FIND MEANING IN COMPLEXITY

© Copyright 2014 by Pacific Biosciences of California, Inc. All rights reserved.

Steve Picton PhD,

Pacific Bioscience - Europe

What could you do with a 10kb read?: SMRT®

sequencing for de novo bacterial assembly and

methylation analysis.

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Close to home………………2013

Close to home………………2013

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What, where (from) ……………….and why. 2011

Cost implications…………….

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• We know <1% of the Earth’s microbiome

• Horizontal gene transfer is wide-spread and frequent

• High-quality, finished genomes are the starting point for:

– Functional genomic studies

– Comparative genomics

– Forensics………………

– Metagenetics

The case for de novo, Finished Microbial Genomes

Chain et al. (2009) Science 326: 236-237 Fraser et al. (2002) J Bacteriology 184: 6403-6405

Finished Genomes to Fight Foodborne Outbreaks

• ~76 million illnesses each year

• ~325,000 hospitalizations

• $78 billion economic loss (US)

• High serotype diversity

• Emerging hypervirulence

• ~76 million illnesses each year

• ~325,000 hospitalizations

• $78 billion economic loss (US)

• High serotype diversity

• Emerging hypervirulence

National Collection of Type Cultures (NCTC)

• Collaboration with Public Health England & the Wellcome Trust

Sanger Institute

• Plan to finish 3000 bacterial genomes

Sanger Sanger/454 454/Illumina Illumina Illumina/PacBio PacBio

2002-2006 2006-2008 2011 2008-2011 2011-2013

Historic timeline of JGI sequencing of bacteria and archaea:

2013-

Return of the Finished Genome?!

$50k $35k $1.5-3k $10k $5k <$2k

No Finishing

49 contigs* 44 contigs 69 contigs 22 contigs 6 contigs 1 contig

*contig counts = medians for all JGI

projects

Manual Finishing ($35k/genome)

$45-85k

A. Copeland (JGI)

FIND MEANING IN COMPLEXITY

© Copyright 2014 by Pacific Biosciences of California, Inc. All rights reserved.

SMRT® technology - Overview

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

PacBio® RS II Trace

SMRT® Cells Zero-Mode

Waveguides

Phospholinked

Nucleotides

PacBio® Advances in Read Length

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Early PacBio chemistries

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

453 1012 1734 LPR

FCR ECR2

C2–C2

P4–C2

Read L

ength

(bp)

2008 2009 2010 2011 2012 2013 2014 2015

P5–C3

Long Read Lengths needed to Resolve Repeats

• E. coli:

>1 kb

>2 kb

>5 kb

Repeats

>99% identical

with length: 5.4 kb repeat

Repeat instance 1:

Repeat instance 2:

Fully Resolving the Legionella

pneumophila rtxA Locus • D4174:

• D4174: 13.8 kb

• LR1342: 17.8 kb

Collaboration with A. Ensminger (U Toronto & PHO) & K. Dewar (McGill)

• LR1342:

25 repeats; 14kb region

550bp

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10

14

18kb region

Resolving the Legionella pneumophila

rtxA Locus • D4174:

Collaboration with A. Ensminger (U Toronto & PHO) & K. Dewar (McGill)

• A single 23 kb PacBio read:

• A single 300bp Illumina read:

The Pertussis Genome is Extremely Repetitive

• Bordetella pertussis: • E.coli:

>1 kb

>2 kb

>5 kb

Repeats

>99% identical

with length:

>8% repeats >500bp

First sequenced 2002, Parkhill

Genome Organization Comparison – Presented AGBT 2013

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

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

1917

3582

1920

3585

3640

3405

3658

3913

3921

2011, CS

2003, Tohama

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

Tracking hospital associated

bacteria with epidemiology and

genomic sequencing

Julie Segre, PhD

Senior Investigator

National Human Genome Research Institute

Presented at the 60th NIH Clinical Center Anniversary, November 6th, 2013

De novo assembly of plasmids from draft genomes is difficult

poorly aligned contig

repeat regions

indel

ind

el

reference sequence

Fold Coverage Alignment to Reference

Fragmented and incomplete information, plasmids unresolved

Targeted sequencing could have given misleading result

Identical

Unrelated

pt #1 pt #53

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Full genome sequencing rules out patient-patient transmission

Klebsiella pneumoniae Klebsiella pneumoniae pKpQIL

KPC-2

KPC-3

Horizontal gene transfer from patient to environment

Klebsiella pneumoniae Enterobacter cloacae Citrobacter freundii

KPC-2

KPC-3

Salmonella De Novo Assemblies

Strain Genome size Additional genomic elements

S. Bareilly (SAL2881) 4,730,611 bp 78,193 bp

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

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

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

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

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

S. St.Paul SP3 4,730,130 bp none

S. St.Paul SP48 4,940,224 bp 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)

• DNA polymerization runs freely (currently at ~3 bases/sec)

• Kinetic information for each nucleotide addition

• Can be used to infer presence of bases different from A, C, G or T

Kinetics in Single-Molecule, Real-Time DNA Sequencing

Interpulse

duration

(IPD)

Epigenome Information

Comprehensive Understanding of Pathogens

The E. coli Outbreak Strain Methylome

E. Schadt (Mt. Sinai), M. Waldor (Harvard) & Rich Roberts (NEB)

Lambda-like phage element specific to

outbreak strain

• Contains stxAB

• Contains putative methyltransferase

and restriction enzyme for CTGCAG

motif 55989

C227-11

C734-09

C35-10

C682-09

C760-09

C754-09

C777-09

Novel sequence motif: CTGCm6AG

Effects of Horizontal Gene Transfer on Pathogenicity

M.EcoGIII

Summary -

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• ‘Whole genome sequencing’ does not always deliver whole genome assembly

• High accuracy , long contiguous reads enable

• Complete genome assembly

• Identification of complex genome rearrangements

• Precise location of phage insertions

• Identity of extra chromosomal elements

• Kinetic data enables identification of methylation events

• Strain specific methylation may provide addition identifiers

Thank you

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