This presentation accompanies a webinar at: https://www1.gotomeeting.com/register/367952841
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Hitachi Solutions has partnered with OpGen to offer MapIt® Optical Mapping Services to our customers. Trevor Wagner, Senior Applications Scientist Manager from OpGen will be our guest presenter. Trevor was part of the team that developed, tested, and released OpGen’s first major product, the Argus Optical Mapping System in 2010.
This webinar will describe:
1. How Optical Mapping technology will benefit you in the following application areas:
-Strain Typing
-Comparative Genomics
-Whole-genome Sequence Assembly
2. How the MapIt Service works.
8. What is Optical Mapping?
Whole genome, ordered restriction maps
• Whole genome analysis of bacteria, yeast, fungi
– High level of precision
– Eliminates high cost of sequencing
• De novo process, no sequencing required
9. Optical Mapping
Locates and measures distance between restriction sites
acagctctcgagaggatcctcgtcgggatccctcgcgctcgagatcgcgtagcgctagagc
gctctagaggctcgcggagagctcgcgcgagtgcgtcggggacacattcgaggatccagtt
agagatcggctcgtgctagaggcctgctcgtagagacacagatagacagatagagcggctcg
ctctcgctgctcggaagtcgctcgcgtaagttcgcgctggatcccacagctcgcgctgacaca
gtcgcgtagagatgcggctgagcgctggcgctgaggctggacagtgctgctgagctcggaca
gctcgtgtggcgcggatccgtgctcggcggatcctagggcgtgtcgcgtgctggatgcgc
tggtgggccccagtttggcggcgctcgcggctcggctgctggtcgcctgcttt
These patterns are specific to individual organisms
- identify, compare microbial isolates
10. How Optical Mapping Works
Cells gently lysed to extract long
genomic DNA molecules, pieces of
microbial chromosomes
DNA is captured in parallel
arrays of single DNA molecules
using microfluidic device
After staining with intercalating dye digestion reveals restriction
cleavage sites as ―gaps‖, under fluorescent microscopy
12. Map Assembly
27.52
40.52
51.99
24.45
58.94
17.93
45.26
28.99
46.25
8.89
7.20
5.52
1.56
8.08
Overlapping single molecule restriction maps are
aligned to produce a map assembly covering an
entire chromosome
13. Map Assembly
consensus map
Patterns of restriction sites highly informative
~ 500 sites per Salmonella genome
Characteristic of microbial species and individual isolates
Use to identify samples to strain level
Overlapping single molecule restriction maps are aligned
to produce a map assembly covering an entire
chromosome
17. Strain Typing
High Resolution Epidemiology with Optical Mapping
Traditional technologies (PFGE, ribotyping & Rep-PCR) provide
limited information, are unreliable for distinguishing closely
related isolates, do not relate to sequencing data
18. Strain Typing
Optical Mapping Compared to PFGE
USA-400(MW2)
GLMC-10
GLMC-10
USA 400
SSCMec VS-alpha PhiSA2
(PVL)
Optical Mapping detects absence of SSCmec, VS-α, PhiSA2/PVL
19. Strain Typing: Ongoing E coli Outbreak
• E coli O104:H4 outbreak reported in Germany, May 2011
– Shiga toxin positive (rare for O104:H4)
– High incidence of hemolytic uremic syndrome (HUS)
– Similar to Enteroaggregative E coli (EAEC) which normally
produces mild illness
• Reports spread to 12 countries, including US, Canada
• Over 3,000 cases reported by June 13, including 35 deaths
20. Strain Typing: Ongoing E. coli Outbreak
Current
Outbreak
2001 HUS outbreak
EAEC Seq. Reference
• Whole genome maps available in 48 hours
• Indicated outbreak was clonal – single source
• Identified genomic islands unique to the outbreak
21. Strain Typing: Ongoing E coli Outbreak
Current
Outbreak
Outbreak Specific
Conserved Region 2
stx2 tehA
Outbreak Specific
Outbreak Specific
Conserved
Conserved Region 1
Region 3
22. Strain Typing: Publication Example
2006 E. coli O157:H7 ―Spinach‖ Outbreak
• 51% hospitalizations v typical 10-20%
• 15% kidney failure v typical 2-7% (and 3 deaths)
• FDA CFSAN used Optical Mapping to identify 13
chromosomal markers that define the outbreak strain
• Outbreak strain contained prophage insertions
carrying extra Shiga toxin genes resulting in increased
pathogenicity
• “Most of the chromosomal changes found by optical
mapping would not have been detected by
microarray-based techniques”
• “Optical mapping ….. provides insights into
chromosomal changes and gene acquisitions that
neither PFGE nor microarray analysis allow”
Kotewicz et al (2008) Microbiology 154: 3518-3528
23. Strain Typing Summary
• Optical Mapping provides required resolution to
differentiate closely related strains: > 90%
sequence similarity
• Other technologies lack resolution and typically
focus on a few loci, may not relate to sequencing
25. Comparative Genomics Background
Definition:
Analysis and comparison of genomes from different strains
and different species to better understand gene function and
relatedness.
Involves:
• Sequence similarity
• Gene location and synteny (order of genes)
• Conserved and non-conserved regions of the genome
26. Comparative Genomics
Comparative analysis of US Vancomycin-resistant
Staphylococcus aureus strains
27. Comparative Genomics
Comparative analysis of US Vancomycin-resistant
Staphylococcus aureus strains
VRSA-6 has
direct repeat
28. Comparative Genomics:
Enterococcus faecalis
• E. faecalis isolates are diverse
at whole genome level with
differences from 0% to 35%
• Strain V583 is whole genome
DNA sequence
• ATCC 49477 is 2.9% different
at the whole-genome level
from V583 using Optical Map
29. Comparative Genomics:
Enterococcus faecalis
ATCC
49477
V583
• van genes are responsible
for vancomycin resistance
in Enterococcus faecalis1
• Optical Mapping can draw
attention to insertions that
confer antibiotic resistance
1Evers & Courvalin (1996). J Bacteriol 178(5):1302-9.
35. Sequence Validation:
Optical Mapping Finished Genomes
• Finished whole-genome DNA sequences are considered the
gold standard (Chain et al. 2009)
• Finished whole-genome DNA sequences provide valuable
insights into organization and structure of the genome that
draft quality sequences cannot offer (Fraser et al. 2002)
• However, currently no strict quality or validation requirement
for submitting a finished whole-genome to GenBank or peer-
reviewed journal
36. Sequence Validation:
Optical Mapping Finished Genomes
• Purpose
– Produce Optical Maps of published and peer-reviewed
finished bacterial genomes to validate quality of the
finished genome
• Hypothesis
– Optical Mapping will identify at least one finished
genome to contain a discrepancy.
37. Sequence Validation:
Optical Mapping Finished Genomes
• Methodology
– Select organisms with finished genomes that are linked to a
specific ATCC submission
– Generate Optical Maps using Argus® Optical Mapping System
– Compare Optical Maps to in silico maps of finished genomes
39. Sequence Validation:
Optical Mapping Finished Genomes
Relative in silico Insertion Discrepancy Example
Finished whole-genome DNA sequence contained 131 Kb
extra DNA that should not be in ATCC 17978
40. Sequence Validation:
Optical Mapping Finished Genomes
Relative in silico Deletion Discrepancy Example
• Finished whole-genome DNA sequence missed a 375 Kb repetitive
region, most likely a ribosomal repeat that the sequence
assembler compressed
• Optical Mapping can span these large regions by using >150 Kb
single molecule restriction maps
41. Sequence Validation:
Optical Mapping Finished Genomes
Relative in silico Inversion Discrepancy Example
• The first V. cholerae finished genome published in 1999
contains a putative inverted misassembly
42. Sequence Validation:
Optical Mapping Finished Genomes
• Optical Map of N16961 compared to finished genome of V.
cholerae M66-2 published in 2009
• M66-2 contains a putative inverted misassembly at the same
locus as the DNA sequence of N16961, and is probably a
resequencing error propagated into M66-2
44. Sequence Assembly: Summary
• Maximum value in contig alignment & gap closure
as well as independently validating sequence
• Optical Mapping works best with larger contigs:
>40 Kb
54. Large Genome Application
• Focus on Super-scaffolding
– Order and orientate contigs or scaffolds by creating
Super-scaffolds with mapping information
• Hybrid approach combining draft sequence and
Optical Mapping
• Uses Argus® System to produce mapping
information with cluster-based data pipeline
55. OpGen Application Accelerates Workflow
Bioinformatics
Bioinformatics
Bioinformatics
Bioinformatics
Library Prep Multiple Paired-end, Construct Construct BAC Marker
Shotgun seq Mate-Pair Libraries Fosmid library Library Analysis
Sequence runs Sequence runs Sequence runs
Multiple rounds YEARS
WEEKS WEEKS
MONTHS
Bioinformatics
Bioinformatics Review sequence
data in SS context
Library Prep Multiple Paired-end,
Shotgun seq Mate-Pair Libraries Optical Map
Sequence runs SUPER-SCAFFOLD
DONE
Multiple rounds
OPTICAL 1 WEEK
PCR, PE libraries if
MAPPING
need more seq info
1 WEEK
Another one of our powerful applications is found in the area of comparative genomics. So, Perhaps, you’ve done some strain typing and would like to now dig into your data a little more.
The Argus system is a complete system for performing optical mapping. And it includes hardware, reagents, consumables and instrumentation. There are 4 Key ComponentsInstrumentation—Some Consumables or Qcards, Mapcards, which have these derivitaized glass surfaces….Reagents (the fluorescent stain and enzyme kit)And our SoftwareMapManager is the System Software and MapSolver is the Software analysis tool.