OPTICAL MAPPING AS A OPTICAL MAPPING AS A METHOD OF WHOLE METHOD OF WHOLE GENOME ANALYSISGENOME ANALYSIS

MAY 4, 2009COURSE: 22M:151

  PRESENTED BY: AUSTIN J. RAMME

Presentation OutlinePresentation OutlineIntroduction to Optical MappingDefinitions for UnderstandingModern Optical Mapping ProcessData Analysis

◦Overview◦Steps to Restriction Map Generation

Applications of Optical MappingConclusions

Optical Mapping (OM) Optical Mapping (OM) IntroductionIntroduction

The number of identified polygenetic diseases is ever increasing

Methods to analyze the entire genome will enhance current diagnostic and treatment methods for a variety of diseases

Patient-specific genomic analysis has become the goal in genetics-based medical research

Optical mapping(OM) is an automated method of ordered restriction map generation with a goal of whole genome analysis that avoids the limitations inherent to traditional techniques

DefinitionsDefinitionsRestriction Enzymes

◦Proteins that cleave DNA molecules based on a specific base pair sequence (e.g. ATCG)

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DefinitionsDefinitionsRestriction Map

◦ Representation of the cut sites on a given DNA molecule to provide spatial information of genetic loci

Optical Mapping◦ Process to generate ordered restriction maps from

single DNA molecules

Optical Map◦ Ordered restriction map of a portion of genomic DNA

DNA strand

[2]

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Computer Representation of Computer Representation of Imaging DataImaging Data

Imaged datasets are converted into barcode patterns corresponding to the cleaved fragments

Lengths are determined using an internal λ standard and fluorescence intensity values

Computer Representation of Ordered DNA Fragments

Imaged Cleaved DNA Fragments[5]

Raw DataRaw DataDescription

◦ Image collection containing genomic restriction fragments of known length deposited in an ordered manner

◦ Fragments represent randomly sheared genomic DNA ◦ Each OM imaging study redundantly represents the

entire genomic region of interestChallenges with analyzing individual DNA

molecules:◦ Extra cut sites - physical breakage◦ Missing cut sites - partial digestion◦ Loss of small fragments ◦ Sizing error◦ Chimeric maps- physically overlapped molecules

Combining multiple OMs gives more accurate restriction maps

Graphing has been used to accomplish this

Steps to Restriction Map Steps to Restriction Map GenerationGeneration1. Calculation of OM Overlaps2. Overlap Graph Construction3. Graph Correction Procedure4. Identification of Islands5. Contig Construction6. Construction of Draft Consensus

Map7. Consensus Map Refinement

Calculation of OverlapsCalculation of OverlapsA multitude of OMs are collected per optical mapping

experimentScoring system used to find overlaps between individual

optical maps:

Scoring system components:Matching sites are rewardedDiscordant sites are penalizedLength similarity is rewarded

[6]

Overlap Graph ConstructionOverlap Graph Construction Overlap Graph = G(V,E)

◦ Literature describes it as a graph, but its technically a digraph

◦ The set of nodes (V) represent individual optical maps◦ The set of edges (E) represent high quality overlaps

between pairs of maps  Weighting and orienting the edges of the graph

◦ Edge weights correspond to genomic distances of the overlapping map regions

◦ Orientation based on the sign of distance measurements from neighboring map centerpoints

Goal: Heaviest weight path represents the most comprehensive genomic restriction map

OM1

OM2

OM3

OM4

…Graph Construction

Optical Mapping Data

Graph Correction Procedure Graph Correction Procedure (1)(1)

False edges correspond to falsely identified overlaps ◦Spurious edges

Connect two nodes forming a cycle which is not possible in linear DNA

◦Orientation consistent false overlaps (cut edge) Edges that connect two

unrelated portions of the genome

[4]

[4]

Graph Correction Procedure Graph Correction Procedure (2)(2)

False Nodes Chimeric maps ◦Consist of two groups of nodes only

connected via a single node (cut vertex)

◦Connect two unrelated portions of the genome

[4]

Identification of IslandsIdentification of Islands Islands correspond to genomic regions spanned

by multiple overlapping optical maps

Contig ConstructionContig ConstructionFor each island, “contigs” are defined as paths

from sources to sinks within the overlap graph for the island

The most complete representation of the genomic region is represented by the heaviest edge path from source to sink

Island 1

Island 2

Island 3

[4]

Construction of Draft Construction of Draft Consensus MapConsensus Map

Using the determined paths, the nodes and edges are used to merge the individual optical maps corresponding to each chosen island component

Each of the individual composite optical maps are stored for further analysis

[4]

Consensus Map Refinement Consensus Map Refinement (1)(1)The draft map may contain errors:

◦ Missing cut sites◦ False cut sites

Hidden Markov Model (HMM) for map refinement◦ Compares draft map to many other optical maps◦ Statistics used to identify matching, deleted, and

inserted cut sites

Hidden Markov Model

[7]

Consensus Map Refinement Consensus Map Refinement (2)(2)

The corrected consensus map for each island pieced back together to form a complete genomic restriction map

Typically takes 13-15 iterations for statistical correction of the draft map

Sample HMM Path

[7]

Applications of Optical Applications of Optical MappingMapping

Identification of genetic insertions, deletions, inversions, and repeats

Establish genotype-phenotype correlations for advancements in diagnosis and treatment of genetic disorders

Reduction of the time needed and the cost to sequence entire strands of DNA

In the future: Patient-specific whole genome analysis

ConclusionsConclusionsOptical mapping is a method of restriction

map generation for whole genome analysis

Applications range from clinical phenotype-genotype correlations to identification of polymorphisms in a variety of diseases

In the future, optical mapping technology will help to realize the goal of patient-specific whole genomic analysis

Optical Mapping is a modern application of discrete mathematics with potential to change medicine

ReferencesReferences1. Samad A, Huff EF, Cai W, Schwartz DC. Optical mapping: A

novel, single-molecule approach to genomic analysis. Genome Res. 1995;5:1-4.

2. Ramme AJ. Personal image collection. .

3. Schwartz DC, Samad A. Optical mapping approaches to molecular genomics. Curr Opin Biotechnol. 1997;8:70-74.

4. Valouev A, Schwartz DC, Zhou S, Waterman MS. An algorithm for assembly of ordered restriction maps from single DNA molecules. Proc Natl Acad Sci U S A. 2006;103:15770-15775.

5. Aston C, Mishra B, Schwartz DC. Optical mapping and its potential for large-scale sequencing projects. Trends Biotechnol. 1999;17:297-302.

6. Valouev A, Li L, Liu YC, et al. Alignment of optical maps. J Comput Biol. 2006;13:442-462.

7. Valouev A, Zhang Y, Schwartz DC, Waterman MS. Refinement of optical map assemblies. Bioinformatics. 2006;22:1217-1224.

Questions?Questions?

Further information available from:1.) Laboratory for Molecular and Computational Genetics (http://www.lmcg.wisc.edu/)2.) Opgen (http://www.opgen.com/)