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

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

Please note: This presentation accompanies the
webinar recording at:
https://www1.gotomeeting.com/register/730705248

November 16, 2011
Presenter: Robert Lynde Deputy Director
Panelist: Hector Salcedo Account Manager

www.miraibio.com | support@miraibio.com | 1-424-237-8524 | © Hitachi Solutions America, Ltd. 2011. All rights reserved.

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!

How can you reduce the cost and length of
your sequencing project by 50% or more?

Whole Genome Mapping (WGM)


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!


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.



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

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



Image Analysis and Markup



Map Assembly

27.52
40.52

51.99

24.45

58.94

17.93
8.89

45.26

28.99

7.20

46.25
5.52
1.56

8.08

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


Map Assembly
consensus map

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



Applications of Whole Genome Mapping

Whole Genome Maps are compared to perform high
resolution epidemiology, discover genetic
variation, and accelerate sequence assembly

Strain Comparative Sequence
Typing Genomics Assembly


Not all Genomes are created equal


When whole genomes matter

• 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



Order
&
Orient

Gaps

Overlaps


Example – Reduce PCR

Finishing genomes with limited resources: lessons
from an ensemble of microbial genomes Nagarajan et al.
BMC Genomics 2010, 11;242

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


Example - Resequencing Validation

• Resequencing sequence
assembly was not validating
phenotypic differences

• Whole Genome Map was
made of sequenced isolate

Anne Buboltz, Microbial Genomics Conference (2009)


Alignment with
MapSolver™: 3 Four Misassemblies
1
2 4

Contig Breakage and
alignment:



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

Advances in New Sequencing Technologies move the
Sequencing Bottleneck

?

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

Sequencing Read Quality=Assembly Quality

• 13 Randomly selected ATCC Reference
Genomes from Genbank
New Data
Presented
SFAF June 2011
• Finished Whole Genome Sequences

• 8 of 13 or 62% Contained Assembly Errors

• 11 Different
Species

• Insertions, Del
etions, Inversio
ns and
Translocations
found


Sequencing Read Quality=Assembly Quality

New Data
Presented
SFAF June 2011

A. baumannii—insertion

S. cerevisiae—deletion

V. cholerae—inversion

V. cholerae—potential
resequencing error?


How does the MapIt service work?

Send us your sample DNA is extracted & used Whole Genome Map is Visualize your Whole
in restriction digest assembled Genome Map


Summary

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


Resources

• Learn More: http://www.miraibio.com/mapit/optical-map-
service
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• Sales: info@miraibio.com or 1-424-237-8524
• MiraiBio Support Menu
• Community
• Knowledge Base
• Blog http://www.MiraiBio.com/blog
• Webinars: http://www.MiraiBio.com/Webinars


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