© 2011 Illumina, Inc. All rights reserved.

Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect,

CSPro, GenomeStudio, Genetic Energy, HiSeq, HiScan, TruSeq, Eco, MiSeq and Nextera are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the

property of their respective owners.

Illumina’s Suite of

Targeted Resequencing

Solutions

Colin Baron

Sr. Product Manager

Sequencing Applications

2

TruSeq™ Sample Prep Solutions Integrated workflow from sample to analyzed data

TruSeq Chemistry Clustering & Sequencing

TruSeq DNA Simple, scalable, and

cost effective

TruSeq RNA Optimized, gel-free, low

input

TruSeq Small RNA High-throughput miRNA

discovery & profiling

TruSeq Custom

& Exome Enrichment Lowest cost and most scalable

targeted resequencing

Nextera Low input, fast

3

TruSeq Exome Enrichment

Targets = 100,000s

TruSeq Custom Enrichment

Targets = 1000s

Multiplexed Amplicons

Targets = 100s

TruSeq Targeted Resequencing The simplest and most scalable targeted resequencing solutions

PCR Amplicons

Targets = 10s

4

TruSeq Targeted Resequencing A broad suite of tools for discovery or validation experiments

Option Amount of

sequence Best for Availability

TruSeq Exome

Enrichment ~62 Mb

Mendelian disease:

case-control exome studies, rarer

variants, causal variants

exome-wide linkage analysis

Now!

TruSeq Custom

Enrichment ~1 to ~10 Mb

GWAS follow-up: validation of

variants, variant discovery, pathways Mid-2011

TruSeq Custom

Amplicon Sub-500 Kb

Amplicon sequencing: high-

throughput CE experiments, ultra

deep seq, variant disc, screening

2H2011

Nextera + PCR

Amplicons

100’s of bp

targets

Amplicon sequencing: ultra-deep

sequencing, validation, screening, CE

replacement

Now!

5

Exome Sequencing Science Magazines “Top Breakthroughs of 2010”

Quantum motion machine

Synthetic Biology

Neanderthal Genome

HIV Prophylaxis

Exome Sequencing/Rare Disease Genes

Molecular Dynamics Simulations

Quantum Simulator

Next-Generation Genomics

RNA Reprogramming

The Return of the Rat

Published on December 16, 2010

6

Exome Approach Success Evident by the Number of Publications

Dramatic increase in number of exome publications over the past 3 years

Major focus has been on study of Mendelian Disease

– 2008: 1 publication

– 2009: 6 publications

– 2010: 66 publications

Huge increase in number of variants

7

TruSeq Exome Enrichment Pre-enrichment pooling and comprehensive coverage for the most

cost-effective exome

Most comprehensive exome solution

– High coverage uniformity

– Lowest DNA input

Plate-based processing for up to

96 samples

– Simple & scalable workflow

Pre-enrichment pooling of up to 6 samples

– Reduced hands-on time

– Decreased costs

– Gel-free protocol

Integrated with TruSeq DNA

Sample Prep Kits

– Optimized workflow

– Internal QC controls

Launch of TruSeq Exome

8

TruSeq Exome Enrichment Kit Most up-to-date and comprehensive exome available

Only empirically tested

probes are included

9

TruSeq Exome Enrichment Workflow Three-day assay with <4 hours hands-on time

TruSeq DNA Sample Prep

(1 ug starting input)

*PCR, cluster generation &

sequencing

* 2 successive rounds of enrichment

10

Internal Quality Controls For TruSeq exome enrichment

Internal controls for sample prep

and exome enrichment with full

software support

Library prep controls:

– Enzymatic activity of End Repair

– A-Tailing

– Ligation reactions

CTO (Custom Target Oligo)

– Set of specialized probes (150) in

capture pool targeting non-

polymorphic regions across high,

med, and low GC classes

– Also target known homo- &

heterozygous SNPs

CTE1 – Control Target End-repair 1

CTE2 – Control Target End-repair 2

CTA – Control Target A-tailing

CTL – Control Target Ligation

CTO – Custom Target Oligo

11

Highest Coverage Uniformity

Coverage uniformity for 6-plex sample pooling*

*HiSeq 2000 run data

% b

ases c

overe

d

>80% of targeted bases covered at

0.2x of the mean coverage

12

High On-Target Enrichment

Enrichment rates* for 6-plex sample pooling

*Percentage of reads mapping to target from total reads

HiSeq 2000 run data. +/- 150bp includes flanking up- & down-stream regions of target

13

Strategically designed to be larger than other commonly used methods

– Reduced cost – less probes; savings passed on to customer

– Better coverage uniformity; can tolerate more variance in fragment size

– Less issues with problematic regions ie. GC content

– Sum length of ~340K x 95-mer probes is only ~32 Mb, the enrichment actually targets

62Mb of the human genome, or 117.5Mb if the 150 bases up/down stream are taken

into account

Probe-fragment-library design Fully optimized with the most efficient design

14

Multiplexed sample enrichment A huge time and cost savings

Developed and optimized for 6 samples per enrichment reaction

– Huge increase in throughput

– Massively reduced FTE time

No impact on ability to call variants

15

Normalized coverage plots Determine amount of sequencing needed to achieve desired coverage

0.2

e.g. 90% of bases

covered at 10x

Mean Norm. Coverage

Desired Coverage =

16

Calculating sequence amount needed

200Gb 600Gb

Library Prep $54 $54

Enrichment $300 $300

Combo total $354 $354

Cluster Gen $259 $87

Sequencing (2 x 100 bp) $359 $120

Total Per Exome $972 $507

Breakdown of TruSeq Exome Costs*

(62Mb x 50 / 0.65) / .90 = 5.3Gb

2.4 exomes / lane at 200Gb = 38

7.2 exomes / lane at 600Gb = 115

*50x coverage per sample, processed on HiSeq at 2 x 100bp; list price.

Sum Regions Size x Coverage

Enrichment % = PF Gb

Eg. 50x

TruSeq

Exome:

Alignment

rate ÷

17

Alignment and variant calling with CASAVA

Target statistics, graphs

Enrichment %, read distribution, coverage,

normalized coverage plots, controls

Genome Studio integration for visualization

TruSeq Exome Data Analysis

18

Coming Soon! TruSeq Custom Enrichment Kits Same proven technology as in the TruSeq Exome Enrichment Kits

Target 1–10 Mb of DNA per sample

– Highest enrichment efficiency and

coverage uniformity

Leverages Illumina’s expertise in oligo

production

– Interactive online design software

– High coverage of targeted regions

Pre-enrichment Sample pooling

– Up to 12 samples per enrichment

reaction

– Reduced hands-on time; increased

throughput

Integrated with TruSeq DNA Sample

Prep Kits

– Fully optimized workflow

– Most cost-effective solution available

Early access

program has begun!

First custom order

expected summer

2011!

19

Coming Soon! TruSeq Custom Amplicon Sequencing Highly multiplexed, targeted amplicon resequencing

*Based on example study of 96 samples and 384 targets

Fully customized target probes and capture

– Based upon GoldenGate® Technology

Interactive probe design and ordering

– Streamlined user interface

– Rapid probe turnaround

Rapid & economical amplicon sequencing

– Up to 384 amplicons per sample

– Plate-based processing; 96 samples per

plate

– Assay time < 8 hours

– No additional hardware requirements

Up to 10× more cost effective than CE*

21

Assay

Biochemistry

TruSeq Custom Amplicon Assay Time 96 samples & 384 targets: from DNA to called variants in ~2 days

8am – Day 1

Hybridization

Setup

Oligos,

universal

reagents

Extension &

Ligation, PCR

Library

Normalization

Create pooled

Library,

normalize

Cluster Gen &

Sequencing

Pre-kitted

sequencing

reagents

Real-time

Analysis

Alignments,

variant calling

2pm – Day 1 5pm – Day 2

<8 hr assay with <3 hr hands-on time

No fragmentation required

No gel purification steps

No additional hardware

22

Simplest workflow and most convenient TRS offering One stop shop for entire targeted resequencing workflow

Fully integrated, end-to-end

solution including probe design,

sample prep, enrichment,

sequencing and data analysis

Multiplexed sample enrichment (up

to 12)

Master-mixed formulations and

plate-based processing for up to

96 samples

Internal quality controls for each

assay step from library prep

through enrichment with full

software support

Sales

Training

Sample Prep

Enrichment

Cluster gen

Seq

Analysis

Support

23

SIMPLE, FAST LIBRARY PREP IN

LESS THAN 2 HOURS

Transposon mediated library preparation

Closed tube DNA fragmentation

Ultra-low input requirements (50 ng)

ENABLES A RANGE OF CE AND NGS

APPLICATIONS

VALIDATED BY LEADING RESEARCHERS

Epicentre Nextera Technology for Library Prep Single Tube, Rapid Library Prep

26

TruSeq Sample Prep Kits for RNA & DNA

Master-mixed formulations & gel-free

RNA protocol

Universal adapter design with embedded index

Plate-based processing up to 96 samples;

volumes optimized for liquid handling

Low price, all-inclusive kit

Internal quality controls

Convenient, one-stop shop

Economical large-scale studies

Flexible Design—one kit for single,

paired-end, and mate paired reads

Robust indexing solution

High-throughput and automation

friendly

Simple workflow with minimal

pipetting and clean-up steps

Sample prep success monitoring

with software support

27

A Sequencer for Every Need. Every Budget. Every Lab.

Two proven technologies.

One powerful platform.

HiScanSQ

The most widely

cited platform, now at

half the price.

GAIIx MiSeq

My Samples. My

Study. MiSeq

Powerful.

Flexible.

Scalable.

HiSeq 1000

Redefining the

trajectory of

sequencing.

HiSeq 2000

28

Questions?

29

Using coverage uniformity curves to calculate output needed

Inputs:

• Size of Exome (no. of targeted bases) = 62Mb

• Enrichment efficiency (fraction of reads on target) = 65%

• Uniformity = 80% of targeted bases at 0.2x mean coverage

• Desired minimum % of bases at specified coverage = 80% at 10x

• Normalized mean coverage = ???

Mean sequencing coverage can be calculated from normalized coverage

plots

(normalized mean coverage) x (mean sequencing coverage) = 10x

(0.2) x (mean sequencing coverage) = 10x; therefore,

Mean sequencing coverage = 10x / 0.2 = 50x

The total amount of sequence can be calculated as follows:

• Total amount of target sequence = 62Mb

• Mean sequencing coverage = 50x

• Enrichment efficiency (fraction of reads on target) = 65%

(62Mb) x (50x) / (0.65) = 4.8Gb

30

Optimizing Coverage for Targeted Resequencing Determining the optimal amount of sequencing to achieve a desired coverage level

TruSeq exome product

specification: >80% of bases

covered at 0.2× mean coverage

– If average/mean coverage is

100x, then >80% of bases are

covered at 20x (20 reads per

base)

– If average/mean coverage is 50x,

then >80% of bases are covered

at 10x (10 reads per base)

Optimize coverage by leveraging:

– TruSeq Exome Scripts

– Mean normalized coverage

curves

Determine number of target bases

Calculate fraction of reads on target

Determine mean sequencing coverage with normalized coverage plots

Calculate required amount of sequencing data

31

TruSeq Exome Enrichment Kits – Pricing

Catalog

Number Product

Reactions

per kit

Samples

per kit (6-

plex)

Kit Price

Price per

sample

FC-121-1008 TruSeq Exome

Enrichment Kit – 8 8 48 $14,400 $300

FC-121-1024 TruSeq Exome

Enrichment Kit – 24 24 144 $39,600 $275

FC-121-1048 TruSeq Exome

Enrichment Kit – 48 48 288 $72,000 $250

FC-121-1096 TruSeq Exome

Enrichment Kit – 96 96 576 $129,600 $225

FC-121-1192 TruSeq Exome

Enrichment Kit - 192 192 1152 $230,400 $200

FC-121-1480 TruSeq Exome

Enrichment Kit - 480 480 2880 $504,000 $175

FC-121-1960 TruSeq Exome

Enrichment Kit – 960 960 5760 $864,000 $150

Orders from Nov 3, 2010; Shipping from Nov 22

33

TruSeq Exome Data Analysis Overview of outputs from TruSeq Exome Scripts―data visualization & QC

GC control probes

– Probes selected to have low, medium

or high GC content

– Independent of the probes targeting

the exome

Read distribution

– Shows the number of reads around the

center of the targeted regions

Coverage & mean coverage levels

– Shows the fraction of targeted bases

that are covered at a given coverage

level

– Compare data on the same scale from

runs that have different mean coverage

levels

Control SNPs

– Targeted at known SNPs that are not

in any targeted region

34

TruSeq Exome Data Analysis Leverage GenomeStudio® software for simple reporting on exome data

BED file seamlessly defines

targeted regions

‘Regions’ table allows for easy

selection of targets

Navigate regions and view

annotation data in

GenomeStudio’s viewer

View multiple samples in single

table or IGV window