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From Exons to Exomes: the Latest DNA

Solutions from Illumina

Colin Baron Market Manager Sequencing Systems

2

Illumina Sample Prep Solutions Integrated workflows from sample to analyzed data

TruSeq Chemistry Clustering & Sequencing

TruSeq DNA LT & HT Simple, scalable, and

cost effective

TruSeq RNA Optimized, gel-free, low input

TruSeq Small RNA High-throughput miRNA

discovery & profiling

TruSeq Custom/Exome New - Nextera

Custom/Exome

TruSeq Custom Amplicon

Coming - TruSeq

FFPE/Stranded RNA

BaseSpace Data storage, analysis

Nextera Low input, fast, versatile

New - Nextera XT Fast Amplicons, Small

Genomes, Plasmids

New– TruSeq ChIP-Seq

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Master Mixed Formulations

Gel-free protocols for exome and

custom enrichment

Robust performance for any NGS

app including

WGRS

Options for 24 & 96

optimized; QC’d

barcodes

Low-price; all-inclusive, end-to-end

solution

TruSeq DNA Sample Preparation Most widely adopted NGS DNA sample prep

4

TruSeq DNA HT Kits & Adapter Plates

TruSeq DNA HT Sample Prep Kits TruSeq DNA Adapter Plate (DAP)

5

Dual-indexes provided in pre-filled plate Achieve 96 total barcodes with only 20 unique indices

Simple assay setup

– Thaw plate and go!

– Create sample sheets with

Illumina Experiment Manager

Easy and highly-specific

multiplexing

– 20 total barcodes provide 96

combinations

– Flexible pooling for <96

samples/lane

– Unique ID of samples with 2

indices

– Full software support

6

Rapid and streamlined workflow

– Complete protocol in < 90 min

– Single well enzymatic reaction both

fragments and adds adapter

– No mechanical fragmentation/shearing

required

– Easy four step protocol

Low DNA input of only 50 ng

High-throughput, high-indexing

– Prepare up to 96 samples per batch

– Master-mixed reagents and automation-

friendly configurations

– 96 indices supported on all Illumina

sequencers

Nextera DNA Sample Prep Sequencing’s fastest and easiest sample prep

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Human resequencing

Targeted Resequencing

Metagenomics (Amplicons)

Haplotyping (BACs)

Many more!

Applications for Nextera DNA Sample Prep Selected publications highlight breadth of applications

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Step 1: Tagmentation of template DNA

9

Step 2: PCR to add adapters and indices

10

Step 3: Cleanup and Sequence

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Sample Normalization is included No library quantification or qPCR is required – go straight to MiSeq!

Completed libraries,

range of yields

Index CV for

20-sample pools

Quant in

triplicate with

qPCR

Calculate

dilutions

Manually

dilute and

pool

Pool A Pool B

15.8% 18.2%

Nextera XT sample pooling is as simple as pipetting 5 µl!

Bead-based

Normalization:

Bind, Wash,

Elute

13.5% 15.5%

5 µl of each desired library

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Applications: from Exons to Exomes

Mendelian diseases -

case-control studies,

rarer variants, causal

variants, linkage

analysis

GWAS validation, large

scale variant discovery

& screening in cancer,

human diseases and

pathway panels

Highly multiplexed,

high TP amplicon

sequencing: Custom

panels for variant

discovery, validation

and screening

Med TP amplicon

sequencing: ultra-deep

seq, validation,

screening, CE

replacement

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CE for Amplicons? Time to try NGS! Nextera XT enables easy transition to MiSeq

FAST

– Prep a sample in <90 min with 15 min hands on time

– Rapid way to add sequencing adapters for ILMN sequencers

EASY

– Prep existing amplicons, including long range >1 kb!

– Simple sample normalization for easy pooling

– Barcoding supports pooling up to 96 samples per lane

ENABLING PRICE

– Affordable Nextera XT preps for a pool of many amplicons

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Miseq Run

Nextera for PCR Amplicons Quick workflow for long or short range PCR

PCR AMP Pool Amplicons,

Nextera XT Pool Samples

90 min total

15 min hands on

7 Exons

255-416 bp range 12 patient samples

2x36 (6.5hrs)

95%>Q30

22 Amplicons

1-6kb range

68.5kb total

90 min total

15 min hands on 5 patient samples

2x76(15hrs)

95%>Q30

Short Range - CFTR

Long Range – BRCA1/2

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Nextera PCR Amplicon Detection of mutations in CFTR with short range PCR

Exon 10 Exon 20

Complete coverage of target regions at >300X depth

Complete variant concordance Collaboration with Steven Abbs and Michale Yau, London

Het deletion

D F508

Het

W1282X

Amplicons

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Nextera PCR Amplicon Detection of mutations in BRCA1 & 2 with long range PCR

Collaboration with Graham Taylor, Leeds, UK

Complete coverage of target regions at >50x depth

Complete variant concordance

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Nextera XT DNA Sample Prep The fastest & easiest prep for small genomes, PCR amplicons and plasmids

Rapid Prep

– 90 min prep, only 15 min of hands on time

Optimized for small genomes, PCR amplicons & plasmids

Innovative sample normalization

– No library quantification needed

Fastest time to results

– DNA to analyzed data in <8 hrs with MiSeq

Ultra low input

– 1ng input DNA

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Sequencing HIV Isolates with MiSeq and Nextera XT

“MiSeq changed the scales.

With Sanger sequencing I

could pick up a 50/50

mutation…maybe 25/75, but

I could never pick up a

minor population around

2% like I can with MiSeq.”

“There’s a vast

improvement in speed.

Using the Nextera XT kit,

our process went from

several days to only half a

day—that’s just 4 hours for

96 samples.”

“We can monitor mutations

pretty rapidly with the

Nextera XT kit and MiSeq.”

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Coming soon: TruSeq Custom Amplicon 1536-plex Amplicon sequencing transformed

Rapid & economical

– Up to 1536 amplicons per sample, 96 samples per

plate; ~400kb content

– <8 hrs from gDNA to sequencing-ready library

– Utilizes standard lab equipment

– BBN included: No quant needed before sequencing

Fully customized target probes & capture

– DesignStudio for interactive design and ordering

– Personalized and easy to use

– Rapid design turnaround

Pre-configured, automated data analysis

147,456 Amplicons in one plate

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DesignStudio for TruSeq Custom Amplicon Easy to use design tool makes creating new projects simple

Supports custom designs and

online ordering

Simple and interactive

interface

– Modifiable design parameters

– Accepts a variety of input

regions

– Automated selection of exons

– Design statistics

– Automated email notification

Dynamic feedback to optimize

designs & target coverage

Integrated help functionality

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250ng

Genomic DNA

TruSeq Custom Amplicon Assay Overview Begin with 250ng genomic DNA

Hybridization

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Genomic DNA

Hybridization

TruSeq Custom Amplicon Assay Overview Using a pair of custom PCR primers, each targeted region is amplified in each

sample

Extension-

Ligation

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Genomic DNA

Hybridization

Extension-

Ligation

TruSeq Custom Amplicon Assay Overview Incorporation of indexed primers followed by sample pooling

Amplification

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TSCA Performance

Parameters

Customer Order #

AVE 1 2 3 4 5 6 7 8 9

# Target Regions 179 29 92 88 14 86 71 46 82 76

Total Bases Submitted (kb)

49.4 6.4 18.5 14.0 33.6 29.1 12.0 19.4 16.5 22

# Amplicons 347 61 158 113 191 210 100 127 163 163

Designability (%) 94.8 100 98.8 95.8 85.7 91.3 92.6 96.9 100 95.1

Dropouts (no reads) 0 0 0 1 0 3 0 0 0 0.4

Specificity(>70%) 96.4 96.8 91.1 95.8 92.8 93.2 94.7 69.0 97.3 91.9

Uniformity(>85%) 96.6 96.7 84.5 94.3 91.1 79.5 95.5 89.0 98.2 91.7

Specificity = on target bases out of total bases

Uniformity = % bases at >0.2x mean coverage

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TruSeq Custom Amplicon Data Analysis Illumina-developed and supported analysis tools

Illumina Experiment Manager

– Wizard-driven tool to set up and

manage sample preparation

plates

– Generation of sample sheet for

the sequencing of pooled samples

MiSeq Reporter

– On-instrument software

– Demultiplex indices/samples

– Perform read alignment

– Report on detected variants

Illumina Amplicon Viewer

– Offline viewer for visualization of

data from multiple runs

– Custom report generation

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TruSeq® Amplicon – Cancer Panel Hundreds of loci. Rapid prep. FFPE-ready.

Comprehensive Content

– >35 kb total including oncogenes

such as BRAF, KRAS & EGFR

– 212 amplicons in one tube; 48 genes

Unrivaled Multiplexing

– Up to 96 sample pooling on MiSeq

– >90% specificity and uniformity

– Detect low frequency variants (<5%)

Unparalleled Workflow

– FFPE-enabled with sample QC Kit

– No qPCR quant needed for

normalization

– Automated paired end sequencing

with MiSeq

– Pre-configured, automated data

analysis

For research use only

ABL1 EGFR GNAS MLH1 RET

AKT1 ERBB2 HNF1A MPL SMAD4

ALK ERBB4 HRAS NOTCH1 SMARCB1

APC FBXW7 IDH1 NPM1 SMO

ATM FGFR1 JAK2 NRAS SRC

BRAF FGFR2 JAK3 PDGFRA STK11

CDH1 FGFR3 KDR PIK3CA TP53

CDKN2A FLT3 KIT PTEN VHL

CSF1R GNA11 KRAS PTPN11

CTNNB1 GNAQ MET RB1

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Reliable somatic mutation detection below 5% BRAF gene, colon cancer sample

2.95%, Q40

>6,000x coverage

The TSACP detected a 2.95% variant (A to T SNV) in the BRAF gene from a colon

cancer sample, with a variant score of Q40, or 99.99% confidence (6,048x coverage).

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Excellent performance with early access users Superior specificity, uniformity and coverage

Run 1 Run 2 Run 3

Samples 20 20 1

MiSeq

Output 1.8 Gb 2.0 Gb 1.9 Gb

Mean

Depth 1,366 1,410 27,019

Specificity 93.7% 94.5% 90.7%

Uniformity 96.2% 96.2% 94.4%

Amplicon

Dropouts 0 0 0

First time user of TruSeq Amplicon

All customer-selected FFPE

samples from range of tumor types

(lung, brain, cell lines)

Zero amplicon dropouts

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Sanger TruSeq Amplicon – Cancer Panel

Mutated

Gene

% Mutation,

Run 1 (Coverage)

% Mutation,

Run 2 (Coverage)

Sample 1 EGFR

8.2% (3238x)

8.0% (3860x)

8.7% (4967x)

7.5% (5748x)

Sample 2 EGFR 37.1% (1387x)

35.3% (1591x)

Sample 3 EGFR

40.5% (941x)

42.2% (1092x)

39.8% (1810x)

41.8% (2123x)

Sample 4 cKIT 46.4% (1030x)

44.3% (1074x)

Sample 5 cKIT 44.4% (2006x)

46.3% (2080x)

Sample 6 BRAF 99.8% (2706x)

99.8% (3262x)

Sample 7 BRAF 53.6% (5130x)

53.1% (4945x)

R² = 0.997

0%

50%

100%

0% 50% 100%

% M

uta

tio

n, R

un

2

% Mutation, Run 1

Highly reproducible detection of known mutations

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Simple and scalable workflow

– Plate based processing and master-mixed reagents

– Gel-free; integrated with TruSeq DNA Sample Prep

– 1ug DNA input

– 3 days total time with less than 4hr hands-on

Pre-enrichment sample pooling

– Dramatically reduces FTE time and increases throughput

TruSeq Exome and Custom Enrichment Simple, integrated and cost-effective

Exome Custom

Region size 62Mb 500kb-25Mb

Pre-enrichment pooling 6 samples 12 samples

Read length 2 x 100 2 x 50-75

Min order 48 samples 48 samples

Test kit available Exome Cancer panel

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TruSeq Exome Enrichment The most comprehensive and cost effective exome

Only empirically tested

probes are included

“TruSeq Exome is the only

platform that is designed to

enrich UTRs, which are almost

completely untargeted by the

other two platforms, and is

therefore the natural choice for

researchers interested in those

regions”

“All of these platforms can detect

disease-associated variants, of

which a small proportion are

unique to each platform”

32

Nature, 2009.

TruSeq Exome Enrichment Content Interrogate more than just coding exons

“the predicted exons and regulatory regions

exhibited more than twice the variant density of

the CCDS exome”

“many exome sequencing studies fail to find

genes associated with disease, which may be

due to the fact that these studies are missing

important regions of the genome”

1. Manolio T, et al. (2009) Finding the missing heritability of

complex disease. Nature 4618:747-753.

2. Ashford, M. (2011) Baylor Team Finds Target Capture

Beyond Consensus Coding Regions Is Difficult, but

Revealing. GenomeWeb.

3. Bainbridge, MN. et. al. (2011) Targeted enrichment beyond

the consensus coding DNA sequence exome reveals exons

with higher variant densities. Genome Biology 12(7):R68.

33

Highest Coverage Uniformity Coverage uniformity for 6-plex sample pooling*

•*% of reads mapping to target from total reads

•HiSeq 2000 run data with v3 chemistry.

• Padded (+/- 150bp) includes flanking up- &

down-stream regions of target

•>80% of targeted bases

covered at 0.2x mean

coverage (58-72x)

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TruSeq Custom Enrichment Cancer Panel Standard panel targeting exons of >370 key cancer genes

Cancer genes from the Sanger Cancer

Gene Census

– 1 Mb total; >370 genes

– Panel created with DesignStudio

Same assay as TruSeq Exome

Enrichment

– 1 ug DNA input for sample prep

– 12-plex sample enrichment

Excellent data quality

– At 100x average depth:

>90% bases covered at 20x depth

Sequence 5+ spls/MiSeq run

*Content obtained from http://www.sanger.ac.uk/genetics/CGP/Census/chromosome.shtml (April 2011)

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Nextera Exome and Custom Enrichment Comprehensive enrichment solutions, now with the easiest prep

One simple bundle

– Sample prep & enrichment

reagents

Simple and scalable workflow

– 50ng DNA input

– Plate-based processing

– Master-mixed reagents

– Gel-free

No need for mechanical DNA

fragmentation (Covaris)

Pre-enrichment sample pooling

(12)

– Reduces FTE time; increases

throughput

Nextera

Exome

Nextera

Custom

Content 62Mb 500kb-

25Mb

Pre-enrichment

pooling (spls) 12 12

Read length 2 x 100

(or less) 2 x 50-75

Min order (spls) 48 48

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Nextera Enrichment Workflow Faster, simpler, lower DNA input requirement than TruSeq

TruSeq Enrichment

Purified Genomic DNA

Fragment Genomic DNA

Repair Ends

Cleanup

Adenylate 3’ Ends

Ligate Indexed Adapters

Cleanup

PCR

Cleanup

Enrichment

Nextera Enrichment

Purified Genomic DNA

Tagment DNA

Cleanup

PCR

Cleanup

Enrichment

Archive samples for future

analyses

Increase sample throughput

Decrease FTE time

Remove need for mechanical DNA

fragmentation

Reduce need for automation

Make process easier!

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Nextera Enrichment Workflow Prepare spls in <3hrs without mechanical shearing; enriched libraries for 96 spls

~2.5 days

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Nextera Exome and Custom Enrichment Sequencing’s fastest and easiest sample prep can soon be used with enrichment

Ultra-low DNA input of only 50ng

Fastest, simplest workflow

– Library prep reduced from 12hrs to <2hrs

– Total workflow <2.5 days

No mechanical DNA fragmentation

High-sample throughput with pre-

enrichment sample pooling

– 12 exomes / enrichment

Current

TruSeq

Modified

Nextera

Library prep input 1ug 50ng

Library prep time (hrs) 12 <2

Total workflow time (d) 3.5 <2.5

0

10

20

30

40

50

60

70

80

90

Pe

rce

nt

En

rich

me

nt

Aligned Padded

0

10

20

30

40

50

60

70

80

90

100

Spl 1 Spl 2 Spl 3 Spl 4 Spl 5 Spl 6 Spl 7 Spl 8 Spl 9 Spl 10Spl 11Spl 12

Pe

rce

nt

of

Ba

ses

Co

vere

d

0.2x 0.5x 1x

39

Nextera Exome Enrichment Kits The most comprehensive exome….now with the simplest prep workflow

Fastest and simplest workflow

– Prep up to 12 samples in <3 hrs

– Total prep & enrich time ~2.5

days

– No need for mechanical DNA

fragmentation

– 12-plex pre-enrichment pooling

Low DNA input of 50ng

One kit does it all

– Sample prep & enrichment

Expansive coding & non-coding

content

Excellent enrichment rates &

coverage uniformity

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Nextera Custom Enrichment Kits Fastest, easiest, lowest input sample prep workflow for custom enrichment

Target 500kb to 25Mb

– High enrichment efficiency &

coverage uniformity

– Customize content with

DesignStudio

Pre-enrichment sample pooling

– Up to 12 spls per enrichment rxn

– Reduce hands-on time; increase

throughput

One kit does it all

– Sample prep and enrichment

Fastest and simplest workflow

– Prep up to 12 samples in <3 hrs

– No need for mechanical DNA

fragmentation

DNA input of 50ng

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Questions?