome

GWAS

Follow-up Exome

Follow-Up WGS

Follow-up GWAS

Clinical Research

Panels

HaloPlex

For research use only

The advantages with Traditional PCR

High Coverage

High Specificity

Fast

Cost Effective

Simple

Well established

HaloPlex

For research use only

The problems with Multiplex PCR

Primer Cross reactivity

Limited in # target regions

Long optimization

Hard to change

Dropouts

Artifacts

HaloPlex

For research use only

HaloPlex Workflow

A Simple & Fast 4-step protocol

2014-03-05

Sample is fragmented using restriction

enzymes

2. Hybridize probes

3. Purify and ligate

targets

4. Amplify targeted

fragments

Probe library is added and hybridized

to the targeted fragments making them

form a Halo shape.

Probe/Fragment hybrids are retrieved

with magnetic streptavidin beads. The

circular molecules are then closed by

ligation

Only circular DNA targets are

amplified. Sample barcodes are

introduced. Final product is ready for

sequencing

1. Digest DNA 1

2

3

4

HaloPlex

For research use only

Each DNA sample is fragmented in eight

double-digest reactions 1. Digest DNA

Define region of interest (ROI)

Genome digested in silico

Fragments chosen for enrichment

Design Wizard - In Silico digestion

and HaloPlex probe design

Molecular Concept

HaloPlex

For research use only

Each DNA sample is fragmented in eight

double-digest reactions 1. Digest DNA

Amplicon tiling improves coverage

by design

Amplicon 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

Molecular Concept

HaloPlex

For research use only

2. Hybridize probes

Fragments are mixed with custom

HaloPlex probes and a Primer Cassette.

Hybridization overnight.

Biotin

Same hybridization requirement as in

regular PCR – high specificity!

Both “primers” on the same oligo – no

cross-reactivity!

Sample barcode is integrated (96 barcodes

available)

Correctly hybridized fragments will form a

nicked dsDNA structure with the HaloPlex

probe

Hybridization guides circularization

Molecular Concept

Red parts – complementary to the targeted fragment

Grey part – contains sequencer specific

motifs and the sample barcode

HaloPlex

For research use only

3. Purify and ligate

targets

Probe/Fragment hybrids are retrieved

with magnetic streptavidin beads. The

circular molecules are then closed by a

DNA ligase.

Biotin

Level of enrichment is increased by

removal of non-targeted fragments

Only perfectly hybridized fragments

will be ligated

Purification and DNA Ligation

Molecular Concept

HaloPlex

For research use only

4. Amplify targeted

fragments

Only circular DNA targets are

amplified. Sample barcodes are

introduced. Final product is ready for

sequencing

1) Bridge PCR primer 2) Sequencing primer

PCR is primed from the Primer

Cassette

Tailed primers add bridge PCR

motifs

Thousands of amplicons – one

primer pair

Molecular Concept

1

2

HaloPlex

For research use only

HaloPlex Technology Overview

Eight different fragments cover each target position. Amplicons are tiled over the region.

Genomic region Target exon

HaloPlex

For research use only

HaloPlex Technology Overview

Genomic region Target exon

A subset covering the target region is selected using HaloPlex probes

More than one different fragment cover the target region

HaloPlex

For research use only

HaloPlex Technology Overview

Genomic region Target exon

HaloPlex baits guide the circularization

HaloPlex

For research use only

HaloPlex Technology Overview

The selected subset is equipped with sequencing primer sequences by the

selector probes.

Genomic region Target exon

1) library bridge PCR primer

2)Sequencing primer+Sample

barcode

1 2 1

HaloPlex

For research use only

HaloPlex Technology Overview

The sequencing primers are used to sequence in from the fragment ends

(paired-end sequencing).

Genomic region Target exon

HaloPlex

For research use only

HaloPlex Technology Overview

Genomic region Target exon

0001111122322222333455422221111222111110000 Read coverage

HaloPlex

For research use only

HaloPlex Technology Overview

By tiling amplicons high target coverage is achieved

Genomic region Target exon

0001111122322222333455422221111222111110000

HaloPlex

For research use only

HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance)

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)

What makes HaloPlex better than other PCR methods!

HaloPlex

For research use only

HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance)

TARGET TARGET

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

What makes HaloPlex better than other PCR methods!

HaloPlex

For research use only

HaloPlex Others (TruSeq, AmpliSeq, Fluidigm, RainDance)

TARGET TARGET

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!

HaloPlex

For research use only

Consistent Results among Different Design Sizes

Extremely high Performance

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Covered by

design

% Bases on

Target

% Bases

covered 1x

% Bases

covered 20x

% Bases

covered 100x

% Bases

covered at 10%

of mean

% Bases

covered at 20%

of mean

4.4Kb

21Kb

60Kb

120Kb

280Kb

396Kb

Don’t limit yourself: thousands of amplicons

25

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Coverage by

design

Specificity Coverage 1x Coverage at

10% of mean

1.5 Mb

3.2 Mb

5.0 Mb

¡73,328 amplicons!

¡100,550 amplicons!

¡193,775

amplicons!

How many amplicons are amplified in a single tube?

HaloPlex

For research use only

Publications

Page 26

HaloPlex

For research use only

SureDesign

Create your design in only 10 minutes

https://earray.chem.agilent.com/suredesign/

1. Input gene

ID/name/coordinate

2. Define regions of interest

(eg. Exons, UTRs, etc)

3. Click “Start Design”

4. Receive design report in

10 minutes

HaloPlex

For research use only

FFPE Samples Using HaloPlex

FFPE Challenges

• DNA quality issues (age, storage, fixation process)

• Sample degradation can impact sequencing quality (sensitivity, quality, allelic balance, etc)

HaloPlex with FFPE samples

• Validated protocol

• Adapted Design for FFPE

• Shorter probes

• Both DNA strands

• Quality Assesment protocol

HaloPlex

For research use only

Target Region Target Region

Normally fragments in the range of 100-400bp are used

For FFPE samples shorter fragments (50-400bp) are included

Standard design

Designed for FFPE compatibility

Exon Exon

FFPE design

HaloPlex

For research use only

One probe is targeting the sense and one the anti-sense strand of the target DNA

Sense Strand

AntiSense Strand

DNA TARGET FRAGMENT

Probes are designed for both target fragment polarities • Improves capture efficiency • Eliminate strand specific FFPE artifacts

Designed for FFPE compatibility

HaloPlex

For research use only

Thank You!

Muchas gracias por su atención

Dirección de contacto: amparo_davila@agilent.com