FIND MEANING IN COMPLEXITY

© Copyright 2013 by Pacific Biosciences of California, Inc. All rights reserved.

10/28/2013

PacBio® SMRT Sequencing Technology,

Applications & Roadmap

Outline

2

Technology RoadMap

Q&A

Applications

General Technology Overview/Current Performance

Single Molecule, Real-Time (SMRT®) DNA Sequencing

3

PacBio® RS Trace

The PacBio® Difference

Observes single molecules in real time to provide high-throughput

SMRT® Sequencing of DNA and base modifications simultaneously

• Generate finished genomes

• Discover a broad spectrum of

base modifications

• Characterize complex variations

• Extraordinarily long read lengths

• Extremely high accuracy

• Exquisite sensitivity

• Direct detection of a broad spectrum of DNA base modifications

• Shortest run time

• Least GC bias

• No amplification bias

Typical PacBio® RS II Results

SMRT® Sequencing Accuracy

Data generated with P4-C2 chemistry on PacBio® RS II;

Analyzed using Quiver with 2.0.1 SMRT® Analysis

Perspective: Understanding SMRT Sequencing Accuracy

Detection of DNA Base Modifications by SMRT®

Sequencing

Flusberg et al. (2010) Nature Methods 7: 461-465

7

Detectable by other Sequencing Methods

Signatures of Different DNA Base Modifications

8

Prokaryotic Eukaryotic DNA Damage

Integrated End-to-End Solutions

Easy, user-friendly,

web-based solutions

Streamlined data analysis

and viewing

Support for novice and

expert users

The PacBio® RS Helps Resolve Genetically Complex Problems

10

Generate finished

assemblies

De Novo Assembly

Comprehensively

characterize

genomic variation

Targeted

Sequencing

Automatically detect

DNA base

modifications

Base Modification

Detection

Microbiology on the PacBio RS

100K FoodBorne

Pathogen genome

project

Increase food safety using microbe systems biology

Discover the genetic constituents that are robust to be predictive biomarkers for specific traits

Rapid ID and tracking

Understand evolution to build more robust detection systems

New isolate emergence and persistence http://100kgenome.vetmed.ucdavis.edu

2012 HHSInnovate Secretary’s Choice Awardee

The Value of De Novo, Finished Microbial Genomes

• Less than 1% of the Earth’s

microbiome is known

• Horizontal gene transfer is

wide-spread and frequent

• High-quality, finished genomes are

the starting point for:

– Functional genomic studies

– Comparative genomics

– Forensics

– Metagenomics

Chain et al. (2009) Science 326: 236-237

Fraser et al. (2002) J Bacteriology 184: 6403-6405

Read Primer: The Value of Finished Bacterial Genomes

New Bioinformatics Solution:

Finishing Genomes Using Only PacBio® Reads

Full push-button solution from beginning to end

• Longest reads for continuity

• All reads for high consensus accuracy

Hierarchical Genome Assembly Process (HGAP)

HGAP Nature Methods (2013)

PacBio® Advantage of Even Coverage

Rubrivivax gelatinosus Gemmatimonadetes species

(*not drawn to scale*)

S. Noble, J. Yu, P. Maness, J. Chen, K. Wawrousek, C.

Eckert (NREL, U Wyoming)

S. Polson, R. Marine, D. Nasko, M. Radosevich, J. DeBruyn, E. Wommack

(U Delaware, U Tennessee)

5,075,070 bp

71.3% GC

5,312,117 bp

72.6% GC

20,957 bp

72.0% GC ~10x

1,106,726 bp

72.7% GC

1,040,999 bp

72.8% GC 235,512 bp

64.9% GC

47,366 bp

73.5% GC ~5x

Improving the Plasmodium Genome (23.3 MB)

Malaria:

– 350-500 million infections per year

– 1 million deaths per year

– 20% average GC content Plasmodium falciparum

454® pyrosequencing Sanger sequencing Illumina® sequencing SMRT® sequencing

Progeny Parents Reference genome 30 SMRT Cells

7C126 SC05 Dd2 HB3 NP-3D7-S NP-3D7-L 3D7

Number of Contigs 9,452 9,597 4,511 2,971 26,920 22,839 98

N50 Contig Size (kb) 3.3 3.3 11.6 20.6 1.5 1.6 1,242

Largest Contig (kb) 36.7 34.4 79.2 111.9 29.1 24.0 2,534

Number of assembled bases (Mb) 20.8 21.1 19.5 23.4 19.0 21.1 23.5

Average Coverage 33× 36× 7.8× 7.1× 43× 64× 155×

Sample provided by the Broad Institute & Sarah Volkmann (Harvard School of Public Health)

Samarakoon et al. (2011) BMC Genomics 12: 116.

Understanding Virulence in Infectious Diseases

Whooping cough

– Highly contagious

– Especially severe in children

– 48.5 million infections per year

– 295,000 deaths per year, 90% in developing countries

– Vaccines ~80% effective, but emergence of vaccine escape strains

– Infections & deaths on the rise in recent years, declared epidemic in California

(2010), Washington & Vermont (2012), UK (2012)

Bordetella pertussis

The Pertussis Genome is Extremely Repetitive

Bordetella pertussis E. coli

>1 kb

>2 kb

>5 kb

Repeats

>99% identical with length:

Collaboration with A. Zeddeman, H. van der Heide, M. Bart & F. Mooi

National Institute for Public Health and the Environment (RIVM), Netherlands

Year Strain Sequencing Genome size Reference

2003 Tohama Sanger:

• 87,500 paired-end reads (1-4kb shotgun libraries)

• 2,560 paired-end reads (10-20kb pBAC library)

• 41,700 sequencing reads during finishing

4,086,186 bp Parkhill et al.

Nature Genetics

35: 32-40

Complete Pertussis Genomes

Year Strain Sequencing Genome size Reference

2003 Tohama Sanger 4,086,186 bp Parkhill et al.

2011 CS 454 & Sanger:

• 329,480 454 reads yielding 287 contigs

• 11,444 paired-end ABI3730 reads

• Filled gaps through sequencing of PCR products

4,124,236 bp Zhang et al.

J Bacteriology

193: 4017-4018

Year Strain Sequencing Genome size Reference

2003 Tohama Sanger:

• 87,500 paired-end reads (1-4kb shotgun libraries)

• 2,560 paired-end reads (10-20kb pBAC library)

• 41,700 sequencing reads during finishing

4,086,186 bp Parkhill et al.

Nature Genetics

35: 32-40

Complete Pertussis Genomes

Year Strain Sequencing Genome size

2013 B1917 6 SMRT Cells 4,102,176 bp

2013 B1920 8 SMRT Cells 4,114,613 bp

2013 B3405 6 SMRT Cells 4,109,986 bp

2013 B3582 8 SMRT Cells 4,104,315 bp

2013 B3585 8 SMRT Cells 4,106,397 bp

2013 B3640 8 SMRT Cells 4,110,999 bp

2013 B3658 6 SMRT Cells 4,103,245 bp

2013 B3913 6 SMRT Cells 4,109,515 bp

2013 B3921 4 SMRT Cells 4,111,519 bp

Finish Challenging Genomes with a Few SMRT® Cells

Complexity of Genome

Collaboration with A. Zeddeman, H. van der Heide, M. Bart & F. Mooi

National Institute for Public Health and the Environment (RIVM), Netherlands

Strains of Bordetella pertussis genomes

>1 kb

>2 kb

>5 kb

Repeats

>99% identical

with length:

Watch Jonas Korlach present the complete story (AGBT 2013)

Compare Genome Organization Between Strains

1917

3582

1920

3585

3640

3405

3658

3913

3921

CS

Tohama

Collaboration with A. Zeddeman, H. van der Heide, M. Bart & F. Mooi

National Institute for Public Health and the Environment (RIVM), Netherlands

Organization of Virulence Genes Differs Between Strains

Collaboration with A. Zeddeman, H. van der Heide, M. Bart & F. Mooi

National Institute for Public Health and the Environment (RIVM), Netherlands

Phylogeny of Sequenced Pertussis Strains

Collaboration with A. Zeddeman, H. van der Heide, M. Bart & F. Mooi

National Institute for Public Health and the Environment (RIVM), Netherlands

High Mobile-Element Diversity

using PHAST (http://phast.wishartlab.com)

Phage Element

To

ham

a

B19

17

B19

20

B34

05

B35

82

B35

85

B3

64

0

B36

58

B39

13

B39

21

Prophage Brucella suis 1330

Prochlorococcus phage P-SSM2

Burkholderia_phage_BcepGomr

Prophage Brucella suis 1330

Feldmannia_species_virus

Lactococcus_phage_bIL312

Cronobacter phage phiES15

Pseudoalteromonas phage H105/1

Spiroplasma_kunkelii_virus_SkV1_CR2_3x

Collaboration with A. Zeddeman, H. van der Heide, M. Bart & F. Mooi

National Institute for Public Health and the Environment (RIVM), Netherlands

Tracing Foodborne Pathogens

Salmonella contributes to many foodborne outbreaks

– ~76 million illnesses each year

– ~325,000 hospitalizations

– ~3000-5000 deaths

Salmonella is particularly devastating:

• $78 billion economic loss (US)

• High serotype diversity

1500 subspecies I serotypes alone

• High mobile-element diversity

Frequent horizontal gene transfer

• Emerging hypervirulence

Assemblies of Salmonella Genomes

• HGAP assemblies of the complete Salmonella genome were

constructed in only a few weeks and revealed additional novel

genetic elements

Strain

Sequencing

(PacBio® RS) Genome size (bp) Additional genomic elements

S. Bareilly (SAL2881) 8 SMRT® Cells 4,730,611 78,193 bp

S. Heidelberg (318_04) 8 SMRT Cells 4,793,478 117,929 bp; 35,296 bp; 3969 bp

S. Heidelberg (2069) 8 SMRT Cells 4,783,941 110,345 bp; 37,704 bp

S. Typhimurium (2048) 8 SMRT Cells 4,967,892 142,804 bp; 48,532 bp

S. Javiana (1992_73) 8 SMRT Cells 4,629,444 24,013 bp; 17,094 bp

S. Cubana (2050) 12 SMRT Cells 4,977,480 166,668 bp; 122,863 bp

S. St.Paul SP3 8 SMRT Cells 4,730,130 none

S. St.Paul SP48 8 SMRT Cells 4,940,224 44,606 bp; 40,801 bp

Collaboration with M. Allard, E. Brown, E. Strain, M. Hoffman, T. Muruvanda, S. Musser (FDA),

R. Roberts (NEB), B. Weimer (UC Davis)

Read about the Genome

Diagnosing Active Salmonella Outbreaks with Finished Genomes

Clinical Arizona isolate from October

2012 produce-related outbreak

Complete process from isolate to finished genomic

sequence in <1 week on PacBio® RS

Finished Assembly Results:

• 1 chromosome

• 2 plasmids containing never-before-seen sequence

• Observed 4 active 6-mA methyltransferases

Collaboration with M. Allard, E. Brown, E. Strain, M. Hoffman, T. Muruvanda, S. Musser (FDA), R. Roberts (NEB), B. Weimer (UC Davis)

Genome Announc. March/April (2013) 1:doi:10.1128/genomeA.00081-13

Methylome of the German E. coli outbreak strain. The

inner and outer red circles show the kinetic signals. The

colored internal tracks show the different methylation motif

distributions.

Genome-wide detection of methylation for the German E. coli outbreak strain.

Characterization of Methylation Profiles

• Methyltransferases bind specifically to DNA motifs in a genome and methylate bases

• PacBio® software locates modified sites and motifs

Case Study: Beyond Four Bases - Epigenetic Modifications Prove Critical to Understanding E. coli Outbreak

CTGCAG Motif is Unique to Outbreak Strain

30

Methyltransferase Motif (weblogo) C227

outbreak 55989 782-09 17-2 734-09 760-09 35-10 042 1010

M.EcoGI Non-specific

M. EcoGII Non-specific

M. EcoGV

M. EcoGVIII

M. EcoGVI

M. EcoGIV

M. EcoGIII

M. EcoGVII

M. EcoGIX Non-specific

M. EcoGDam

CTGCAG Methylation Affects Gene Expression

31

Up-regulated

Down-regulated

New Bacterial Modification Systems Identified

Methyltransferase specificities for bacteria

sequenced recently on the PacBio® RS System

Type of Methyltransferase

(sequenced) I II III

Biochemically / Genetically

Characterized 69/20 854/722 22/21

Putatives from GenBank 3,480 15,226 1,600

Previously known

Previously unknown

Listeria monocytogenes Epigenomes

• Characterize strain methyltransferase diversity

• Identify novel methyltransferases

Serogroup 1/2a 1/2b 4b

Methyltransferase

Specificity

Modified

Base 861

878

899

1846

2074

2625

2626

2676

859

867

911

2624

G4599

1493

1494

1495

5'-GATC-3'

3'-CTAG-5' m6A

5’-GATC-3’

3’-CTAG-5’ X

5'-GACN5GGT-3'

3'-CTGN5CCA-5' m6A

5'-GAN6TGCG-3'

3'-CTN6ACGC-5' m6A

5'-TACN7GTNG-3'

3'-ATGN7CANC-5' m6A

5'-TAGRAG-3'

3'-ATCYTC-5' m6A

5'-GTATCC-3'

3'-CATAGG-5' m6A

complete methylation

partial methylation

5’-GAxTC-3’

5’-Gm6

ATC-3’

Collaboration with C. Tarr (CDC), H. den Bakker (Cornell U),

R. Roberts (NEB), B. Weimer (UC Davis)

Requirements for Achieving High-Quality Finished Genomes

1. High Consensus Accuracy

– Lack of systematic bias

2. Lack of sequence context bias

– GC content

– Low complexity sequence

3. Long sequence reads

– Resolve repeats, plasmids

Finished Microbial Genomes & Epigenomes on the

PacBio® RS II

35

PacBio® Benefits for Microbial Genomes

• Highest consensus accuracy (>99.999%)

• Complete bacterial chromosomes with minimal or no gaps

• Capture associated phage or plasmid elements

• Epigenomic characterization

• Simple sample preparation

• Rapid results in one week

• Full push-button bioinformatics solutions

• Cost effective

PacBio® Microbial Applications

Solutions for Plant & Animal Genomes

Discover Biology with Extraordinary Read Lengths

Complete microbial genomes and improve assemblies of larger organisms

• Highest N50

• Fewest fragments

• Detect structural variation

• 99.999% consensus accuracy

Read lengths up to 20 kb, unbiased genome coverage, and high accuracy

Finished bacterial genome

www.pacb.com/denovo

Improve and Finish Genomes with the PacBio® System

De novo Assembly

Complete genomes with

PacBio reads alone

Combine technologies

for best of both worlds

2 3

2 3

1

1

Scaffold

Establish framework for genome

and resolve ambiguities

Span Gaps

Polish genomic regions with up to

10x improvement

• HGAP

• PacBio2CA /

Celera® Assembler

• Others (Mira,

Cerulean, etc)

• AHA

• PB Jelly 2

• PB Jelly

Long Reads Span Difficult Genomic Regions

Address genomic challenges

with longer read lengths

• Resolve long palindromes

• Identify structural variants

• Obtain accurate microsatellite lengths

• Span homopolymeric, low-complexity, and

highly repetitive regions

• Delineate tandem repeats

Loomis et al. (2013) Sequencing the unsequenceable: Expanded CGG-repeat alleles of the fragile X gene.

Genome Research, 23(1):121-8

Fragile X gene with >2 kb of repeat regions

PacBio® reads span extreme

CGG repeats and AT-rich regions

Improving Atlantic Cod (G. morhua )

Genome with PacBio® Data

http://www.slideshare.net/flxlex/combining-pacbio-with-short-read-technology-for-improved-de-novo-genome-assembly

http://www.slideshare.net/flxlex/a-different-kettle-of-fish-entirely-bioinformatic-challenges-and-solutions-for-whole-de-novo-

genome-assembly-of-atlantic-cod-and-atlantic-salmon#btnNext

“When we looked at these PacBio

reads mapping to the assembly, we

saw them crossing large gaps of

even multiple kilobases. I could

see that the problem of STRs

and heterozygosity could be

addressed by this technology.”

Lex Nederbragt, Ph.D.

Research Fellow

University of Oslo

Case Study: Long Reads Offer Unique Insight

English et al. (2012) Mind the Gap: Upgrading Genomes with Pacific Biosciences RS Long-Read

Sequencing Technology. PLoS One.

Towards Gap-Free Reference Genomes

D. melanogaster (139.5 Mb)

D. pseudoobscura (176.04 Mb)

M. undulatus (1.23 Gb)

C. atys (2.82 Gb)

Original PacBio Original PacBio Original PacBio Original PacBio

Gap Count 4651 311 6026 1852 49,376 39,204 186,841 66,211

Total Gap Size (Mb) 3.19 0.54 6.67 3.61 154.9 134.6 197.5 79.3

Contig N50 (kb) 64 723.6 53 224.4 134.4 233.27 34.92 128.38

Contig N50

Improvement

1030.6%

(11.3x) 323.4%

(4.2x) 73.6%

(1.74x) 267.6%

(3.68x)

Improve Assemblies with Low PacBio® Coverage

“With the RS, the contigs from

our de novo assembly of the 400

Mbp rice genome are several fold

better than the state-of-the-art

ALLPATHS-LG assembly using

short reads”

Michael C. Schatz, Ph.D.

Assistant Professor of Quantitative Biology

Cold Spring Harbor Laboratory

Rice Genome Assembly (Oryza sativa pv Nipponbare: 400 MB)

Contig N50

HiSeq® Fragments 50x 2x100bp @ 180

3,925

MiSeq® Fragments 23x 459bp

8x 2x251bp @ 450

6,332

Illumina® Mates 50x 2x100bp @ 180

36x 2x50bp @ 2100

51x 2x50bp @ 4800

18,248

PBeCR + Illumina reads 7x 3500bp ** MiSeq reads for

correction

50,995

PBeCR + Illumina reads 19x ** MiSeq reads for correction

155 kb

http://schatzlab.cshl.edu/presentations/2013-04-10.UVA.De%20novo%20assembly%20of%20complex%20genomes.pdf

http://schatzlab.cshl.edu/presentations/2013-06-18.PBUserMeeting.pdf

Case Study: The Next Frontier in Assembly – Long Reads Offer Finished Genomes

Hybrid Assembly for Improving Larger Genomes

Melopsittacus

undulatus

(parrot)

vs.

Taeniopygia

guttata

(Zebra finch) ®

®

More Gene Content in

Assemblies with PacBio®

Long Reads

Koren et al. (2012) Hybrid error correction and

de novo assembly of single-molecule

sequencing reads.

Nature Biotechnology 30, 693–700.

Tackle De Novo Assembly of Large Genomes

Novel Rumen Fungal Genome (100.95 MB) (Orpinomyces sp. Strain C1A)

• Anaerobic fungal cultures from Angus steer

• Motivation to understand biomass degradation

• 10x PacBio® sequencing improved Illumina® assembly

Illumina platform

(29 Gb) Illumina + PacBio platforms

(1 Gb)

Genome Assembly Size 105.1 MB 100.85 MB

# ambiguous bp 91,688 bp 0

# Contigs 82,325 32,574

N50 1 KB+ Contigs 2,226 bp 3,373 bp

N90 1 KB+ Contigs 1,072 bp 1,829 bp

Avg. Gene Model Length 903 1,623

# introns 2,458 35,697

# gene models 14,594 16,437

% GC Content 15.8 17

Youssef NH et al. (2013) The Genome of the Anaerobic Fungus Orpinomyces sp. Strain C1A Reveals the Unique Evolutionary History of a

Remarkable Plant Biomass Degrader. Appl Environ Microbiol, 79(15):4620-34

Significant improvements in gene models and transcript alignment.

Detect Transposable Elements

Improved genome assemblies

allows for transposable

element analysis.

Transposable element breakdown

for fungus Orpinomyces sp. Strain

C1A.

46

TE Class Detected occurance

Total Length (bp) % Genome Coverage

Cla

ss I

LTR

Copia 2,482 1,197,759 1.19

Gypsy 2,752 1,533,722 1.58

Pao 49 5,972 0.01

no

n-L

TR :

LIN

ES L1 77 38,441 0.04

L2 76 28,505 0.03

CR1 3 492 0.00

Rex 8 3,937 0.00

RTE 157 40,204 0.04

RTE-X 545 214,720 0.21 C

lass

II hA 9 1,926 0.00

MuDR 495 269,488 0.27

Pdinton 11 1,552 0.00

Total 6,664 3,336,718 3.31%

Youssef NH et al. (2013) The Genome of the Anaerobic Fungus Orpinomyces sp. Strain C1A Reveals the Unique Evolutionary History of a

Remarkable Plant Biomass Degrader. Appl Environ Microbiol, 79(15):4620-34

Resolve Difficult BACs

Aluminum tolerance in maize is important for drought resistance and

protecting against nutrient deficiencies

• Segregating population localized a QTL on a BAC, but unable to genotype

with short-read sequencing because of high repeat content and GC skew

• BAC assembly with PacBio® long reads revealed a triplication of the

ZnMATE1 membrane transporter

Genomic organization of the MATE1 locus

Maron, LG et al. (2012) A rare gene copy-number variant that contributes to maize aluminum tolerance and adaptation to acid soils. PNAS

PacBio® Long Reads Span Full Length Transcript

• Recover missing exons

• Gene structure annotation

• Identify gene isoforms and splicing events

• New gene identification in absence of reference genome

Koren et al. (2012) Hybrid error correction and de novo assembly of single-molecule sequencing reads.

Nature Biotechnology 30, 693–700.

Example Annotation with Corn Transcriptome

Arabidopsis Assembly Offers Glimpse of De Novo SMRT

Sequencing for Larger Genomes

Sample Ler-0 Ler-0 Short Read

Assembly (2011)

Assembl

y Total

Size

124,572,7

84

110,357,1

64

Missing

significan

t chunk

# contigs 540 4,662 8.6X

more

Contig

N50

6,190,353 66,600 ~1/90th

PacBio’s

assembly

Max

Contig

Length

12,982,39

0

462,490

~ 1/30th of

PacBio’s

assembly

General Conclusion:

PacBio’s data provides a more complete assembly.

Pacific Biosciences’ Customers are Improving Large

Genome Assemblies

Large genomes assemblies being improved using

SMRT® Sequencing:

– Cotton

– Rice

– Wheat

– Salmon

– Sea Bass

– Medaka

– Pig

– …and more

PacBio® Benefits for Large Genomes

• PacBio complements short reads to improve

new and existing de novo assemblies

• Improve N50 contig length with modest

5x coverage

• Scaffold PacBio® long reads to set framework

for genome completion

• Resolve troublesome gaps with low-complexity

and repetitive genomic regions

• Increase annotations of gene structure with

transcripts

• Catalog transposable elements

PacBio® De Novo Assembly Homepage

PacBio® Targeted Biomedical Research

Applications

Targeted Sequencing: High-Resolution Insights

Exquisite sensitivity and specificity to fully

characterize genetic complexity

– Multi-kilobase reads

– 99.999% consensus accuracy

– Linear variant detection to <0.1% frequency

– Access to the entire genome

SNP Detection and Validation Repeat Expansions

Full-Length Transcripts and Splice Variants

Compound Mutations and Haplotype Phasing Minor Variants Detection

www.pacb.com/target

HLA Complex

54

PacBio® SMRT ® Full-Length Gene HLA Typing

55

Long-Read SNP Phasing

• Long reads provide haplotype directly

• Example: heterozygous SNPs across 5 kb amplicon at 20x coverage

maternal:

paternal:

maternal:

paternal:

vs.

~5 kb

Phasing:

A--C

T--A

A--A

T--C

…AGACACGACATGCG… …TCTGCACCGGCCT…

…GACTTGTCCGCGTT… …CAGCTTGAGGATA…

…AGACACGACATGCG…

…GACTTGTCCGCGTT…

…CAGCTTGAGGATA…

…TCTGCACCGGCCT…

56

Long-Read SNP Phasing

• Long reads provide haplotype directly

• Example: heterozygous SNPs across 5 kb amplicon at 20x coverage

• Phasing:

9 A--A

7 T--C

0 A--C

0 T--A

1 A--del

1 del--C

1 T--del

1 C--C

~5 kb 57

Full Phasing Information for HLA Haplotyping

• Amplified HLA-A region via long-range PCR (~3,000 bases)

• SMRT® sequencing of the full gene (all exons & introns) with long reads

• Phase and compare to HLA database:

Sample 1 HLA-A Type Comment

Best Match A*02:05:01

2nd Best

Match

A*02:06:01 5 SNPs from

the best match

Sample 2 HLA-A Type Comment

Best Match A*02:01:01:01

2nd Best

Match

A*02:07:01 only one SNP

from the best

match

Differ by 7 SNPs over

3 kb region

58

FLT3 Compound Mutations and Haplotype Phasing

• FLT3 mutations impact acute myeloid leukemia treatment

• Activating internal tandem duplication (ITD) mutations in FLT3 detected in ~

20% of AML patients and associated with a poor prognosis

• Potential resistance mutations located > 800 bp away from ITD region

F691 D835 Y842 E608

ITD

(20-100 bp repeat) > 800 bp

One PacBio® Read Spans Region

Smith et al. (2012) Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukemia. Nature

485, 260–263.

Case Study: A New Hope in Acute Myeloid Leukemia Treatment

Secondary and Rare Polyclonal Mutations for Resistance Identified

Pre-Treatment Relapse Normal Control #1

Subject

Number Mutation

Native

Codon

Alternative

Codon

Observed

Alternative

Codon

Frequency in

ITD+

Sequences

Total Number

of ITD+

Sequences

Sampled

Observed

Alternative

Codon

Frequency

In ITD+

Sequences

Total Number

of ITD+

Sequences

Sampled

Observed

Alternative

Codon

Frequency

Total Number

of Sequences

Sampled

1009-003 D835Y GAT TAT 0.21% 482 8.4% 332 0.00% 768

D835V GAT GTT 0.00% 482 3.3% 332 0.13% 768

D835F GAT TTT 0.00% 482 10.2% 332 0.00% 768

1011-006 D835Y GAT TAT 0.00% 196 41.0% 402 0.00% 768

1011-007 F691L TTT TTG 0.18% 561 6.2% 341 0.22% 450

D835Y GAT TAT 0.00% 930 3.0% 436 0.00% 768

D835V GAT GTT 0.43% 930 29.6% 436 0.13% 768

1005-004 F691L TTT TTG 0.00% 496 29.6% 513 0.22% 450

1005-006 D835Y GAT TAT 0.00% 171 39.5% 261 0.00% 768

D835F GAT TTT 0.00% 171 2.7% 261 0.00% 768

1005-007 D835Y GAT TAT 0.00% 57 4.0% 378 0.00% 768

D835V GAT GTT 0.00% 57 47.4% 378 0.13% 768

1005-009 D835Y GAT TAT 0.00% 19 50.6% 445 0.00% 768

1005-010 F691L TTT TTG 0.00% 387 25.3% 150 0.22% 450

Coupled Secondary Mutations Rare Polyclonal Mutations (<5%)

Smith et al. (2012) Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukemia. Nature

485, 260–263.

Trinucleotide Repeat Disorders

• Set of genetic disorders caused by trinucleotide repeat expansion

• A mutation where trinucleotide repeats in certain genes exceed the normal,

stable, threshold, which differs per gene

• Disease Examples:

– Autism

– Mental retardation (especially males)

– Huntington’s disease

– Fragile X syndrome

AGGTAT CGGCGGCGGCGGCGGCGGCGGCGGCGG AGATC …

AGGTAT CGGCGGCGGCGGCGGCGGCGGCGGCGG AGATC …

120

600+

A First Look into a Repeat Expansion Disorder

• Repeat regions sequenced accurately

• Sequence through up to 750 CGG repeats

Loomis et al. (2013) Sequencing the unsequenceable: Expanded CGG-repeat alleles of the fragile X gene. Genome Research, 23(1):121-8

Case Study: SMRT® Sequencing Provides a First Look at Repeat Expansion Disorder Sequence

Filling the Genomic Gap in MUC5AC Gene using PacBio Long

Reads and De Novo Assembly

63

SMRT® Sequencing of Intact, Full-Length HIV-1 Genomes From

Single Molecules to Study HIV Transmission

• Complete HIV-1 genomes from single

molecules

– Sanger-quality, fully phased

– Samples of complex mixtures

• Eliminate need for Single Genome

Amplification

• Full genomic characterization of clinical

transmission events

Donor (chronic infection)

Recipient (acute infection)

Full HIV Genome – 9, 084 bp

Collaboration with CFAR site at Emory University

Poster: Rapid Sequencing of HIV-1 Genomes as Single Molecules from Simple and Complex Samples

Reliably Detect Variant Mutations Below 0.1% Frequency

All minor variants

reliably detected

down to 0.08%

L180M

254 C A

S202G

320 A G

M204V

326 A G

Single SMRT® Cell provides enough data to detect HBV minor variants:

Poster: Sensitive Detection of Minor Variants and Viral Haplotypes

Complete Capture of Full-Length, No Assembly Transcripts

• Recover missing exons

• Gene-structure annotation

• Identify gene isoforms and splicing events

• New gene identification

Single PacBio read covers 32-exon mRNA:

Poster: Full-Length cDNA Sequencing on the PacBio® RS

• Entire length of human glutamyl-prolyl-tRNA synthetase mRNA

• cDNA primers observed at both ends

• PolyA tail observed at the 3’ end indicates correct end capture

A Strength of PacBio Long Read Technology is the

Unambiguous Detection of Splice Isoforms

CCS alignments uncover multiple isoforms of the CDK4 transcript. Exons 2, 3, 4, 5, 6, and 7 are

skipped in various combinations. The 5’ end also shows variable transcription start sites.

Survey of the human transcriptome using PacBio

68

Validation of Illumina® SNPs with SMRT® Sequencing

• Whole‐exome hybrid capture and deep sequencing to identify somatic

mutations in 92 primary medulloblastoma‐normal pairs

• All SNPs studied were validated (including 2 bp deletion in CTDNEP1)

69

PacBio®

Illumina®

PacBio®

Illumina®

Pugh et al. (2012) Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations. Nature 488, 106-110.

Accurate SNP Detection

Discordant PacBio® data confirmed by

PCR-Sanger to be 100% correct

Genomic Regions Associated

with Mental Retardation

• Comparison of a BAC libraries thought to

be highly similar to HG19 reference

genome

• Discordant bases (~30 sites) between

PacBio data and HG19 (Sanger) data

were identified

• PacBio calls 100% confirmed by PCR-

Sanger

Clone HG19 Ref PacBio Sanger

BAC 1

T G G

T -- --

T A A

G T T

C T T

C T T

G C C

C G G

BAC 2

C T T

C G G

A G G

G A A

T C C

T C C

C T T

BAC 3

T -- --

T -- --

T C C

T C C

G T T

A C C

BAC 4

A G G

T C C

A T T

T -- --

T G G

In collaboration with Evan Eichler (HHMI, University of Washington)

Implementation of cancer sequencing in the clinic

Using PacBio sequencing in CLIA setting

72

Pathogenic Repeat Panels Carrier Screening

73

TNR

74

Beyond Targeted Sequencing: SMRT® Sequencing of Whole Human Genome Reveals Undetected Variations

Mt. Sinai human-genome sequencing (NA12878 from CEPH)

• Detect clinically significant variants not detected with short-read technologies

• Identify unexplored structural variants across the genome

• Develop new, clinically relevant gene panels only identifiable using PacBio® technology

454® Reads

Number of reads ~100M

Mapped coverage ~15X

Single and Paired-End Illumina® Reads

Number of reads ~100s of M

Mapped coverage ~30X

PacBio® Reads

Number of Reads ~12M

Mapped coverage 10X+

Mean subread length 2,766

Mean unrolled read length 4,066

95th Percentile 11,630

Accuracy (error-corrected reads) >99%

Watch Eric Schadt present the complete story

PacBio® Benefits for Targeted Biomedical Research

• Achieve >99.999% consensus accuracy (QV 50)

• Direct strand-specific haplotype phasing with multi-kilobase reads

• Resolve troublesome gaps with low-complexity and repetitive genomic

regions

• Improve gene-structure annotation with intact, full-length transcripts

• High-resolution detection of low-frequency minor variants

• Finish human genomes

PacBio® Targeted Sequencing Applications Page

Recent Publications

1) Efficient and accurate whole genome assembly and methylome profiling of E. coli

Authors:Jason G Powers, Victor J Weigman, Jenny Shu, John M Pufky, Donald Cox and Patrick Hurban

2) An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome

Authors:Marco Ferrarini, Marco Moretto, Judson A Ward, Nada Surbanovski, Vladimir Stevanovic, Lara Giongo, Roberto Viola,

Duccio Cavalieri, Riccardo Velasco, Alessandro Cestaro and Daniel J Sargent

3) Reducing assembly complexity of microbial genomes with single-molecule sequencing

Authors: Sergey Koren, Gregory P Harhay, Timothy PL Smith, James L Bono, Dayna M Harhay, Scott D Mcvey, Diana Radune,

Nicholas H Bergman and Adam M Phillippy

4) Genome Reference and Sequence Variation in the Large Repetitive Central Exon of Human MUC5AC

Authors: Xueliang Guo, Shuo Zheng, Hong Dang, Rhonda G Pace, Jaclyn R Stonebraker, Corbin D Jones, Frank Boellmann, George

Yuan, Prashamsha Haridass, Olivier Fedrigo, David L Corcoran, Max A Seibold, Swati S Ranade, Michael R Knowles, Wanda K

O'Neal, and Judith A Voynow

5) Comparing the genomes of Helicobacter pylori clinical strain UM032 and Mice-adapted derivatives

Authors: Yalda Khosravi, Vellaya Rehvathy, Wei Yee Wee, Susana Wang, Primo Baybayan, Siddarth Singh, Meredith Ashby, Junxian

Ong, Arlaine Anne Amoyo5, Shih Wee Seow, Siew Woh Choo, Tim Perkins, Eng Guan Chua, Alfred Tay, Barry James Marshall, Mun

Fai Loke, Khean Lee Goh, Sven Pettersson and Jamuna Vadivelu

6) The advantages of SMRT sequencing

Authors: Richard J Roberts, Mauricio O Carneiro and Michael C Schatz

7) McGill University Team Develops Rapid Genome Sequencing Technique for Outbreak Monitoring 77

FIND MEANING IN COMPLEXITY

© Copyright 2013 by Pacific Biosciences of California, Inc. All rights reserved.

PacBio® Roadmap

PacBio® Advances in Chemistry and Software

79

Early PacBio chemistries

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

q108 q208 - 453

q308 q408 q109 q209 -

1012

q309 -

1734

q409 q110 q210- lpr

q310 q410 q111 q211 - fcr

q311 q411 - ecr2

q112 q212 - c2

q312 q412 - xl

q113 q213 q313 q413 -

p5c3

453 1012

1734 LPR

FCR

ECR2

C2–C2

P4–C2

P5–C3

8,500 bp

Read L

ength

(b

p)

2008 2009 2010 2011 2012 2013

BluePippin™ System for Size Selection

Pol Protecting

Scaffold

Dye

Pol

Polymerase

surface that

dye can

access

Dye cannot

access

polymerase

surface

Photodamage Mitigation: Photo-Protected Analogs

Large macromolecule scaffold can prevent the dye from touching the polymerase

Dye

81

BluePippin™ Size-Selection Protocol

P5-C3, E coli 20 kb BluePippin™ size-selected library, 3-hour movie

P5-C3, E. coli 10 kb library, 3-hour movie

Avg Subread lengths: 3,427 bp

Subread N50: 5,607 bp

Mapped Reads: 47,321

Avg Subread lengths: 7,537 bp

Subread N50: 10,725 bp

Mapped Reads: 41,301

Official Procedure for 20 kb Template Preparation

Impact of Improvements for Microbial Assembly

0

4

8

12

16

20

24

28

32

Jan 2012

C2 Release Sept 2012

MagBead

Dec 2012

XL Release

32 SMRT Cells

Two SMRTbell™ libraries

DevNet tools - hybrid

Degree of Completeness & Quality

SM

RT

® C

ells

<35 contigs,

Q40

<5-10 contigs, Q50,

plus methylome

16 SMRT Cells, 2 libraries

Hybrid - Celera® Assembly

Identify base modifications

Quiver for consensus (DevNet)

4 - 8 SMRT Cells

Single SMRTbell library

HGAP assembly pipeline w/ Quiver

Automated methylation detection

Q2 2013

150K, Size

Selection

1 SMRT Cell

Size-selected library

HGAP w/ Quiver

Single contig per

chromosome, Q50,

plus methylome

Examples based on E. coli

Upcoming Improvements Will Make Generation of 10X

PacBio® Coverage of 3 GB Genomes More Economical

84

Estimated per

SMRT® Cell

Throughput (MB)*

Estimated Number

of SMRT® Cells

per 10X

Instrument

Run Time

(days)

Beginning of 2013 ~100 300 38

Today’s

Throughput

-150K

~200 150 19

Optimally loaded

size-selected

libraries

~400 75 7

Photo-protected

Analogs ~800 38 4

one SMRT Cell =

one microbial

de novo genome

& epigenome

FIND MEANING IN COMPLEXITY

© Copyright 2013 by Pacific Biosciences of California, Inc. All rights reserved.

Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, and SMRTbell are trademarks of Pacific Biosciences in

the United States and/or other countries. All other trademarks are the sole property of their respective owners.