S

1
S M A S H

• Single
• Molecule
• Approach
• to
• Sequencing-by-Hybridization

2
Bud Mishra

• Professor of Computer Science, Mathematics and
  Cell Biology
• Courant Institute, NYU School of Medicine, Tata
  Institute of Fundamental Research, and Mt. Sinai
  School of Medicine

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Reading DNA Sequences
5
How Does Nature Do It?
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Tools of the trade

• Where we collect three important tools from
  biotechnology scissors, glues and copiers

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Scissors

• Type II Restriction Enzyme
• Biochemicals capable of cutting the
  double-stranded DNA by breaking two -O-P-O
  bridges on each backbone
• Restriction Site
• Corresponds to specific short sequences EcoRI
  GAATTC
• Naturally occurring protein in bacteriaDefends
  the bacterium from invading viral DNABacterium
  produces another enzyme that methylates the
  restriction sites of its own DNA

Tools of the Trade
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Glue

• DNA Ligase
• Cellular Enzyme Joins two strands of DNA
  molecules by repairing phosphodiester bonds
• T4 DNA Ligase (E. coli infected with
  bacteriophage T4)
• Hybridization
• Hydrogen bonding between two complementary single
  stranded DNA fragments, or an RNA fragment and a
  complementary single stranded DNA fragment
  results in a double stranded DNA or a DNA-RNA
  fragment

Tools of the Trade
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Copier

• DNA Amplification
• Main Ingredients Insert (the DNA segment to be
  amplified), Vector (a cloning vector that
  combines with an insert to create a replicon),
  Host Organism (usually bacteria).

Tools of the Trade
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Copier

• PCR (Polymerase Chain Reaction)
• Main Ingredients Primers, Catalysts, Templates,
  and the dNTPs.

Tools of the Trade
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Science by Stamp Collecting

• How biotechnology relates to the classical
  coupon collectors problem

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Sir Ernest Rutherford
For Mikes sake, Soddy, dont call it
transmutation. Theyll have our heads off as
alchemists. Rutherford, winner of 1908 Nobel
prize for chemistry for cataloging alpha and beta
particles

• All science is either physics or stamp
  collecting.

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Sanger Chemistry
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A Challenge

• Prize for DNA Decoding Aims to Fuel Innovation
• first team that completely decodes the DNA of 100
  or more people in 10 days

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Sequencing-by-SynthesisPyro-Sequencing

• BASE EXTENSION A single-stranded DNA fragment,
  known as the template, is anchored to a surface
  with the starting point of a complementary
  strand, called the primer, attached to one of its
  ends (a). When fluorescently tagged nucleotides
  (dNTPs) and polymerase are exposed to the
  template, a base complementary to the template
  will be added to the primer strand (b). Remaining
  polymerase and dNTPs are washed away, then laser
  light excites the fluorescent tag, revealing the
  identity of the newly incorporated nucleotide
  (c). Its fluorescent tag is then stripped away,
  and the process starts anew.

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The Middle Way

• Combining Two Extremes
• Indexing For each character b in the genome,
  make a list of each position where it occurs.
• Shotgunning For each long sentence in the
  genome, select it with low probability
  (o(lgn/n)), and then read it reasonably
  accurately.
• Where is the middle???

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The Middle Way

• Character Index
• A 1, 11,
• T 2, 3, 8, 9, 12
• C 4, 5, 9, 10, 13
• G 6, 7, 8, .

• Sentences w/o Index
• ATTCCGGG
• GGGCCATCGT
• CGTCATTCC

ATTCCGGGCCATC
ATTCCGGGCCATC

• Words w/ approx. Index
• ATTC 2..4
• TCGG 6..8
• GGGC 7..9
• GCCA 10..12

ATTCCGGGCCA
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Comparison

• Shotgun Assembly of Short Reads from
  Sequencing-by-Synthesis/Ligation
• Bottom-up
• Short high-quality reads without location
  information
• Shot-gun Assembler
• Must use maps or paired-reads
• Error Prone
• Genotypic Sequence
• Less Flexible

• SMASH Single Molecule Approach to
  Sequencing-by-Hybridization
• Top-down
• Shorter low-quality reads with location
  information
• Bayesian Assembler
• Haplotypic Sequence
• Flexible
• Gap-less Sequencing
• Large high-quality contigs
• Haplotype phasing
• Structural changes (e.g., chromosomal aberrations)

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Outline

• Physical Mapping Sequencing
• Map
• assign physical locations to important markers
  (e.g., restriction sites or hybridization
  probes).
• Sequence
• align short sequence reads to the markers
  (map-based sequence assembly) or
• align long sequence reads to each other (shotgun
  assembly)
• Optical Mapping
• Sequencing

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Optical Mapping

• Where we map by watching the genome

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Probing a Single Molecule

• Standard ensemble measurements yield only average
  values of a parameter for a large number of
  identical copies of macromolecules.
• Single molecule measurements provide rich set of
  information
• dependence of a parameter on its nano-environment
• statistical distribution
• dynamics (temporal effects)

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Optical Approaches

• Single DNA molecule on a surface can be explored
  by nanometer scale with tunneling electrons,
  forces from sharp tips or magnetic resonance
• STM (scanning tunneling microscope)
• AFM (atomic force microscopy)
• MRFM (magnetic resonance force microscopy)
• Optical Approaches non-invasive, avoids
  synchronization, need not be real-time.

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Optical Approaches are Inherently Noisy!

• Since many biological macromolecules are smaller
  than the Raleigh limit, the optical approaches
  involve attaching single fluorescent probes to
  specific macromolecules.
• Controlling Noise
• Magnitude of Stoke-shift
• Steric hinderance
• Absorption cross-section
• Point spread function (PSF)
• Image Processing

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Optical Mapping
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Optical Mapping

1. Capture and immobilize whole genomes as massive
   collections of single DNA molecules

Cells gently lysed to extract genomic DNA
DNA captured in parallel arrays of long single
DNA molecules using microfluidic device
Genomic DNA, captured as single DNA molecules
produced by random breakage of intact chromosomes
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Optical Mapping
2. Interrogate with restriction
endonucleases 3. Maintain order of restriction
fragments in each molecule
Digestion reveals 6-nucleotide cleavage sites as
gaps
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Optical Mapping
4. Determine size of fragments
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Optical Mapping
5. GENTIG Robust Bayesian Map Assembler to
make whole-genome restriction map
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Computational Analysis
Single DNA molecule on Optical Chip after
digestion, staining

• Image analysis software measures size and order
  of restriction fragments
• Overlapping single molecule maps are aligned to
  produce a map assembly covering an entire
  chromosome

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Map Assembly
Overlapping single molecule maps are aligned to
produce a map assembly covering an entire
chromosome
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Error Sources

• Sizing Error
• (Bernoulli labeling, absorption cross-section,
  PSF)
• Partial Digestion
• False Optical Sites
• Orientation
• Spurious molecules, Optical chimerism, Calibration

Image of restriction enzyme digested YAC clone
YAC clone 6H3, derived from human chromosome 11,
digested with the restriction endonuclease Eag I
and Mlu I, stained with a fluorochrome and imaged
by fluorescence microscopy.
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E. coli Shotgun Map
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Shotgun Mapping

• Large fragments of genomic DNA of length from 2Mb
  to 12Mb are optically mapped
• The resulting ordered restriction maps are
  automatically contiged by Gentig
• The consensus map computed by Gentig is free of
  errors due to partial digestion, sizing error and
  false cuts

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Taming the noise

• Where we examine many noise sources in optical
  mapping

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Optical MappingInterplay between Biology and
Computation
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How Errors Complicate the Mapping
Correct Restriction Map

• Accuracy in Sizing
• False Cuts
• (governed by surface conditions, illuminations,
  optic, imaging,)
• Partial Digestion

Error Sources

• Orientation
• Spurious Data
• Crossing
• Breakage
• Missing Fragments

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Complexity

• Where we admit defeat with combinatorial
  algorithmic approaches

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Complexity Issues
Various combinations of error sources lead to
NP-hard Problems
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SMRM(Single Molecule Restriction Map)
DRj
Dj
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SMRM(Single Molecule Restriction Map)
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Problem 2 (Sizing Error)
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Problem 2 is NP Complete
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Example
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Probabilistic Analysis

• Where we design the experiments to generate easy
  instances of a difficult problem

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Sir Ernest Rutherford

• If your experiment needs statistics, you ought
  to have done a better experiment.

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Combinatorial Structure
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Flips Flops
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Intuition
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Other Error Sources
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Discretization
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Sizing Error
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Prediction
The probability of successfully computing the
correct restriction map as a function of the
number of cuts in the map and number of molecules
used in creating the map
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Experimental Results
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Bayesian Methods

• Where we rely on statistical models of the error
  sources to map correctly

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Bayesian Approach
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Robustness of Optical Mapping Algorithm

• BAC Clones with 6-cutters
• Average Clone size 160 Kb Average Fragment
  Size 4 Kb, Average Number of Cutsites 40.
• Parameters
• Digestion rate can be as low as 10
• Orientation of DNA need not be known.
• 40 foreign DNA
• 85 DNA partially broken
• Relative sizing error up to 30
• 30 spurious randomly located cuts

Algorithm Design and Analysis jointly with
T.S.Anantharaman
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Experimental Results
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Bayesian Inference
Pr(H D) Pr(D H) Pr(H)/ Pr(D)
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Bayesian Model
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Multiple Alignement
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Bayesian Optimization
Gradient search for good parameters
Local gradient optimization
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Y

• From a genes point of view, reshuffling is a
  great restorative
• The Y, in its solitary state disapproves of such
  laxity. Apart from small parts near each tip
  which line up with a shared section of the X, it
  stands aloof from the great DNA swap. Its genes,
  such as they are, remain in purdah as the
  generations succeed. As a result, each Y is a
  genetic republic, insulated from the outside
  world. Like most closed societies it becomes both
  selfish and wasteful. Every lineage evolves an
  identity of its own which, quite often, collapses
  under the weight of its own inborn weaknesses.
• Celibacy has ruined mans chromosome.
• Steve Jones, Y The descent of Men, 2002.

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Mapping the DAZ locus on Y Chromosome
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GENomic conTIG

• Where we map large genomes

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Plasmodium falciparum

• Malaria Parasite
• Deadliest of all the human Malaria parasites
• P. falciparum
• P. vivax
• P.malariae
• P. ovale
• Responsible for 1.5-2.7 million deaths in 1997.

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Gentig MapsPlasmodium falciparum

• A. Gap-free consensus BamHI NheI maps for all
  14 chromosomes.
• B.BamHI map
• C. NheI map
• D.NheI map of Chromosome 3 displayed by ConVEx

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Gentig MapDeinococcus radiodurans
Nhe I map of D.radiodurans generated by Gentig
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Gentig MapE. coli

• Whole genome XhoI restriction map of E. coli O157
  generated by Gentig software of Anantharaman
  Mishra.
• The outer circle represents an in silico XhoI
  digest of the sequence.
• The second outermost circle shows the consensus
  optical map.
• The inner circles represent the individual
  molecule maps from which the consensus map was
  generated.

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Complexity, Revisited

• Where we admit defeat again(?)
• Can Gentig assemble large Eukaryotic Genomes??

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GCP is NP-Complete

• Transformation from Hamiltonian Path Problem
  restricted to cubic graphs.

Choose p 3/4 k M
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Comparing MapsEffect of Partial Digestion

• Parameters
• Partial digestion probability, p
• Relative sizing error, b
• Restriction fragments, n
• Overlap threshold ratio, q
• m n p Expected detected restriction
  fragments.
• Controlling False Negative
• K 5 np4 q/2 and r k1/p4, k1 ¼ 2
• If in fact the clones A and B overlap then we
  will detect it with a probability, at least
• (1-exp(-k1)) (1 exp(-n p4 q/8))

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Experiment Design

• Relation among the error parameters
• 3b n p /4 5 k 5 n p4 q/2
• ) p (3 b/2 q)1/3
• Parameter choice for shotgun-mapping. Make the
  partial digestion probability rather high (close
  to 1) or the relative sizing error as low for
  instance by using a rare cutter.

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Contour Plot as a Function of Sizing Error
(x-axis) and Digestion Rate (y-axis)

• The calculation is for the human genome, G 3,300
  mb.
• The average molecule length 5 mb, with an
  overlap of 1 mb
• The average restriction fragment length 25 kb
• For a sizing error of 3 kb, the required
  digestion rate is 94
• If the sizing error is reduced to 2 kb, the
  required digestion rate drops to 88
• If the sizing error is reduced to 1 kb, the
  required digestion rate drops to 80

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Sir Ernest Rutherford

• You should never bet against anything in science
  at odds of more than about 1012 to 1.

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How does Gentig Work

• Gentig uses a purely Bayesian Approach.
• It models all the error processes in the prior.
• It initially starts with a conservative but fast
  pairwise overlap configuration, computed
  efficiently using Geometric Hashing.
• It iteratively combines pairs of maps or map
  contigs, while optimizing the likelihood score
  subject to a constraint imposed by a
  false-positive constraint.
• It has special heuristics to handle non-local
  errors.

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The problem Data error

• Miss restriction site rate
• Gaussian sizing var
• False restriction sites rate
• Small fragments missing rate
• Multiple chromosomes mixed together.

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Solution Bayesian probability maximization
H
f(HD1, L, Dm c f(H) f(D1, L, Dm H)
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Conditional Probability sum of alignments right
of I,J
H
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Example increase interval in H
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Sir Ernest Rutherford

• You should never bet against anything in science
  at odds of more than about 1012 to 1.

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HAPTIG HAPlotypic conTIG

• HOOPLAS, HYPES HAPLOTYPES
• Replacing Gentig
• Faster More Accurate

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Single Molecule HapoltypingCandida Albicans

• The left end of chromsome-1 of the common fungus
  Candida Albicans (being sequenced by Stanford).
• You can clearly see 3 polymorphisms
• (A) Fragment 2 is of size 41.19kb (top) vs
  38.73kb (bottom).
• (B) The 3rd fragment of size 7.76kb is missing
  from the top haplotype.
• (C)The large fragment in the middle is of size
  61.78kb vs 59.66kb.

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Goals

• Identify and phase polymorphism in data.
• Problem
• Similar to Gentig Problem
• if distributions of restriction site polymorphism
  (due to SNPs) and restriction fragment length
  polymorphism (due to indels) are known
• How can one separate partial digestions from
  restriction site polymorphism and sizing error
  from restriction length polymorphism?
• Solution
• Bayesian Analysis with Parameterized Priors
  Fast Dynamic Programming Implementation.

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Comparison on 3.4 GHz CPU
Chr1 length Coverage USC time NYU time
12 Mb 20x 3431 secs 448 secs
12 Mb 100x 17052 secs 2818 secs
30 Mb 20x 28993 secs 1444 secs
30 Mb 100x 124194 secs 9308 secs
100 Mb 20x NA 25465 secs
100 Mb 50x NA 74025 secs
200 Mb 20x NA 88835 secs
200 Mb 50x NA 259024 secs
Note Haptig times based on 2.4 GHz CPU
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Comparison on 3.4 GHz CPU
20 X coverage
100 X coverage
Unlike the USC algorithm (by Waterman et al.),
Gentig/Haptig algorithms easily scale to human
genome sized problems. Gentig is routinely used
by OpGen and other labs to map.
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Other Interesting Applications

• Phylogeny
• Sequence Validation
• Haplotyping
• Sequencing
• Comparative Genomics
• Rearrangement events
• Hemizygous Deletions
• Epigenomics
• Characterizing cDNAs
• Expression Profiling
• Alternate Splicing

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Sequencing
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Joint Work with

• T.S. Anantharaman, NYU, NY.
• V. Demidov, UCLA, CA.
• A. Lim, NYU, NY.
• S. Paxia, NYU, NY.
• J. Reed, UCLA, CA.
• C. Cantor, Sequenom, Inc., CA
• J. Gimzewski, UCLA, CA.
• M. Teitell, UCLA, CA.

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OverviewTechnology

• How does it work?
• Optimal integration of several technologies based
  on manipulation of single molecules on a surface.

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Goal

• Sequence a human size genome of about 6
  Gbinclude both haplotypes.
• Integrate three component technologies
• Optical Mapping to create Ordered Restriction
  Maps with respect to one or more restriction
  enzymes,
• Hybridization of a pool of short nucleobase
  probes (PNA or LNA oligomers) with Single Genome
  dsDNA molecules on a surface, and
• Efficient polynomial time algorithms to solve
  localized versions of the PSBH (Positional
  Sequencing by Hybridization) problems over the
  whole genome.

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SMASH

• Genomic DNA is carefully extracted from small
  number of cells of an organism (e.g., human) in
  normal or diseased states. (Fig 1 shows a cancer
  cell to be studied for its oncogeneomic
  characterization.)

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SMASH

• LNA probes of length 6 8 nucleotides are
  hybridized to dsDNA (double-stranded genomic DNA)
  in a test tube (Fig 2) and the modified DNA is
  stretched on a 1 x 1 chip that has microfluidic
  channels manufactured on its surface. These
  surfaces have been chemically treated to create a
  positive charge.

DNA samples are prepared for analysis with LNA
probes and restriction enzymes.
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SMASH

• Since DNA is slightly negatively charged, it
  adheres to the surface as it flows along these
  channels and stretches out. Individual molecules
  range in size from 0.3 3 million base pairs in
  length.
• Next, bright emitters are attached to the probes
  on the surface and the molecules are imaged (Fig
  3).

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SMASH

• A restriction enzyme1 is added to break the DNA
  at specific sites. Since DNA molecules are under
  slight tension, the cut fragments of DNA relax
  like entropic springs, leaving small visible gaps
  corresponding to the positions of the restriction
  site (Fig 4).
• 1. A restriction enzyme is a highly specific
  molecular scissor that recognizes short
  nucleotide sequences and cuts the DNA at only
  those recognition sites.

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SMASH

• The DNA is then stained with a fluorogen (Fig 5)
  and reimaged. The two images are combined to
  create a composite image suggesting the locations
  of a specific short word (e.g., probes) within
  the context of a pattern of restriction sites.

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SMASH

• The intensity of the light emitted by the dye at
  one frequency provides a measure of the length of
  the DNA fragments.
• The intensity of the light emitted by the
  bright-emitters on probes provides an intensity
  profile for locations of the probes.
• Images of each DNA molecule are then converted
  into ideograms, where the restriction sites are
  represented by a tall rectangle and probe sites
  by small circles (Fig 6).

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SMASH

• The steps above are repeated for all possible
  probe compositions (modulo reverse
  complementarity).
• Sutta software then uses the data from all such
  individual ideograms to create an assembly of the
  haplotypic ordered restriction maps with
  approximate probe locations superimposed on the
  map.

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SMASH

• Local clusters of overlapping words are combined
  by Suttas PSBH (positional sequencing by
  hybridization) algorithm to overlay the inferred
  haplotypic sequence on top of the restriction map
  (Fig 7).

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Sir Ernest Rutherford

• We haven't the money, so we've got to think."

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Hybridization
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Thermal Stability of LNA

• LNA/DNA or LNA/RNA duplexes have increased
  thermal stability compared with similar duplexes
  formed by DNA or RNA.
• The LNA modification has been shown to increase
  the biological stability of nucleic acids.
• Fully modified LNA oligonucleotides are resistant
  towards most nucleases tested.

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Experiments with Probes

• To test the feasibility of hybridization of a
  modified PNA, we first tested hybridization of
  PNA probe to lambda DNA molecules.
• We were able to measure the degree of
  hybridization by digesting lambda DNA, after it
  was hybridized with the PNA probe, with PmlI
  restriction enzyme and run the sample on a 4
  PAGE gel and found the degree of hybridization to
  be greater then 90.

Bound
Unbound
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Probe Map

• Overlayed fluorescent images of labmda DNA
  molecules using a FITC filter (white) and CY5
  filter (red), showing the position of the probes
  on the lambda DNA molecules.

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Final Probe Map

• We next combined all probe maps from around 20
  image pairs using a Bayesian algorithm to compute
  the most likely consensus map.
• For one set of 20 image pairs, a total of 512 DNA
  molecules with a total of 678 probes were
  identified and combined into a consensus map with
  2 probe locations at 14.8 and 52.4 of the DNA
  length.
• The orientation of the DNA cannot be determined
  from optical maps.
• Thus our result is in close agreement with the
  correct map with probes at 50.2 and 85.7 known
  from the sequence and the implied probe
  hybridization rate of 42 is also quite good.

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Positional SBH Algorithm
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Algorithmic Problem

• The correct DNA sequence is s
• Computable
• Correct Haplotypic Restriction Maps (with respect
  to multiple enzymes)
• Positional Spectra For a position x, wx wcx,
  the two k-mers at position x on the watson- and
  crick-strands.
• Data
• Map Errors Errors in sizing, false positives and
  negatives in cleavage
• Spectral Errors Errors in position and false
  positives and negatives in hybridization
• Compute a sequence t.
• How closely does t approximate s?

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Anomalies

• Irresolvable Ambiguities
• From assemblies based on 6bp probes
• Error Pattern s w sRC
• Correct Pattern s wRC sRC
• s tcgcc (any 5 bases)
• sRCggcga (Reverse compliment of X)
• w CCCCTAAC (any short sequence under 50bp)
• wRC GTTAGGGG (Reverse compliment of Y)

AssemblytcgccCCCCTAAC ggcga
Correct
tcgccGTTAGGGGggcga
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A Library of Anomalies

• Irresolvable Ambiguities Unavoidable Error
  Patterns
• Assuming assemblies with K-bp probes
• Most common s w sRC vs s wRC sRC
• Also common s w s t s vs. s t s w s
• Many more complicated patterns (but extremely
  rare)
• Here
• s any K-1 bp sequence
• w, t any short sequence under 50bp
• The probabilities of such patterns can be reduced
  exponentially with gapped probes without
  increasing the costs.

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Complexity

• Overcoming the complexity issues
• Spectrum of k-mer probes constraints on the
  location of the k-mers in the sequence
• However if the location constraints are in the
  form of k contiguous locations then the
  reconstruction problem is exponential in k,
  rather than the sequence length m.
• NP-Complete, if k 3

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Solution

• Complexity Issue
• a Experiment Design Powerful Heuristics
• Accuracy Issue
• a Combinatorial Design of Probes (Gapped Probes
  using universal Bases)

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Gapped Probes

• Mixing solid bases with wild-card bases
• E.g., xxxxxx (9-mers) or xxxxxxxx (14
  mers)
• An inert base
• Universal In terms of its ability to form base
  pairs with the other natural DNA/RNA bases.
• Applications in primers and in probes for
  hybridization
• Examples
• The naturally occurring base hypoxanthine, as its
  ribo- or 2'-deoxyribonucleoside
• 2'-deoxyisoinosine
• 7-deaza-2'-deoxyinosine
• 2-aza-2'-deoxyinosine

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2'-Deoxyinosine derivatives

• 2'-Deoxyinosine derivatives can be used as
  universal DNA analogues.

Loakes, D. Nucl. Acids Res. 2001 292437-2447
doi10.1093/nar/29.12.2437
114
Other Chemistries

• The bases 3-nitropyrrole and 5-nitroindole have
  also been incorporated into peptide nucleic acids
  (PNA)
• In PNADNA duplexes, it was found that the T_m
  range was narrower than that found for
  corresponding DNADNA duplexes, although
  significantly higher than the corresponding
  DNADNA duplex containing an AT base pair.

Loakes, D. Nucl. Acids Res. 2001 292437-2447
doi10.1093/nar/29.12.2437
115
Approximation

• A special case of the PSBH problem,
• Separate constraints for each of the multiple
  instances of each k-mer.
• By focusing on a small window of about 2k-1bp, in
  which most k-mers will occur only once, we hope
  to approximately solve the standard PSBH problem
• Here separate constraints for multiple instances
  of each k-mer are not important.
• Amortize over many instances

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Multiple PSBH problem

• Data set
• Probe maps for all K-mers (with gaps)
• We have multiple instances of each k-mer, (for
  k6, about one every 4Kb on each strand of the
  DNA in the sequence),
• For each instance we can constrain the location
  to within about 100 base pairs depending on the
  optical resolution.
• Beam Search Based Heuristics

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Simulation Results

• Probe Map Assumptions
• For single DNA molecules
• Probe location Standard Deviation 240 bases
• Data coverage per probe map 50x
• Probe hybridization rate 30, and
• false positive rate of 10 probes per megabase,
  uniformly distributed.
• Analytically estimation of the average error rate
  in the probe consensus map
• Probe location SD 60 bases
• False Positive rate lt 2.4
• False Negative rate lt 2.0.

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Simulation Results

• Using estimated error rates, randomly introduced
  errors at the calculated rates into each of the
  2080 simulated probe consensus maps (for the
  above example).
• Ran our sequence assembly algorithm.
• Aligned the sequence produced with the originally
  assumed correct sequence using Smith-Waterman
  alignment.
• Counted the total number of single base errors
  (mismatches deletions insertions).
• Repeated this experiment until a total of 200,000
  bases of sequence had been simulated and computed
  the average error rate per 10,000 bases.

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Simulation Results

• We first tried un-gapped probes with 5,6,7 and 8
  bases respectively and got error rates per 10,000
  bases of 1674, 255, 39.6 and 3.7 bases
  respectively.
• Our machine ran out of memory trying to simulate
  a 9 base ungapped probe, but from the trend it is
  clear that the goal of 1 base error per 10,000
  bases would most likely be achieved by a 9 base
  ungapped probe.

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Results
UNGAPPED
GAPPED
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1000 Rupees Genome
22.67 US for 6 billion bases 135 billion US
for the entire human population
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Sir Ernest Rutherford

• I have become more and more impressed by the
  power of the scientific method of extending our
  knowledge of nature.
• Experiment, directed by the imagination of either
  an individual, or still better of a group of
  individuals of varied mental outlook is able to
  achieve results which far transcend the
  imagination alone of the greatest natural
  philosopher.

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Sir Ernest Rutherford

• Experiment without imagination, or imagination
  without recourse to experiment, can accomplish
  little. But for effective progress, a happy blend
  of these powers is necessary

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the end