Reduce cost and length by 50% or more on whole-genome sequencing projects

www.miraibio.com | [email protected] | 1-424-237-8524 | © Hitachi Solutions America, Ltd. 2011. All rights reserved. 0

www.miraibio.com | [email protected] | 1-424-237-8524 | © Hitachi Solutions America, Ltd. 2011. All rights reserved.

Presenter: Robert Lynde Deputy Director

Panelist: Hector Salcedo Account Manager

Webinar: Reduce cost and length of whole-genome sequencing by 50% or more

November 16, 2011

Please note: This presentation accompanies the

webinar recording at:

https://www1.gotomeeting.com/register/730705248

https://www1.gotomeeting.com/register/730705248


A little bit about Hitachi and what we do…

• MiraiBio Group has been serving life

science customers since 1991

• Part of the Hitachi family of companies

that have been around for more than 100

years

• #52 on the Global 500

• Everything from bullet

trains to wet lab services

• DNASIS – Sequence analysis software

released in 1983!

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How can you reduce the cost and length of

your sequencing project by 50% or more?

Whole Genome Mapping (WGM)

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Agenda

• What is Whole Genome Mapping (WGM)?

• When whole genomes matter

• How WGM can reduce the cost and time of a

sequencing project by 50% or more

• Examples

– Close your gaps with less PCR

– Resequencing Validation

– Sequence Read Quality = Assembly Quality

• MapIt Service – Get your own WGM!

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What is Whole Genome Mapping?

Whole Genome Mapping is a de novo process that

generates whole genome, ordered, restriction maps with no

requirement for previous sequence information & provides

a comprehensive view of genomic architecture.

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After staining with intercalating dye, the digestion reveals restriction cleavage sites as ―gaps‖, under fluorescent microscopy

Cells gently lysed to extract long genomic DNA molecules, pieces of chromosomes

DNA is captured in parallel arrays of single DNA molecules using microfluidic device




Image Analysis and Markup


40.52

1.56

51.99

8.08

24.45

58.94

17.93

8.89

45.26

28.99

7.20

46.25

5.52

27.52

Map Assembly

Overlapping single molecule restriction maps are aligned to produce a map assembly covering an entire chromosome



Map Assemblyconsensus map

Overlapping single molecule restriction maps are aligned to produce a map assembly covering an entire chromosome



Whole Genome Maps are compared to perform high resolution epidemiology, discover genetic

variation, and accelerate sequence assembly

Comparative Genomics

Strain Typing

Sequence Assembly


Applications of Whole Genome Mapping


Not all Genomes are created equal

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When whole genomes matter

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• Gene dosage

– Duplications

• Genome variation

– Repetitive regions

• Location of genes across the genome

– Operon Structure

– Gene regulation

– Locality of genes working together

– Genomic rearrangements

• Accuracy and quality

• Missing sequence or genes

Confidential—not for customers


Sequence Finishing

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Sequence Finishing

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Order&

Orient

Gaps

Overlaps



Example – Reduce PCR

Whole Genome Mapping reduces the number of PCRs

needed

• ―From a finishing perspective, these scaffolds are particularly

useful, as for a set of n contigs, they help reduce the number of

PCR experiments needed from roughly n2 to n [23].” P 7

• Example: ―Using only 43 PCR experiments and 26 sequencing

reactions 33 of the gaps were closed, leaving only 7 gaps to close.

In contrast, working with the original assembly (59 large contigs)

could have necessitated on the order of 592 ≈ 3000 PCR

experiments (see Table 1).‖ P 4

Finishing genomes with limited resources: lessons from an ensemble of microbial genomes Nagarajan et al.

BMC Genomics 2010, 11;242


Example - Resequencing Validation

Anne Buboltz, Microbial Genomics Conference (2009)

• Resequencing sequence assembly was not validating phenotypic differences

• Whole Genome Map was made of sequenced isolate


Alignment with MapSolver™: Four Misassemblies

Contig Breakage and alignment:

12 4

3



Inversion Identified—Comparison to the in silico reference strain sequence

Inversion Validated—Comparison to Whole Genome Map of reference strain



We have a data problem

Remains

“As next-generation sequencing generates more data, managing, storing and analyzing this

data will become a major sequencing bottleneck.” BCC 2010 Sequencing Market Research Report

?

Advances in New Sequencing Technologies move the Sequencing Bottleneck


Sequencing Read Quality=Assembly Quality

• 13 Randomly selected ATCC Reference Genomes from Genbank

• Finished Whole Genome Sequences

• 8 of 13 or 62% Contained Assembly Errors

New DataPresented

SFAF June 2011

• 11 Different Species

• Insertions, Deletions, Inversions and Translocations found


A. baumannii—insertion

S. cerevisiae—deletion

V. cholerae—inversion

V. cholerae—potential

resequencing error?

New DataPresented

SFAF June 2011

Sequencing Read Quality=Assembly Quality


How does the MapIt service work?

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Send us your sample DNA is extracted & used in restriction digest

Whole Genome Map is assembled

Visualize your Whole Genome Map


Sequence only the really important strains

• Save Time• Save Money• Publish Faster

Improve your sequence assembly pipeline

• Order and Orient your contigs to a whole genome scaffold

• Reduce the number of PCRs and sequencing reactions

Sequence Independent Validation

• Restriction sites are in the order that they appear in the genome—generating a whole genome scaffold

• Depth of SMRM coverage ensures accuracy

Summary


Resources

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• Learn More: http://www.miraibio.com/mapit/optical-map-

service

• Support: [email protected] or 1-650-615-7680

• Sales: [email protected] or 1-424-237-8524

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• Knowledge Base

• Blog http://www.MiraiBio.com/blog

• Webinars: http://www.MiraiBio.com/Webinars

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Technology

Reduce cost and length by 50% or more on whole-genome sequencing projects