agilent target enrichment solutions for ngs · haloplex for research use only sureselectxt2 -...

HaloPlex

For research use only

Agilent Target Enrichment Solutions for NGS

Winfried van Eyndhoven Genomics FAS, BeNelux/Iberia

HaloPlex


Agenda

4 Whole Methylome Sequencing

NGS Target Enrichment Solution 1

5 Custom SureSelect & HaloPlex Panels

Targeted RNA Sequencing 6

7 Automation

Whole Exome Sequencing V4 & V4+UTR 3

2 QC workflow

HaloPlex


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

HaloPlex


Library Preparation

0.5µg

Hybridization / Capture 24 hours

Baits:

- cRNA probes

- Long (120bp)

- Biotin labeled

- User-defined (eArray)

- SurePrint synthesis

Bead Separation

Wash / Elution / Amp

Basic SureSelect

Target Enrichment Workflow

HaloPlex


Key Benefits – Agilent SureSelect

PROVEN Technology

COMPLETE solution

FLEXIBLE solution

High PERFORMANCE

HaloPlex


SureSelect Portfolio

What do

you want to

capture?

DNA RNA

Human Human

Non

Human Non

Human

Animal

Models Bovine

All Exon

*

Zebrafish

All Exon

*

Dog

All Exon

*

Mouse

All Exon

Custom

Capture

Custom

Capture

Kinome

RNA

X

Chrom

Whole

Exome

All Exon

V4 *

(51Mb)

All Exon

50Mb

Custom

Capture

Kinome

DNA

Custom

Capture

All Exon

V5*

(71Mb) * NEW

HaloPlex


SureSelect All Exon – also non-Human Species

All Exon Bovine:

64Mb design. The library is based on University of Maryland build 3.1 against NCBI

exons as well as predicted exons, UTRs, and miRNAs. Worked with USDA on

design.

All Exon Canine:

54Mb design. UCSC tracks from CanFam2 for ensembl as well as Refseq, human

protein alignments, and spliced ESTs that lie outside of ensembl.

All Exon Zebrafish:

96Mb design. Zv9 library is in Emsembl and is annotated with gene and exon

coordinates. Worked with Sanger on design.

All Exon Mouse:

49.6Mb design. Exon definition derived from Ensembl + RefSeq, designed against

mm9 reference from UCSC

Note: All Exon Bovine, Canine and Zebrafish are being provided in Early Access Format

HaloPlex


SureSelectXT – Complete Workflow Solution

SureSelectXT/XT2

Target Enrich. Library

Prep

Herculase

enzyme

HaloPlex


APPENDIX – SURESELECTXT2

SureSelectXT2

A more Efficient Workflow:

Pre-Capture Pooling

Streamlined workflow

Pool More, Pippete Less

HaloPlex


SureSelectXT2 - Pre-Capture Pooling

Pre-capture pooling of:

8 Samples for All Exon Kits

16 Samples for Custom Kits

MasterMixed Reagents:

Streamlined workflow – 3x less tubes

Less Pipetting, no gel size-selection

96 indexes:

Scalable

Availability:

Illumina NGS Platforms

Low Starting DNA input

1ug DNA only

Individual

Library Preps

Sample Pooling

Pooled Capture

Multiple Capture

Pooling

Prep for

Sequencing

Pre-Capture Pooling

HaloPlex



PROVEN Technology

COMPLETE solution

FLEXIBLE solution

High PERFORMANCE

HaloPlex


SureSelect kits are platform agnostic!

Illumina GAIIx

HiSeq 2000 SOLiD 4

5500

454 FLX

GS Junior

Pacific

Biosciences*

Ion Torrent

PGM

MiSeq*

* -coming soon!

From High throughput to desktop sequencers

NEW!

NEW!

SureSelect and 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


SureSelect Custom Designs

For all sizes and needs

1Kb-

500kb

10Mb-

20Mb

5Mb-

10Mb

500kb-

5Mb

HaloPlex


Different paths to create your custom design

eArray Web Portal -you

create the design

Design Services We create the

design for you

Your Custom

SureSelect Kit A bait library to capture

your regions of interest

Genomic

Coordinates Regions you want

to capture

NEW!

HaloPlex



PROVEN Technology

COMPLETE solution

FLEXIBLE solution

High PERFORMANCE

HaloPlex


SureSelect: Enabling Scientific Discovery

… and many more to

come

>200 Publications…

HaloPlex


Unlocking the Cancer Genome

Breast and Ovarian

Leukemia

Renal

Pancreatic

Colorectal

Melanoma

Hepatocellular

HaloPlex



PROVEN Technology

COMPLETE solution

FLEXIBLE solution

High PERFORMANCE

HaloPlex


Performance of a Target Enrichment technology

• Mappability

•% on-target

• Uniformity

• Coverage (% bases at 20x)

MOST IMPORTANT

• SNPs!

• Indels!

• CNVs!

• Allelic Balance

Longer probes will

perform better!

Overall

Performance Mapping

Metrics Sensitivity

Metrics

Operational

Excellence

• Automation?

• # indexes

• Compatibility

• Protocol

Don’t forget to look at the whole picture!

HaloPlex


SureSelect Baits

Better sensitivity

Feature Benefits

Ultra-Long RNA Baits

Only 24hr hybridization compared to 72hrs for

competitor

Capture small and large genetic variations:

SNP, InDels, CNV, fusions

Low # PCR cycles, less artifacts

Better Allelic Balance - Better Uniformity

Low input DNA needed

Binding strength

RNA:DNA DNA:DNA >

HaloPlex


Really Long RNA Baits Tolerate Mismatches (even long ones)

Longer baits = Better Sensitivity

Allelic Balance

post-capture

Allele 1 Allele 2 - SNP

0.5 0.5

120-mer

bait

Smaller Probes will poorly hybridize!

Allele 1 Allele 2 – 25bp deletion

0.5 0.5



25bp deletion

7bp deletion

10bp deletion

11bp deletion

SureSelect – Proven high sensitivity Capture not only SNPs and Indels, but also CNVs!

Large deletions and duplications

HaloPlex


Agenda





7 Automation


2 QC workflow



Get the best data and know it

PCR: Agilent Surecycler

Automation:

Agilent NGS Automation

qPCR: Agilent Mx3005P

QC: Agilent Bioanalyzer

QC: Agilent Tapestation

HaloPlex


Quality Control steps in SureSelect workflow

Shear Genomic

DNA

Repair Ends

3’-dA Addition

Adapter

Ligation

PCR

Prepped

Library

SureSelect

Oligo Capture

Library Library

Hybridization

Magnetic

Bead Capture

PCR

Sequencer

SureSelectXT

Library Prep

SureSelect

Target Enrichment

Automated

Bead Capture

QC

QC

QC

HaloPlex


Typical vs. Atypical Post-Shearing Profile: Bioanalyzer DNA 1000 Assay

Typical

• Sample volume • Too much DNA • Wrong buffer • Bubbles

Atypical

HaloPlex


Agenda





7 Automation


2 QC workflow

HaloPlex


Most Updated Content

Better Performance

Complete Workflow

Human All Exon V4 and V4+UTR kits

Discover More, Faster!

NEW!

HaloPlex


30

SureSelect All Exon V4 and V4+UTR Choose the design that is right for your research

Coding

Exons

Human

All Exon V4

(51Mb)

Sequence 4Gb

Coding

Exons +

UTRs

Human

All Exon

V4+UTR

(71Mb)

Sequence 6Gb

HaloPlex


SureSelect All Exon V4 and V4+UTR Most updated content; best performance, complete workflow

Improved design algorithm: captures difficult regions

better

Designed against the most updated databases

Coding content only (V4) or with UTRs (V4+UTR’s)

Improved

Design

Better Uniformity and % Target

Less Sequencing: 4Gb for V4 / 6Gb for V4+UTR

Improved Sample Throughput per Run

Better

Performance

Only 1x24hr Hyb – Achieve your results faster!

Complete Workflow with SureSelectXT

Better

Workflow

HaloPlex


32

SureSelect All Exon V4 and V4+UTR Better Performance, achieve results faster with 20

less sequencing

0,00%

10,00%

20,00%

30,00%

40,00%

50,00%

60,00%

70,00%

80,00%

90,00%

100,00%

% Reads On-

Target

% Reads On-

Target ±

100bp

% bases >1x

cov.

% bases

>10x cov.

% bases

>20x cov.

50Mb (5Gb)

V4 - 51Mb (4Gb)

V4+UTR - 71Mb (6Gb)

Improved

% on-target

Improved Coverage, with less sequencing

HaloPlex


33

SureSelect All Exon V4 and V4+UTR Improved Uniformity – Less Sequencing Needed

0

1

2

3

4

5

6

7

8

9

10

0

15

30

45

60

75

90

105

120

135

150

165

180

195

210

225

240

255

270

285

300

New SureSelect All Exon V4/V4+UTR

More Uniform Coverage – Less Sequencing Needed

Higher Sample throughput, Faster time to Results!

All Exon 50Mb

HaloPlex


Agenda





7 Automation


2 QC workflow

HaloPlex


• Hypomethylation

Under methylated regions

Can lead to genomic instability – Changes in the chromosome structure

• Hypermethylation

Over methylated regions

Leads to transcriptional repression of genes

In tumor suppressor gene (TSG i.e. p16 and Sox17) – Leads to cancer

DNA Methylation: Role in Cancer

Robertson K. Nature Genetics, 2005

HaloPlex


Methylation Effects on Other Diseases

Fragile X Syndrome

Rett Syndrome

Autistic Patients and their Parents

Schizophrenia and Bipolar Disorder

Bipolar Disease

Dementia

Alcoholism

Suicide

Alzheimer’s Disease

Imprinting disorders

Prader-Willi syndrome

Angelman syndrome

Beckwith-Wiedemann syndrome

• Epigenetic modifications in disease extends beyond cancer

• Many epigenetic changes are associated in mental diseases

Epigenetics Market Trend 2011, SelectBioSciences

HaloPlex


Genomic locations of CpG dinucleotides

CpG islands

• High frequency of CpG dinucleotides

– > 500bp & GC content >55% & observed/expected CpG ratio > 0.65

• In or near about 40% of promoters of mammalian genes

Promoters

• 75% transcription start site have CpG-rich regions

• 88% active promoters are associated with CpG-rich sequences

HaloPlex


Common Targets for DNA Methylation

Differentially methylated regions (DMR)

• CpG islands (e.g. 4~8 % tissue-specific differentially methylated regions or T-DMR)

• CpG island shores (~2kb away from islands, e.g. 76% of T-DMRs in shores)

• CpG island shelves (~4kb away from islands)

Irizarry RA et al. Nature Genetics 2009

HS3ST4 :

heparan sulfate D-

glucosaminyl 3-O-

sulfotransferase 4

HaloPlex


SureSelectXT Human Methyl-Seq

•Discovery Tool - Probes are not methylation

state dependent so you do not need to have

prior knowledge of the methylation states of

the regions that you want to target

•Comprehensive design - Not limited to CpG

Islands. Comprehensive targeting key

methylation regions: CpG Islands, Promoters

and DMRs

DESIGN CONTENT - 84 Mb

Design, 3.7M CpGs

CpG islands

Cancer, Tissue-specific DMRs

GENCODe promoters

DMRs or regulatory features in:

CpG Islands, shores and

shelves ±4kb

DNAseI hypersensitive sites

Refseq Genes

Ensembl Regulatory Features

The First Comprehensive Methylation Discovery System

HaloPlex


DNA

Shearing, End

Repair & ‘A’

addition

SureSelect

Hyb (24hr)

SureSelect Methyl-Seq Protocol

mAdapter

ligation

Bisulfite

treatment

me me me me

me me me me

A A

Library

Quant and

PCR

Sequence

Bisulfite treatment is performed after

hybridization to maximize sample complexity

No PCR before Bisulfite treatment

to preserve the Methylation state

HaloPlex


Whole genome data vs. SureSelect Methyl-Seq

R = 0.927

Excellent concordance with whole genome bisulfite sequencing data.

SureSelect vs. Whole-genome bisulfite sequencing

Agilent SureSelect™ Platform

Enabling Products for the Next-

Generation Sequencing Workflow

0

10

20

30

40

50

60

70

80

90

100

1X 5X 10X 15X 20X 25X 30X

Coverage of at least...

Coverage vs. % CpGs covered in targeted regions (~3.7 million CpGs)

Per

cen

t C

pG

s co

vere

d

SureSelect Methyl-Seq

Whole-genome sequencing

10Gb input (raw)

4Gb (filtered)

180Gb input (raw)

91 Gb (filtered)

HaloPlex


Highly sensitive and accurate methylation detection after SureSelect target enrichment demonstrated DNA methylation differences between HCT116 human colon cancer cells and its methyltransferase double-knockout (DNMT1-/- and DNMT3b-/-).

Proof of concept (HCT116 vs.

Methyltransferase DKO)

HaloPlex


Proof of concept (TIMP3: Metalloproteinase inhibitor 3)

HaloPlex


SureSelectXT Human Methyl-Seq

The First Comprehensive Methylation Discovery System

•Covers more individual CpGs compared to Methylation

microarrays

•Increases throughput, reduces costs compared to Whole

Genome Bisulfite Sequencing

•Reveals methylated regions undetected by RRBS and Me-Dip

HaloPlex


Methylation RNA DNA

SureSelect: Complete “Omics” Solution

DNA: Genetic variation

RNA: Gene Expression

Methylation: Effects on Gene Expression

HaloPlex


Agenda





7 Automation


2 QC workflow

HaloPlex


Custom content from <200Kb up to 34Mb Capture only what

you want

>1,000 samples/run with 96 indexes! Higher sample

throughput per run

Up to 10 times less expensive than exome sequencing – save on enrichment and on sequencing

Cost effective

Faster time from sample to results

Results in hours with desktop sequencers

Easier Data Analysis

Why use SureSelect Custom panels?

HaloPlex


All 54 known

deafness genes

The Power of Smaller Panels

21 breast and ovarian

cancer genes

Detects fusions for 90

human tyrosine

kinases Exons – Chrom 7

437 Genes related to

Mendelian Disorders

3.19Mb

Custom Design

HaloPlex


ANNOUNCING HaloPlex Next Gen PCR Target Enrichment System

HaloPlex


HaloPlex – Next Generation of PCR

Macro scale PCR

Physical separation

Hundreds of parallel reactions

Micro scale PCR

Physical separation

Thousands of parallel reactions

Nano scale PCR

Molecular separation

Millions of parallel amplifications

HaloPlex


HaloPlex – Simplicity of Next Generation PCR

SIMPLE

Library-Prep free Target

Enrichment in less than a day

All in a single tube!

96 indexed samples ready for

sequencing

Simple Design Wizard

Any Sequencer, Any Targets

(up to 500kb) A whole lab in a single tube!

HaloPlex


HaloPlex – Performance of Next Generation PCR

PREMIUM

PERFORMANCE

Low DNA Input

High Coverage

Uniform Amplification

High Specificity

Sensitive variant detection

*Ion Torrent compatibility coming soon

HaloPlex


HaloPlex Design Wizard

Create your design in only 10 minutes

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

Gene

Panel 1

HaloPlex


HaloPlex Workflow

A Simple & Fast 4-step protocol

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


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


Agenda





7 Automation


2 QC workflow

HaloPlex


The caveats of analyzing whole

transcriptomes through NGS RNA-Seq

Expensive:

o The expenditure necessary to obtain sufficient sequencing coverage for several research

and potential clinical applications is still prohibitive

o Calling genotypes with high confidence requires coverage levels of at least fivefold to 20-

fold, that can only be obtained with several million reads

Difficult to scale-up

o Limited sample throughput with current NGS systems and sequencing requirements

Data analysis

o Still a challenge

Overall coverage per region may not be high

enough

HaloPlex


RNA Target Enrichment

Enrich your sample for a subset of transcripts of interest

to…

Get deeper coverage per transcript

Discover splice junctions, fusions, SNPs, allelic expression

Quantify gene expression with sensitivity similar to qPCR

Have more cost-effective and faster experiments

Easier data analysis

HaloPlex


SureSelect RNA Target Enrichment Protocol

Start with <1ug of total RNA or mRNA*

Similar process to DNA Target enrichment,

except working with a

cDNA NGS library

Complete protocol takes ~ 3-4 days

Protocols available for Illumina and SOLiD,

for individual or multiplexed samples

*see protocol for details and recommendations

HaloPlex


RNA Target Enrichment in Cancer

RNA Target Enrichment of 467 Cancer Genes (~all Tyrosine

Kinases + Genes from Cancer Gene Census);

Overall >800 target transcripts

Sequencing Libraries: K-562 chronic myeloid leukemia (CML) cell

line cDNA libraries: with and without target enrichment

HaloPlex


What only RNA enrichment Reveals!!!

Feature Without RNA Target

Enrichment

With RNA Target

Enrichment

Avg. Coverage 14.4x 606x

Fold-Enrichment N/A 42x

Regions with 20x cov. 13% 63%

Common SNPs 76 257

Novel Variants 4 16

Splice-Junctions 2958 4720

Alternatively spliced-

genes 52 177

Gene Fusions 2 6

HaloPlex


Performance of SureSelect RNA Target Enrichment

Enri

che

d li

bra

ry

Unenriched library

Gene Expression Levels

R2=0.885

High correlation to unenriched RNA library = enrichment process doesn’t create any bias – reliable gene expression results!

HaloPlex


Agenda





7 Automation


2 QC workflow

HaloPlex


Automation of Agilent SureSelectXT for NGS

Increase Throughput and

Reproducibility

Improve Efficiency

Process up to 192 Samples

Per Week

Validated and ready to run

SureSelectXT

NEW! SureSelectXT AUTO

kits in 96, 480 or 960

formats

HaloPlex


Automation of Next-Gen Sequencing Using SureSelect

Shear Genomic

DNA

Repair Ends

3’-dA Addition

Adapter

Ligation

PCR Enrichment

Prepped

Library

SureSelect

Oligo Capture

Library Library

Hybridization

Magnetic

Bead Capture

PCR

QA

Sequencer

SureSelectXT

Library Prep

SureSelect

Target Enrichment

Automated

Bead Capture

Automated

Purification

Automated

Purification

Automated

Purification

Automated

Purification

Automated

Purification

Automated

Purification

HaloPlex


High Performance and Reproducibility

High Sensitivity and Specificity

HaloPlex


Comparison of Timing and Throughput of

Manual vs. Bravo SureSelect Sample Processing

Up to 10x

throughput per

week vs. manual

processing!

HaloPlex


Agenda





7 Automation


2 QC workflow

HaloPlex


Thank You!

agilent target enrichment solutions for ngs · haloplex for research use only sureselectxt2 -...

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