eseminario introducción a captura de secuencias con haloplex en … · 2018. 8. 8. · haloplex...

HaloPlex

For research use only 1

eSeminario

Introducción a captura

de secuencias con

Haloplex en NGS

Amparo Dávila Especialista Producto Genómica para Iberia 4 abril 2013

HaloPlex

For research use only

• Key Benefits 1

• How it Works 2

• Design a panel 3

• Recent Advancements 4

• Performance And Applications 5

• New Cancer Panel 6

• Data Analysis 7

Agenda – HaloPlex

HaloPlex


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


FOLLOW-UP CLINICAL

From Discovery to Clinical Research

All Solutions currently

available are based on

Multiplex PCR Needs:

Fast turnaround Time

Flexibility in panel size

Simple workflow

High Coverage and Specificity

High Throughput

HaloPlex


The advantages with Traditional PCR

High Coverage

High Specificity

Fast

Cost Effective

Simple

Well established

HaloPlex


The problems with Multiplex PCR

Primer Cross reactivity

Limited in # target regions

Long optimization

Hard to change

Dropouts

Artifacts

HaloPlex


HaloPlex – Simplicity of Next Generation PCR

SIMPLE

Library-Prep free Target

Enrichment in as little as 6

hours

All in a single tube!

Simple SureDesign

No capital equipment, simple

to implement

A whole lab in a single tube!

HaloPlex


HaloPlex – Simplicity of Next Generation PCR

FLEXIBLE

Any targets (sizes can be up

to 5Mb depending on platform)

96 indexed samples ready for

sequencing

Compatible with Ion Torrent

and Illumina Platforms! Any sequencer, Any targets

HaloPlex


HaloPlex – Performance of Next Generation PCR

PREMIUM

PERFORMANCE

Low DNA Input (200ng)

High Coverage

Uniform Amplification

High Specificity

Sensitive variant detection

HaloPlex


• Key Benefits 1

• How it Works 2





• Data Analysis 7

Agenda – HaloPlex

HaloPlex


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


HaloPlex Workflow

A Simple & Fast 4-step protocol

2013-04-04

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


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


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


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


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


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


HaloPlex Technology Overview

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

Genomic region Target exon

HaloPlex




A subset covering the target region is selected using HaloPlex probes

More than one different fragment cover the target region

HaloPlex




HaloPlex baits guide the circularization

HaloPlex



The selected subset is equipped with sequencing primer sequences by the

selector probes.


1) library bridge PCR primer

2)Sequencing primer+Sample

barcode

1 2 1

HaloPlex



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

(paired-end sequencing).


HaloPlex




0001111122322222333455422221111222111110000 Read coverage

HaloPlex



By tiling amplicons high target coverage is achieved


0001111122322222333455422221111222111110000

HaloPlex


Pileup in Integrative Genome Viewer

2013-04-04

Outstanding coverage of targeted bases; highly specific coverage

HaloPlex


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)

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!

HaloPlex


Streamlined Workflow

From Sample to Sequencing in a single day!

1 2 3

Design your gene

panel using the

HaloPlex Design

Wizard

Prepare samples in

less than 6 hours

with HaloPlex Kit

Sequence with a

desktop or high

throughput sequencer

~10 mins <6 hours <1 day

HaloPlex


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

HaloPlex


• Key Benefits 1

• How it Works 2





• Data Analysis 7

Agenda – HaloPlex

HaloPlex


SureDesign

Agilent’s Next Generation Custom Design Software

•3 Easy Steps to Your Targeted Design

•Steamlined Workflow and Sleek User Interface

•Collaboration tools enabling sharing of pre-designed panels

•Both SureSelect and HaloPlex NGS platforms to choose from

www.agilent.com/genomics/suredesign

HaloPlex


• Key Benefits 1

• How it Works 2





• Data Analysis 7

Agenda – HaloPlex

HaloPlex


NEW! HaloPlex for Ion Torrent

Featuring…

Paired-End

by Design!!!

Sequence Single-End…

Get Paired-End Coverage of your

targets!

HaloPlex


How Paired-End by

Design Works

For each target, 2 probes are created.

One probe is for the sense strand and

the other for the antisense strand

After the PCR step with universal

primers…

Amplicon products now have

different ends next to the sequencing

primer.

When generating SINGLE-END reads,

reads will be generated from BOTH

ENDS of the target

Sense Strand AntiSense Strand

Seq primer

Seq primer

DNA TARGET

HaloPlex


Why is HaloPlex Breaking Barriers?

Up to 5Mb capture

Protocol time

(days)

Capture Size

(Mb) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

1

2

3

4

Short protocol, small capture size

Long protocol, large capture size

For the first time ever, a Target

Enrichment technology can at the

same time be FAST and allow

LARGE CAPTURE SIZE

Multiplex

PCR

Hybrid Capture

HaloPlex


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


HaloPlex Compatibilituy with FFPE Samples

Cancer Panel developed by OICR

19 Genes, ~60Kb size

• High Coverage

• Consistent Results

• FFPE Samples Compatibility

• Great variant calling sensitivity

POSTER FILE AVAILABLE – PLEASE

CONTACT US

HaloPlex


• Key Benefits 1

• How it Works 2





• Data Analysis 7

Agenda – HaloPlex

HaloPlex


HaloPlex performs extremely well at larger sizes!

38

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 does

each design amplifies in the same tube???

HaloPlex


Taking the Technology to the next level

Low DNA Input – down to 50ng with same performance

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% reads ontarget

Cov 1x Cov 10x Cov 20x Cov 100x

Panel - 400kb (1,954 targets)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

% reads ontarget

Cov 1x Cov 10x Cov 20x Cov 100x

Panel - 340kb (3,078 targets)

800ng

400ng

200ng

100ng

50ng

0%

20%

40%

60%

80%

100%

800ng 400ng 200ng 100ng 50ng

HET Concordance (HapMap)

400kb 340kb

0%

20%

40%

60%

80%

100%

800ng 400ng 200ng 100ng 50ng

HET Sensitivity (HapMap)

400kb 340kb

800 400 200 100 50

0.0

0.2

0.4

0.6

0.8

1.0

Agilent (82−1)

Sample Input (ng)

AB

800 400 200 100 50

0.0

0.2

0.4

0.6

0.8

1.0

Larry (45−1)

Sample Input (ng)

AB

0.0 0.2 0.4 0.6 0.8 1.0

02

46

81

01

2

AB

De

nsity

Sample Input

800 ng400 ng200 ng100 ng50 ng

0.0 0.2 0.4 0.6 0.8 1.0

02

46

81

01

2

AB

De

nsity

Sample Input

800 ng400 ng200 ng100 ng50 ng

Allelic Balance – 400kb Panel Allelic Balance – 340kb Panel

HaloPlex


• Design Input:

– List of 78 genes

• Design Output:

– Total design coverage 95.5%

of 197391 bp (CCDS)

41

Sten Linnarsson

Dept of Medical Biochemistry and

Biophysics

Molecular Neurobiology

HaloPlex Cancer Panel Pilot Study – Compatibility with 100 Cells+WGA

HaloPlex

For research use only 4 April 2013 Una Kjällquist 42

6% 2%

95% 98%

0%

20%

40%

60%

80%

100%

Targets Bases

0 reads

1+ reads

3% 1%

97% 99%

0%

20%

40%

60%

80%

100%

Targets Bases

0 reads

1+ reads

1M cells, unamplified 100 cells, GenomiPhi

HaloPlex Cancer Panel Little Loss due to WGA

HaloPlex


HaloPlex Cancer Panel Analysis of Cell populations in Leukemia

Detection of rare alleles

HaloPlex


Application: GWAS follow up

Hypertension

Dr. Anders Dahm Department of Hematology Ullevaal University Hospital Oslo, Norway

Four linkage regions were identified in a GWAS

study of female hypertension patients.

Challenge: Target continuous regions in a large

patient cohort

HaloPlex


High Uniform Coverage among all target regions

Design success in silico

Coverage by design:

150 kbp (93,2%)

Repeated bp in ROI: 38%

Assay success after sequencing

97.4% of sequencing reads on amplicons

96.6% or reads on target

>90% of target region sequenced

Targeted region: 4 regions of in total 160 kbp

• Comprehensive Coverage • Uniform Coverage • High Coverage

HaloPlex

For research use only 2013-04-04

Cystic Fibrosis CFTR gene

4443 bp, 27 exons

Covered by design: 100%

Covered by sequencing: 99.8%

Specificity (on target reads): 99.97%

All variants detected

Berivan Baskin, MD, PhD Clinical Molecular Geneticist Uppsala University

Peter Ray , PhD, FCCMC, FACMG Head, Molecular Genetics, The Hospital for sick children

Application – Clinical Research Panel

Cystic Fibrosis

HaloPlex


Uniform Coverage among all target exons

Example of enrichment of all exons within CFTR gene, for 5 different samples:

• Comprehensive Coverage

• Uniform Coverage

• High Coverage • Enrichment of

exons of any size

HaloPlex


Detection of all types of genetic variants

Effective detection of 4bp indel

after enrichment with HaloPlex:

• Top: heterozygote sample

• Bottom: homozygote sample

Effective detection of single

nucleotide variant (C/T) after

enrichment with HaloPlex:

HaloPlex


• Key Benefits 1

• How it Works 2





• Data Analysis 7

Agenda – HaloPlex

HaloPlex


HaloPlex Cancer Research Panel

Catalog kit

10Kb design

47 genes targeting COSMIC mutations o Designed with relevant cancer targets

Designed for FFPE compatibility o Includes shorter fragments

o Design with both polarities

Analyze data in SureCall

Gene List

ABL1 JAK2

AKT1 JAK3

ALK KIT

AR KRAS

ATM MAP2K1

BRAF MAP2K4

CDKN2A MET

CSF1R NOTCH1

CTNNB1 NPM1

EGFR NRAS

ERBB2 PDGFRA

ERBB4 PIK3CA

FANCA PIK3R1

FANCC PTEN

FANCF RET

FANCG RUNX1

FGFR1 SMAD4

FGFR2 SMO

FGFR3 SRC

FLT3 STK11

HRAS TP53

IDH1 VHL

IDH2 WT1

MAP2K2 http://www.sanger.ac.uk/genetics/CGP/cosmic

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



HaloPlex


HaloPlex Cancer Research Panel

HaloPlex


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


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


• Key Benefits 1

• How it Works 2





• Data Analysis 7

Agenda – HaloPlex

Introduction to SureCall

• Agilent SureCall software was developed

for clinical researchers looking for a fast, accurate and

easy-to-use analysis tool for HaloPlex panels

• SureCall reduces the time to results from days to hours by

providing an end-to-end data analysis solution: from

alignment to categorization of mutations

• Agilent SureCall is free-of-charge for HaloPlex users

For Research Use Only. Not Approved for

Use in Diagnostic Procedures

Agilent SureCall Software Resolves data analysis bottleneck

An easy-to-use software for end-to-end data analysis from

alignment to categorization of mutations

• Easy to Implement

• Easy to Use with a

Streamlined Workflow

• Faster Time to Results

• Cost-Effective



SureCall makes NGS data

analysis easier, faster,

cheaper

www.agilent.com/genomics/surecall

Steps included in SureCall

Report generation

Categorization Table output Visualization Links to external databases

Post alignment processing

Identify mutations Mutation impact analysis QC reports

Read alignment

Adapter removal BAM file generation

Sequence generation

De-multiplexing Base caller, quality assessment

Sample

DNA isolation Target enrichment



Adapted from: Frederick National Laboratory for Cancer Research

Simple 3-step workflow-based data analysis



1. Import Samples

2. Describe Samples

3. Run Analysis

Sample review - Triage View



genome

viewer

mutation

table

What you can do from the Mutation Table

1. Suppress mutations

2. Add notes

3. Change categorization

4. Link out to external databases: OMIM, GeneCard, dbVar, NCBI, Ensembl,

and Uniprot



1

2 3 4

Genome Viewer



Go to next/

previous

variant

Zoom in/out Disable/enable

text in grey boxes

Allele fraction

+ genotype

Variants from

VCF

Coverage

from BAM

Regions and

amplicons

from BED

80 bp around variant,

color-coded by category

Links to external databases available from the

Mutation Table



Right click on column

in SureCall Database

ID dbSNP

Gene Name OMIM

GeneCard

Position dbVar

Transcript NCBI

Transcript ID Ensembl

Uniprot Uniprot

All changes that are made are tracked in the

Audit Trail



Data Search functionality



Find other samples containing the same

mutation(s) stored in the system

Choose VCF file or folder of VCF files to add to

the VCF search index (for example a file which

contains known benign variants)

VCF = Variant Call Format = a specification for

storing gene sequence variations

Reports



1-page

mutation

report

QC

report

Make your

own

report

HaloPlex


A simple workflow providing superior data quality

HaloPlex Next Gen PCR Summary

Single tube protocol, ideal for desktop sequencing

Customize gene panels easily using online Design Wizard

Superior coverage enabling comprehensive mutation detection

HaloPlex


Gracias por asisitir

Para más información: [email protected]

Promoción especial: 1304_WEBNGS_SP

Page 73

SureCycler 8800

Kit de 96 HaloPlex

Panel Cancer 25% OFF

mailto:[email protected]

HaloPlex


Thank You!

eseminario introducción a captura de secuencias con haloplex en … · 2018. 8. 8. · haloplex...

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