but FEW - Agilent are Called... · but FEW are CHOSEN . For Research Use Only. Not for use in diagnostic procedures SureSelect ... Screen candidate genes in the locus Mutation Detection

For Research Use Only.

Not for use in diagnostic procedures

Many are called…

but FEW

are CHOSEN



SureSelect

Focused Exome

A Highly Targeted Design

for Deep Coverage

Background:

There are about 7,200 known rare disorders and only 4,000

have known underlying genetic cause

Disease-associated variants are identified in only 50% of

samples analyzed* = extended diagnostic odyssey

WHAT DOES “RARE” MEAN?

• “Rare” diseases affect 8% of the population



8% of 7 billion people = 560 million

*Zemojtel T. Sci. Transl. Med., 2013

Out of 3 billion base pairs organized into ~20,000 genes– how do you look for the one variant linked to disease?

November 13, 2014

Confidentiality Label

4

TRIVIA:

Odds of winning the

Powerball Lottery???

1 in 175 million!!!

• Biochemical assays

• PKU:

- AR; phenylalanine hydroxylase deficiency leading to

accumulation of phenylalanine

- Untreated, may lead to profound mental retardation

PITFALLS

1. Requires a priori knowledge of specific disorder

• Symptoms would support results

2. Laborious and could get expensive if multiple

rounds are done when inconclusive

Is there a better way of finding the needle in the haystack?

Traditional Approach



• Karyotyping

• Technique used to look for large-scale chromosomal

abnormalities (eg. trisomies)

PITFALLS

1. Lack of resolution (limit 4-5Mb)

• Cannot identify causal single-nucleotide variants or

microdeletions

2. Highly dependent on level of experience of

cytogeneticist or technologist





• aCGH

• Used to look for chromosomal abberrations such as

copy number changes

PITFALLS

1. Inability to detect other chromosomal

abberrations without copy number changes

• Balanced translocations

• Inversions

• Mosaicism





• Positional Candidate Gene Discovery

• Linkage mapping combined with Sanger sequencing





4) Study gene product function

A) Normal biology

B) Disease pathogenesis

3) Screen candidate genes in the locus

Mutation Detection

1) Identify large and/or many families with the disease

agcttgaa…(GATC)n…..aggccta

telomeric

centromeric

2) Identify disease gene locus

agcttgaa…(GATC/GATC/GATC)…..aggccta

agcttgaa…(GATC )…..aggccta

agcttgaa…(GATC /GATC)…..aggccta

http://www.clker.com/cliparts/w/e/0/B/W/j/homologous-chromosomes-md.png







PITFALLS

1. Requires a high number of informative meiosis

2. Locus heterogeneity leads to decreased LOD

score

3. Laborious and could get expensive if

sequencing many candidate genes

4. May result in work that takes several months to

years to complete







2005 2006

• Candidate gene

(Sanger) sequencing

started

• Samples collected

from 9 families

• ~5Mb disease

locus identified on

chromosome 5

2009

• STILL sequencing…

EXAMPLE:

Disease Gene Discovery for Infantile Myofibromatosis

- Rare AD proliferative disorder; benign tumor formation on skin, muscle

viscera and bone

???







EXAMPLE:


CHALLENGES:

• Phenotype with reduced penetrance

• Possible locus heterogeneity

NEED:

• Unbiased way to interrogate genomic regions

NGS enables increased identification of disease-causing genes

13

Boycott M. et al. Nat. Rev. Genet. 2013

Gnirke A et al. Nat. Biotech. 2009

SureSelect was introduced



FACT:

>50 Mendelian disease genes

identified with SureSelect







2005 2006

• Candidate gene

(Sanger) sequencing

started

• Samples collected

from 9 families

• ~5Mb disease

locus identified on

chromosome 5

2009

• STILL sequencing…

EXAMPLE:


2012

• exome sequencing with SureSelect

• 2 disease genes identified:

1. PDGFRB (chr 5): 2 missense

variants present in 8 families

2. NOTCH3 (chr 19): missense

mutation in 9th family Martignetti et al. AJHG Jun 2013

15

But what about benchtop sequencer-compatible solutions?



http://arazaan.com/wp-content/uploads/2014/01/Man-Thinking-02.png

Benchtop sequencers cannot provide sufficient exome coverage due to limited output

16




17

More and more sequence their own samples using benchtop sequencers in their labs




18

Targeted solution for

constitutional disease

research

Compatibility with

benchtop sequencing

Highly targeted design + Efficient Workflows

= Reduced turn around time, high performing enrichment,

faster sample to data



SureSelect

Focused Exome When time to answers matters



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1. Focused Coverage

~20,000 genes

~5,700 genes

• 16Mb total capture size

• OMIM, HGMD and ClinVar

Deep coverage of disease-

associated targets with

minimal sequencing for

faster data analysis

Capture size compatible with

benchtop sequencers for

faster sequencing

Key Benefits: Highly targeted design for faster time to answers

21



1. Focused Coverage

~20,000 genes

~5,700 genes

• 16Mb total capture size

• OMIM, HGMD and ClinVar

Cost-effective parallel

interrogation of thousands of

genes: lower cost compared

to running multiple panels

when result is inconclusive

Study of rare disorders:

comprehensive analysis of

disease-associated regions


IDEAL FOR:

22



2. Sample to Sequencing in a Day

with SureSelectQXT

- 1.5 hr lib prep + 90 min hyb

- Single day sample to sequencing

- 30% less hands-on time

- only 50ng input

Faster sample to sequencing


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3. Complete and flexible solutions

from sample to data

- SureCall data analysis support from raw

variants to categorized mutations

- Customization support through SureDesign

- Library prep, sample QC, automation support

Faster sample to

variants report


SureSelectXT Focused Exome Deep coverage of disease-associated targets: 98% at 20x with 3Gb

2x100bp

3Gb

90%

91%

92%

93%

94%

95%

96%

97%

98%

99%

100%

NA10831 NA18507 NA12155 NA12156 NA18997 NA12891 NA18953 NA12878

1X

10X

20X

On-target +/- 100bp: 76.5%

Dup rate: 6.4%



SureSelectQXT Performance: Excellent performance, 99% at 20x, 3.5x FASTER

90%

91%

92%

93%

94%

95%

96%

97%

98%

99%

100%

NA10831 NA18507 NA12155 NA12156 NA18997 NA12891 NA18953 NA12878

1X

10X

20X

On-target +/-100bp: 82.4%

Dup rate: 12%



2x100bp

3Gb

Highly sensitive and accurate variant calling

90%

91%

92%

93%

94%

95%

96%

97%

98%

99%

100%

90%

91%

92%

93%

94%

95%

96%

97%

98%

99%

100%

XT

QXT

CONCORDANCE >98%

90%

91%

92%

93%

94%

95%

96%

97%

98%

99%

100%

90%

91%

92%

93%

94%

95%

96%

97%

98%

99%

100%

SENSITIVITY >99%



2x100bp

Agilent

Confidential

Normalized to 3Gb

90%

91%

92%

93%

94%

95%

96%

97%

98%

99%

100%

1X 10X 20X

2x76

2x100

MiSeq Read Length Compatibility Comparable results between 2x100bp and 2x76bp

2x76bp



SureSelectQXT Focused Exome More relevant targets captured, more comprehensive analysis

*analysis based on total capture design

Accounts only for bases within the target

overlap with padded bait BED



SureSelect

Focused Exome Competitor I

Target Size 12Mb 12Mb

DATABASES COVERED

HGMD_cds 76.80% 73.63%

OMIM_cds 45.45% 41.01%

ClinVar 83.45% 81.54%

Customization Yes No

1 day workflow Yes No

SureSelectXT Focused Exome Deeper target coverage at equivalent sequencing, better variant calling

* Based on publicly-available design files

** analysis based on regions that overlap between the designs



SureSelect

Focused Exome Competitor I

Design Size 16Mb 12Mb*

Recommendation 3Gb

2x100bp

2.5Gb

2x150bp

% targeted bases at 1x 99.87% 97.59%




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Our Approach….



• Compatibility with low sample input

• Streamlined and scalable workflow

• Fast turn-around time

WORKFLOW + DESIGN

• Performance-optimized

designs with relevant targets

• Sensitivity and Accuracy

COMPLETE AND ACCURATE VARIANT PROFILING

faster sample to data

and

31

DESIGN WORKFLOW +

=


Our Approach….



32

• Targeted solution for constitutional disease research

• Compatibility with both high-throughput and benchtop sequencing

SureSelect Clinical Research Exome SureSelect Focused Exome

AND



33

DESIGN WORKFLOW +

=


Our Approach….



COMPLETE and FLEXIBLE Solution

Superior PERFORMANCE

FASTEST Workflow

• 90 minute hybridization, fastest in the market

• Enables single day sample to sequencing

• 3.5x faster than closest competitor

• Excellent on-target and target coverage

• Highly sensitive and accurate variant calling

• Support for exomes or custom captures

• Supported by SureDesign, SureCall and NGS

library prep QC solutions



SureSelectQXT

35

SureSelect: The ONLY complete solution

Enabling constitutional disease research,

accelerating discoveries



SureSelect Competitor I Competitor L Competitor N

Designs optimized for deep coverage of

ONLY regions that matter

= COST-EFFECTIVE ANALYSIS

Compatibility with both high output and

benchtop sequencers Single day sample to sequencing workflow

Clinical Research Exome (for high output)

OR

Focused Exome (for benchtop)

SureSelectQXT +

FOLLOW-UP CLINICAL

RESEARCH

SureSelect: Enabling discovery, advancing clinical research

Compatibility with low sample input

Performance-optimized designs with relevant targets

Streamlined and scalable workflow

Fast turn-around time

Sensitivity and Accuracy

DISCOVERY

Reduced time from sample to data





Documents

but FEW - Agilent are Called... · but FEW are CHOSEN . For Research Use Only. Not for use in diagnostic procedures SureSelect ... Screen candidate genes in the locus Mutation Detection