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Optical Mapping as a Method of Whole Genome Analysis. May 4, 2009 Course: 22M:151 Presented by: Austin J. Ramme. Presentation Outline. Introduction to Optical Mapping Definitions for Understanding Modern Optical Mapping Process Data Analysis Overview - PowerPoint PPT Presentation
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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]
Slide Removed for Online Slide Removed for Online PostingPosting
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/)
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