Next Generation Sequencing• Huge improvements in the technology over the

past two years has resulted in the routine abilityto generate about 20-30 billion bases ofsequence information from each machine eachweek.

• Major technologies• Illumina (GAIIx)• ABI (SOLID V3plus)• 454/Roche• Helicos• Pacific Biosciences

Next Generation Sequencing1) whole genome and whole exome sequencing,

economical large scale targeted sequencing forvariant discovery,

2) CHIP_SEQ for full access to genomic bindingtargets,

3) RNA_SEQ for more complete surveys oftranscription on a genome scale,

4) Methyl_SEQ providing genomic assessment ofmethylation status at base resolution.

5) Small_RNA SEQ

Next gen sequencing

• Steps– Obtain genomic DNA (gDNA)– Fragment the genomic DNA– Repair the ends of fragmented DNA, ligate to

specific primers– Sort the fragments so that there is one

fragment per location– PCR amplify the ligated fragment– Sequence the PCR amplified DNA

Illumina TECH OVERVIEWgDNA

Illumina TECH OVERVIEW

Illumina TECH OVERVIEW

ABI SOLID Tech OVERVIEW

ABI SOLID TECH OVERVIEW

SOLiD Users Group Meeting |11/18/09

Bead Preparation and Deposition

Sample Input and Fragmentation

Library Preparation

One Fragment = One Bead

emPCR (Emulsion PCR) Amplification

One Bead = One Read

Data Analysis

Roche454sequencing

Technology comparisons• Tech• ABI 50b $680 ~30Gb/wk• Illumina 75b $700 20Gb/wk• 454 400b $>10,000 3-6Gb/wk

SOLiD Users Group Meeting |11/18/09

How Many Reads Do I Need to Surveythe Transcriptome?

Application Complexity Reads Estimate mappablereads needed

SamplesSOLID 3

Small RNADiscovery

Low 35bp ~10M Up to 20/slide

SAGE Low 35bp 5M 40/ slide

Expression ofannotatedgenes

Mid 50bp Minimum 50M(human

Up to 4/slide

WholeTranscriptomeDiscovery

High (alternativetranscripts & splicing

50 bp Minimum 100million (human)

2/slide

Allele SpecificExpression

High (variants to bedefined)

50 bp > 150 million(human)

1/slide

The number of reads needed is dictated by the complexity of application

* Current best estimates from literature and internal research

Acute myelogenous leukemia

• Clonal hematopoietic disease caused byinherited and acquired genetic alterations.

• Current AML classification and prognosticsystems incorporate genetic information,but are limited to known abnormalities thathave been identified by cytogenetics,CGH, expression profiling, and generesequencing.

Acute myelogenous leukemia• Karyotype remains the most powerful predictor of

outcome.• Sub-cytogenetic lesions using CGH and SNP arrays

have been employed but remain investigational.• Expression profiling has revealed candidate genes which

are currently being examined.• Candidate gene resequencing has identified recurrent

mutations that can be used to stratify patients withnormal cytogenetic profiles to risk and to identifypatients for targeted therapy.

• These revised classification systems are imperfect,suggesting that important genetic factorsfor thepathogenesis of AML remain to be discovered.

Acute myelogenous leukemia

• Within this background the authorsdescribe their 2nd patient with AML-M1(minimal maturation) for which the entireAML genome has been sequenced.

• The genes identified in their sequencingstudy were then examined in 187additional AML samples.

The patient

• 38 yo male, european descent, who presentedwith fatigue, cough.

• WBC 39.8x103, Hg 8.9, and Plt 38x103

• Bone marrow revealed 90% cellularity, with 86%of the blasts staining for myeloperoxidase.

• Karyotype was normal.• Treatedwith cytarabine, daunorubucin, followed

by consolidation treatement with cytarabine.• Complete remission for 3 years.

Genetic studies

• Paired bone marrow sample and skin biopsy.• Affymetrix Genome Wide Human SNP Array 6.0• Affymetrix U133 Plus 2.0 expression arrays• 69.9 billion bp sequencing of the bone marrow

sample• 63.9 billion bp sequencing of the skin sample• Characterized 4 tiers of single nucleotide

variants (SNV)

Tiers of single nucleotide variants

• Tier 1- amino acid changes in coding regions ofannotated exons, consensus splice site regions,and RNA genes.

• Tier 2- changes in highly conserved regions ofthe genome that have regulatory potential

• Tier 3- mutations in the non-repetitive part of thegenome that does not meet criteria for tier 2.

• Tier 4- the remainder of the genome

Conclusion• Next gen sequencing allows for full genome sequencing

of individual samples.• Becoming more affordable and accessible.• Allows for relatively complete genomic evaluation (98%

coverage), with fewer sequencing runs and fewer falsepositives (47%).

• 20,000 novel SNV described; of these 750 are found incoding or regulatory regions of the genome

• 12 nonsynonymous mutations specific to the tumor aredescribed, 3 of which are found in other AML samples.

• One of these SNV codes for IDH1, which previously hadnot been described in AML patients.

Conclusion

• Available• Powerful• Expensive• Results are limited• Must be combined to other technologies

which interrogate other aspects of generegulation (chromatin).

ABI SOLID TECH OVERVIEW

SOLiD Users Group Meeting |11/18/09

Agenda Item0102 Agenda Item

03 Agenda Item

The SOLiD™ 3 Plus System

SOLiD Users Group Meeting |11/18/09

SOLiD Kits

ChIP Kit & Antibodies (#1) LAUNCHED - Mar 15

Eco Mate-Pair Kit LAUNCHED - Mar 15

Super SAGE Kit LAUNCHED - May 15

Whole Transcriptome (WT) Kit LAUNCHED - June 8

LMP Mate-Pair Kit LAUNCHED - July 15

Fast Strips -1x35, 2x35 LAUNCHED - July 16

SOLiD 16 Barcode Kit

QC Tools

ChIP Kit & Antibodies (#2)

SOLiD Methylation kit

SOLiD 96 Barcode Kit

SOLiD 3.0

SOLiD 3 Plus 2009

SOLiD 3 Plus 2010

LAUNCHED – Sept 15