find meaning in complexity © copyright 2013 by pacific biosciences of california, inc. all rights...

FIND MEANING IN COMPLEXITY

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

Kevin Corcoran, SVP Market Development

PacBio User Group Meeting September 18, 2013

PacBio User Meeting

WelcomeWest Coast User Group Meeting

2

Thank You!Your Success

IsOur Success

More than 50 customer publications

Special Thanks to our Reception Sponsors

4

Latest Advances – Last 9 Months

5

HGAP & Quiver PacBio® RS II P4 Enzyme


6


Finishing Genomes Using Only PacBio® Reads

• Utilizes all PacBio data from single, long-insert library

– Longest reads for continuity

– All reads for high consensus accuracy

Hierarchical Genome Assembly Process (HGAP)

Quiver: A New Consensus Caller for PacBio® Data

• Can achieve accuracy >Q50 (i.e. > 99.999%) using only PacBio reads

• How Quiver works

– Takes multiple reads of a given DNA template, outputs best guess of template’s identity

– QV-aware hidden Markov model to account for sequencing errors; a greedy algorithm to find the maximum likelihood template

– Similar underlying algorithm as currently used for CCS generation

• Links:

– www.pacbiodevnet.com/quiver

– https://github.com/PacificBiosciences/GenomicConsensus

8

https://github.com/PacificBiosciences/GenomicConsensus




9

SMRT® Sequencing Accuracy

0 10 20 30 40 50 60 70 80 90 100

Coverage

Co

nco

rdan

ce -

A

ccu

racy

(Q

V)

99.99999% (QV 70)

99.9999% (QV 60)

99.999% (QV 50)

99.99% (QV 40)

99.9% (QV 30)

99% (QV 20)

90% (QV 10)

Data generated with P4-C2 chemistry on PacBio® RS II; Analyzed using Quiver with 2.0.1 SMRT® Analysis

E. coli

R. palustris

S. aureus

Perfect consensus


10


PacBio® RS II

Pre- and Post-PacBio® RS II Upgrade

Throughput effectively doubles with no significant changes to other sequencing metrics

Organism Chem Insert Size

System Model

Filtered Reads

Filtered Bases (Mb)

Mapped Subreads

Avg Subread Length

Avg Read Length

95th % Read

Length

Max Read Length

Single Pass

Accuracy

B. subtilis C2/C2 10 kb RS II 68,475 221.8 91,071 2,000 2,729 7,215 21,060 86.90%

R. palustris C2/C2 10 kb RS II 63,115 208.4 81,998 2,104 2,784 7,457 19,798 85.50%

E. coli C2/C2 10 kb RS II 73,820 293.9 107,520 2,172 3,234 8,446 20,195 85.00%

E. coli C2/C2 10 kb RS 32,407 107.9 46,894 1,938 2,784 7,237 19,006 85.00%

E. coli E. coli B. subtilis R. palustrisE. coli E. coli B. subtilis R. palustris0

50

100

150

200

250

300

350

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

Megabases per SMRT® Cell Reads per SMRT® Cell

Meg

abas

es p

er

SM

RT

® C

ell

Rea

ds

per

SM

RT

® C

ell

DNA polymerase from Bacillus subtilis phage phi29 • B-family polymerase • Highly processive• High fidelity (3’ to 5’ exonuclease)• Good strand displacement activity• Single subunit (66 kDa)

Berman, A.J. et al., EMBO J. (2007) 26(14):3494-505

Phi29 DNA Polymerase

P4 Polymerase Performance

• Performance summary of current chemistries

• Key Points

– P4 polymerase shows similar consensus accuracy as C2 polymerase

– P4 polymerase shows similar read lengths as XL polymerase

*E. coli 10 kb library, PacBio® RS II, Stage Start, 120 minute movies

Enzyme Sequence Chemistry

QV50Coverage

Mean Mapped Read Length

P4 C2 32x 3,995

C2 C2 32x 3,235

XL C2 70x 4,012

Typical Results: Accuracy with P4 Polymerase

Consensus Accuracy

XL-C2

C2-C2

P4-C2

Coverage

QV

*E. coli 10 kb library, PacBio® RS II, Stage Start, 120 min movies, C2 sequencing chemistry, SMRT® Analysis 2.0.1

• Key Points

– P4 polymerase shows similar consensus accuracy as C2

– P4 polymerase outperforms XL

Increased Read Length Beyond P4 Enzyme

Photodamage Impacts Read Length

Pol Protecting Scaffold

Dye

Pol

Polymerase surface that

dye can access

Dye cannot access

polymerase surface

Photodamage Mitigation: Photo-Protected Analogs

A large macromolecule scaffold can prevent the dye from touching the polymerase

Dye

Early PacBio chemistries

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

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

453 1012

1734LPR

FCR

ECR2

C2–C2

P4–C2

P5–C38,500 bp

Rea

d Le

ngth

(bp

)

2008 2009 2010 2011 2012 2013

P5-C3 Chemistry Average Read Lengths

Throughput: ~ 350-400 Mb

New Products and Features

Reagents• DNA Sequencing Reagent 3.0 • DNA/Polymerase Binding Kit P5

Instrument control Software 2.1• 3 hour movies• CCS processing off instrument• Reliability enhancement: Instrument adjustment to SMRT Cell SNR

SMRT Analysis 2.1• Amplicon Haplotype phasing• Diploid Quiver • BridgeMapper

Binding Calculator• Support for Size Selected libraries

New Protocol to support Sage size selection• Fully supported 20kb size selected library protocol



Instrument Control Software 2.1• 3-hour movies• CCS processing off instrument• Reliability enhancement: Instrument adjustment to SMRT® Cell SNR

SMRT Analysis 2.1• Amplicon Haplotype phasing• Diploid Quiver • BridgeMapper





Instrument Control Software 2.1• 3-hour movies• CCS processing off instrument• Reliability enhancement: Instrument adjustment to SMRT Cell SNR

SMRT® Analysis 2.1• Diploid Quiver• Amplicon Haplotype Phasing • BridgeMapper







Binding Calculator• Support for size-selected libraries






Binding Calculator• Support for size-selected libraries

New Protocol to support Sage Science’s BluePippin™ size selection• Fully supported 20 kb size-selected library protocol

Social Media Policy

• Talks are ‘tweetable’ unless speakers says otherwise

• Use #PacBioUGM for hashtag

• Follow us: @PacBio

• Subscribe to our Blog at:

Blog.pacificbiosciences.com@PacBio

Morning Agenda

Time Agenda

9:00 – 9:15 a.m. Opening RemarksKevin Corcoran, Senior Vice President, Market Development, Pacific Biosciences

9:15 – 9:35 a.m. Overview of JGI’s Microbial and Fungal Reference Assembly PipelineAlex Copeland, DOE - Joint Genome Institute

9:35 – 9:55 a.m. The Epigenomic Landscape of BacteriaMatthew Blow, Ph.D., DOE - Joint Genome Institute

9:55 – 10:20 a.m. Population Genomics & Molecular Diagnostics: 100K Pathogen GenomesBart Weimer, Ph.D., UC Davis - School of Veterinary Medicine

10:20 – 10:45 a.m. Population Genomic and Epigenomic Study of Arabidopsis thaliana with SMRT Sequencing Chongyuan Luo, Ph.D., Salk Institute for Biological Studies

10:45 – 11:20 a.m. Coffee Break

11:20 – 11:45 a.m. A Comparison of 454 and PacBio RS in the Context of Characterizing HIV-1 Intra-host DiversityLance Hepler, Ph,D.,Center for AIDS Research, UC San Diego

11:45 – 12:10 p.m. PacBio Meets the MicrobiomeGeorge Weinstock, Ph.D., Washington University St. Louis

12:10 – 12:35 p.m. Genomic Architecture of the KIR and MHC-B and -C Regions in OrangutanLisbeth Guethlein, Ph.D., Stanford University School of Medicine

Afternoon Agenda

Time Agenda

12:35 – 1:55 p.m. Lunch

1:55 – 2:20 p.m. Reconstructing Complex Regions of Genomes Using Long-read Sequencing Technology John Huddleston, M.S., University of Washington

2:20 – 2:45 p.m. Taking Advantage of Long RNA-Seq ReadsVince Magrini, Ph.D., Washington University St. Louis

2:45 – 3:10 p.m. Chicken in Awesome Sauce: A Recipe for New Transcript IdentificationAlisha K. Holloway, Ph.D., Gladstone Institutes

3:10 – 3:45 p.m. Break

3:45 – 4:10 p.m. Gene Isoform Identification by Error-corrected PacBio DataKin Fai Au, Ph.D., Stanford University

4:10 – 4:35 p.m. Optimizing BluePippin Size Selection for Increased Subread Lengths on the PacBio RS II Bobby Sebra, Ph.D., Mt. Sinai School of Medicine

4:35 – 5:00 p.m. Looking Ahead: Improving Workflows for SMRT SequencingJonas Korlach, Ph.D., Pacific Biosciences

5:00 – 6:30 p.m. Reception - Sponsored by Sage Science

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.

find meaning in complexity © copyright 2013 by pacific biosciences of california, inc. all rights...

Documents

pacbio slide

length slide

polymerase dye slide

xl slide

mb slide

c2 polymerase p4 polymerase

phi29 dna polymerase

c2 p4 polymerase