revolutionize your clinical research - agilent genomics at agbt 2013...haloplex for research use...

HaloPlex

For research use only 1

Revolutionize Your

Clinical Research

Olle Ericsson, PhD, Marketing Director DNA sequencing

HaloPlex

For research use only

Complete Clinical Research Solution

• SureDesign – High confidence custom designs

• Haloplex – Fast, easy and high performance workflow

• SureCall – From raw-data to mutation report

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


SureDesign v1.1

HaloPlex advanced design wizard

1. Custom designs based on HaloPlex Exome probes

2. FFPE optimized designs

3. Improved coverage of pseudogenes

4. Improved design visualization

1. Direct link to UCSC browser to review target regions and individual amplicons

2. Preview sequencing data from exome probes in SureCall

5

www.agilent.com/genomics/suredesign

HaloPlex


Exon 1 Exon 2

All HaloPlex exome probes overlapping with defined targets will be selected Exome probes do not exist for non-coding regions no probes will be selected for these regions

SureDesign v1.1

Use amplicons from HaloPlex Exome

1. Define target regions

2. Use all exome probes for coding regions

3. Combine with custom design for non coding regions

HaloPlex


Review Your Designs in Progress

Direct links to UCSC Genome Browser

• Link out to tiling results from Design Summary

• Review amplicon structure and coverage in high-resolution

7

HaloPlex


Exome probes - Beta-program Visualize sequencing data and QC performance report in SureCall

8

HaloPlex


SureDesign v1.1 - Optimize for FFPE

9

For FFPE optimized designs 1. The design will include shorter fragments (50-400bp) 2. Both target polarities will be selected

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


FFPE design optimization results

12

Standard FFPE optimized

Coriell DNA

FFPE DNA

Fraction of target region covered at 10% of mean

HaloPlex


Pseudogene designs

- Probe Stringency alteration

13

1. Maximize Specificity - Requires probe with only one unique hit in the genome 2. Balanced – Allows selection of probes with 2 hits in the genome. 3. Maximize Coverage – Allows up to 5 hits in the genome.

HaloPlex


Pseudogene Designs

- Probe Stringency Alteration

14

Balanced probe selection – 100% coverage of MYH6

Standard probe selection – 96.6% coverage of MYH6

HaloPlex






HaloPlex


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

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, no flexibility

Dropouts

Artifacts

HaloPlex


HaloPlex Target Enrichment

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 Target Enrichment System

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


HaloPlex Other PCR based solutions

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

Amplicon redundancy

HaloPlex


High Coverage and Uniformity Across Target Sizes

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

Exome average – 90% of 37 Mbp covered 10X usign 4 Gbp of sequencing

HaloPlex






HaloPlex


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

HaloPlex


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

Not approved for use in diagnostic

Adapted from: Frederick National Laboratory for Cancer Research

HaloPlex


Simple 3-step workflow-based data analysis


1. Import Samples

2. Describe Samples

3. Run Analysis

HaloPlex


Sample review - Triage View


genome

viewer

mutation

table

HaloPlex


Reports


1-page mutation report

QC report

Make your own report

HaloPlex



• SureDesign

• Optimize for FFPE samples and pseudogenes

• Leverage 2.5 million all exon probes

• Haloplex

• Single day, easy workflow

• High target coverage and amplicon redundancy

• SureCall

• From raw-data to categorized mutation report

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

32

HaloPlex


HaloPlex Cancer Research Panel Coverage

33

HaloPlex


Yong Yi

Associate Marketing Director

Agilent Next Generation Sequencing

Solutions for Gene Regulation

HaloPlex


Complete Solutions for Gene Regulation Studies Looking at the complete picture

Gene Regulation

RNA • Novel Gene Discovery

• Gene Expression

DNA • Human Exome

Methylation • DNA Methylation

Investigating only one may prevent identification of a novel aberration which could be the real driving force behind a disease/phenotype of interest.

HaloPlex


SureSelect - Most Complete Workflow Solutions From Sample to Analysis

MX3000 SureCycler 8800

Agilent NGS

Automation

2100 Bioanalyzer 2200TapeStation

GeneSpring

NGS

HaloPlex


SureSelect RNA-Seq Whole Transcriptome and Targeted RNA-Seq

NGS Gene Regulation Solutions

HaloPlex


SureSelect Strand Specific RNA Library Prep

Better Performance and Highly Strand-Specificity

High Strand Specificity and Fast Workflow

Strand specific for deeper understanding of gene regulation

Exceptional RNA-Seq Performance with Low RNA Input

Greater uniformity across the entire transcriptome and higher

complexity

Complete Gene Regulation Solutions from Agilent

Targeted RNA-Seq, Methyl-Seq, GE arrays, qPCR, GeneSpring NGS

Automation for high throughput sites

HaloPlex



Strand-Specificity Increases Understanding of Gene Regulation

Identify Overlapping Transcripts

Discern transcripts nested in other genes

Enable Easier Analysis of Data

Increase alignable reads and more accurate annotation

Antisense Expression Can Play a Key Role

Discover antisense transcripts and measure antisense expression

HaloPlex



High Strand Specificity and Outstanding RNA-Seq Performance

– Strand-specificity provides greater understanding of gene regulation

– High uniformity and library complexity

*Top 1000 Expressed Transcripts. 50ng of Human UHRR samples Data normalized to 20 million reads. 2x100bp sequencing

95

96

97

98

99

100

Agilent - Replicate 1



300

320

340

360

380

400

420

440




Mean per Base Coverage Strand Specificity

HaloPlex


*200ng of Human UHRR samples Data normalized to 20 million reads. 2x100bp sequencing


Outstanding RNA-Seq Performance and High Strand Specificity

– Improved performance (Top 1000 Expressed Transcripts)

300

350

400

450

500

550

Agilent Strand-Specific TruSeq Stranded

Mean per Base Coverage

30% greater coverage compared to other strand specific kits

HaloPlex



Outstanding RNA-Seq Performance and High Strand Specificity

– Less 5’-3’ transcript bias

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0

4

8

12

16

20

24

28

32

36

40

44

48

52

56

60

64

68

72

76

80

84

88

92

96

10

0

No

rmal

ize

d c

ove

rage

Normalized Distance Along Transcript (5’-3’)

Agilent

TruSeq Stranded

HaloPlex



Excellent Correlation to Gene Expression Microarrays

– Ideal for follow up and companion studies

R=0.903 R=0.894

RN

ASe

q:

log2

fo

ld c

han

ge B

rain

/UH

RR

Microarray: log2 fold change MAQC_B/MAQC_A

200 ng 50 ng

Microarray: Human catalog 60K array.

Microarray: log2 fold change MAQC_B/MAQC_A

RN

ASe

q:

log2

fo

ld c

han

ge B

rain

/UH

RR

HaloPlex


SureSelect Targeted RNA-Seq

High Performance for Whole Transcriptome and Targeted RNA-Seq

– RNA Kinome kit - targets transcripts of over 600 kinase and kinase related genes

60

70

80

90

100

Breast-T Human RNA kinome

Breast-N Human RNA kinome

Per

cen

t

% on Target

Strand Specificity

*Total RNA input: 200 ng

HaloPlex


Demonstrate ability to capture gene fusions using targeted RNA-Seq

• Target set of 913 human kinase genes (~3.77Mb)

– target ABL1 transcripts

– exclude BCR baits

– look for BCR-ABL1 fusions

– compare whole transcriptome vs. targeted RNA Capture

BCR-ABL1 cDNAs

Fragmentation

ABL1-baits

BCR-ABL1 fusion genes

present in the

transcriptome

SureSelect baits

developed against BCR

transcripts - No baits designed to

BCR

SureSelect ABL1 baits

pull down both ABl1

fragments and BCR-

ABL1 fusions

Efficiency of SureSelect baits capture both BCR and BCR-ABL1 fusions

HaloPlex


SureSelect Targeted RNA-Seq – Discovery More

KCL-22-SR (cell line) Targeted Non-targeted

Fusions 72 Not reported

ABL specific fusions 45 0

Alternatively Spliced Variants 79 3

Non-Synonymous SNPs 156 19

Whole Transcriptome vs. Targeted RNA Capture

Comparable amount of filter passed reads ~40 Million reads

• 88% of captured cDNAs mapped to the target regions using SureSelect

• ~7% of cDNAs from whole transcriptome sample mapped to target regions

Increased depth enables researchers to Discover More!

> 45X enrichment

HaloPlex


SureSelect Human Methyl-Seq

NGS Gene Regulation Solutions

HaloPlex


Target Known Methyl Regions

9-18x more efficient with greater depth per region

Less than 3% of the genome is reported to vary

it’s methylation state

• Focus your research with SureSelect Methyl-Seq

Whole Genome

Bisulfite Sequencing SureSelect Human

Methyl-Seq

3-6 lanes for one sample

90-180Gb per sample

3 samples per lane

10Gb per sample

HaloPlex


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 The First Comprehensive Methylation Discovery System

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

Discovery Tool

• Probes independent of methylation state

• Determine methylation state of all

methyl sites in region

Comprehensive Design

• CpG Islands

• Promoter regions

• DMRs (Differentially Methylated

Regions)

HaloPlex


SureSelect vs. Whole-Genome Bisulfite Sequencing

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


Good Concordance between WGBS & SureSelect Methyl-Seq

R = 0.927

Excellent concordance with whole genome bisulfite sequencing data.

HaloPlex


Proteomics

Metabolomics

SureSelect QC

DNA-Seq

RNA-Seq

ChIP-Seq

Methyl-Seq

miRNA-Seq

Multi-Omic Analysis

Canonical Pathways

Computational Network Discovery

mRNA & miRNA expression arrays

Exon arrays

SNP arrays for GWAS & CNV

GeneSpring 12.5 Analysis Software

HaloPlex


MeDip-Seq & Reduced Representation BiSulfite Sequencing

(RRBS)

• Cannot target specific regions (i.e. DMRs in Shelf and Shore regions) • Biased towards methylated regions, Repeat sequences & CpG-rich sequences • Can miss under-methylated regions • Difficult to design since knowledge of methylation state for the target region is

needed

Limitations

HaloPlex


More Coverage of CpGs Compared to Infinium 450K Array

SureSelect Human Methyl-Seq

Infinium 450K

480,000 CpGs

3,700,000 CpGs

HaloPlex


SureSelect Human All Exon V5

HaloPlex


SureSelect Human All Exon V5 and V5+UTRs

– Updated to the latest genomic databases,

including CCDS, GENCODE, RefSeq,

TCGA

– Less sequencing than other exomes, only

4Gb for V5 and 6Gb for V5+UTRs

– Low overall costs, less sequencing and

greater sample throughput

– 16 hr (overnight) hybridization protocol

– Fastest turnaround time for projects

Ready for Sequencing the Next Day

The Best Performance

HaloPlex


The Best Performance Outstanding coverage with less sequencing

30%

40%

50%

60%

70%

80%

90%

100%

% Reads On-Target +/-100bp % bases >10x cov. % bases >20x cov.

SureSelect V5 (4Gb)

Nimblegen Exome (4Gb)

Nextera Exome (4Gb)

HaloPlex


The Fastest Workflow From sample to sequencing the next day

Library Preparation

6 hours

Capture

16 hours

Wash and PCR

4 hours

Overnight Hybridization Enables Sequencer Ready Samples the Next Day

HaloPlex


The Fastest Workflow Less sequencing, faster time to result

Time to sequence 150 samples on a HiSeq based on recommended sequencing amounts

0 5 10 15 20 25 30 35

Nextera Exome

Nimblegen V3

SureSelect V5

Days

HaloPlex


HaloPlex Exome Early Access

HaloPlex


HaloPlex Exome

HaloPlex

Panels HaloPlex

Fast

HaloPlex

Large

HaloPlex

Agilent Innovation for Target Enrichment

HaloPlex


HaloPlex Exome

Fast and simple workflow

Minimal pipetting steps and no library prep

Generate results in 3 days

Only 3 days from sample to data

Low DNA input

Only 200ng of DNA required

Cover all coding content in the genome

Designs against the most recent databases

HaloPlex


Comprehensive Human Exome Design

What if I could

research all

diseases at the

same time?

Target Size 37 Mb

# Genes ~21,500

# Targeted Exons ~357,000

Exome Content

Databases

CCDS, RefSeq,

GENCODE, miRBase,

TCGA, UCSC, VEGA

HaloPlex Exome Content

HaloPlex


The greatest coverage with less sequencing Only 4Gb of sequencing to achieve 80% at 20X coverage

HaloPlex Exome – Premium Exome Performance

30%

40%

50%

60%

70%

80%

90%

100%

% bases covered at 10X

% bases covered at 20X

HaloPlex Exome

Nextera Exome

HaloPlex


Complete Solutions for Gene Regulation Studies Looking at the complete picture

Gene Regulation

RNA • Novel Gene Discovery

• Gene Expression

DNA • Human Exome

Methylation • DNA Methylation

revolutionize your clinical research - agilent genomics at agbt 2013...haloplex for research use...

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