DNA Sequencing

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DNA Sequencing
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DNA sequencing

• How we obtain the sequence of nucleotides of a
  species

ACGTGACTGAGGACCGTG CGACTGAGACTGACTGGGT CTAGCTAGAC
TACGTTTTA TATATATATACGTCGTCGT ACTGATGACTAGATTACAG
ACTGATTTAGATACCTGAC TGATTTTAAAAAAATATT
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Which representative of the species?

• Which human?
• Answer one
• Answer two it doesnt matter
• Polymorphism rate number of letter changes
  between two different members of a species
• Humans 1/2,000
• Other organisms have much higher polymorphism
  rates

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Human population migrations

• Out of Africa, Replacement
• Single mother of all humans (Eve) 150,000yr
• Single father of all humans (Adam) 70,000yr
• Humans out of Africa 40000 years ago replaced
  others (e.g., Neandertals)
• Evidence mtDNA
• Multiregional Evolution
• Fossil records show a continuous change of
  morphological features
• Proponents of the theory doubt mtDNA and other
  genetic evidence

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Why humans are so similar

• A small population that interbred reduced the
  genetic variation
• Out of Africa 40,000 years ago

Out of Africa
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Migration of human variation

• http//info.med.yale.edu/genetics/kkidd/point.html

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Migration of human variation

• http//info.med.yale.edu/genetics/kkidd/point.html

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Migration of human variation

• http//info.med.yale.edu/genetics/kkidd/point.html

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Human variation in Y chromosome
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DNA Sequencing Overview
1975

• Gel electrophoresis
• Predominant, old technology by F. Sanger
• Whole genome strategies
• Physical mapping
• Walking
• Shotgun sequencing
• Computational fragment assembly
• The futurenew sequencing technologies
• Pyrosequencing, single molecule methods,
• Assembly techniques
• Future variants of sequencing
• Resequencing of humans
• Microbial and environmental sequencing
• Cancer genome sequencing

2015
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DNA Sequencing

• Goal
• Find the complete sequence of A, C, G, Ts in
  DNA
• Challenge
• There is no machine that takes long DNA as an
  input, and gives the complete sequence as output
• Can only sequence 500 letters at a time

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DNA Sequencing vectors
DNA
Shake
DNA fragments
Known location (restriction site)
Vector Circular genome (bacterium, plasmid)


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Different types of vectors
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DNA Sequencing gel electrophoresis

• Start at primer (restriction site)
• Grow DNA chain
• Include dideoxynucleoside (modified a, c, g, t)
• Stops reaction at all possible points
• Separate products with length, using gel
  electrophoresis

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Electrophoresis diagrams
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Challenging to read answer
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Challenging to read answer
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Challenging to read answer
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Reading an electropherogram

• Filtering
• Smoothening
• Correction for length compressions
• A method for calling the letters PHRED
• PHRED PHils Read EDitor (by Phil Green)
• Several better methods exist, but labs are
  reluctant to change

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Output of PHRED a read

• A read 500-700 nucleotides
• A C G A A T C A G A
• 16 18 21 23 25 15 28 30 32 21
• Quality scores -10?log10Prob(Error)
• Reads can be obtained from leftmost, rightmost
  ends of the insert
• Double-barreled sequencing (1990)
• Both leftmost rightmost ends are sequenced,
  reads are paired

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Method to sequence longer regions
genomic segment
cut many times at random (Shotgun)
Get one or two reads from each segment
500 bp
500 bp
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Reconstructing the Sequence (Fragment Assembly)
reads
Cover region with 7-fold redundancy (7X)
Overlap reads and extend to reconstruct the
original genomic region
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Definition of Coverage
C

• Length of genomic segment L
• Number of reads n
• Length of each read l
• Definition Coverage C n l / L
• How much coverage is enough?
• Lander-Waterman model
• Assuming uniform distribution of reads, C10
  results in 1 gapped region /1,000,000 nucleotides

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Repeats

• Bacterial genomes 5
• Mammals 50
• Repeat types
• Low-Complexity DNA (e.g. ATATATATACATA)
• Microsatellite repeats (a1ak)N where k 3-6
• (e.g. CAGCAGTAGCAGCACCAG)
• Transposons
• SINE (Short Interspersed Nuclear Elements)
• e.g., ALU 300-long, 106 copies
• LINE (Long Interspersed Nuclear Elements)
• 4000-long, 200,000 copies
• LTR retroposons (Long Terminal Repeats (700 bp)
  at each end)
• cousins of HIV
• Gene Families genes duplicate then diverge
  (paralogs)
• Recent duplications 100,000-long, very similar
  copies

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Sequencing and Fragment Assembly
3x109 nucleotides
50 of human DNA is composed of repeats
Error! Glued together two distant regions
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What can we do about repeats?

• Two main approaches
• Cluster the reads
• Link the reads

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What can we do about repeats?

• Two main approaches
• Cluster the reads
• Link the reads

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What can we do about repeats?

• Two main approaches
• Cluster the reads
• Link the reads

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Sequencing and Fragment Assembly
3x109 nucleotides
ARB, CRD or ARD, CRB ?
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Sequencing and Fragment Assembly
3x109 nucleotides
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Strategies for whole-genome sequencing

• Hierarchical Clone-by-clone
• Break genome into many long pieces
• Map each long piece onto the genome
• Sequence each piece with shotgun
• Example Yeast, Worm, Human, Rat
• Online version of (1) Walking
• Break genome into many long pieces
• Start sequencing each piece with shotgun
• Construct map as you go
• Example Rice genome
• Whole genome shotgun
• One large shotgun pass on the whole genome
• Example Drosophila, Human (Celera),

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Hierarchical Sequencing
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Hierarchical Sequencing Strategy
genome

• Obtain a large collection of BAC clones
• Map them onto the genome (Physical Mapping)
• Select a minimum tiling path
• Sequence each clone in the path with shotgun
• Assemble
• Put everything together

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Methods of physical mapping

• Goal
• Make a map of the locations of each clone
  relative to one another
• Use the map to select a minimal set of clones to
  sequence
• Methods
• Hybridization
• Digestion

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1. Hybridization
p1
pn

• Short words, the probes, attach to complementary
  words
• Construct many probes
• Treat each BAC with all probes
• Record which ones attach to it
• Same words attaching to BACS X, Y ? overlap

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Hybridization Computational Challenge
p1 p2 .pm
0 0 1 ..1

• Matrix
• m probes ? n clones
• (i, j) 1, if pi hybridizes to Cj
• 0, otherwise
• Definition Consecutive ones matrix
• 1s are consecutive in each row col
• Computational problem
• Reorder the probes so that matrix is in
  consecutive-ones form
• Can be solved in O(m3) time (m gt n)

C1 C2 .Cn
1 1 0 ..0
1 0 1...0
pi1pi2.pim
1 1 1 0 0 0..0
0 1 1 1 1 1..0
0 0 1 1 1 0..0
Cj1Cj2 .Cjn
0 0 0 0 0 01 1 1 0
0 0 0 0 0 00 1 1 1
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Hybridization Computational Challenge
pi1pi2.pim
pi1pi2.pim
1 1 1 0 0 0..0
0 1 1 1 1 1..0
0 0 1 1 1 0..0
Cj1Cj2 .Cjn
Cj1Cj2 .Cjn
0 0 0 0 0 01 1 1 0
0 0 0 0 0 00 1 1 1

• If we put the matrix in consecutive-ones form,
• then we can deduce the order of the clones
• which pairs of clones overlap

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Hybridization Computational Challenge
p1 p2 .pm

• Additional challenge
• A probe (short word) can hybridize in many
  places in the genome
• Computational Problem
• Find the order of probes that implies the
  minimal probe repetition
• Equivalent find the shortest string of probes
  such that each clone appears as a substring
• APX-hard
• Solutions
• Greedy, probabilistic, lots of manual curation

0 0 1 ..1
C1 C2 .Cn
1 1 0 ..0
1 0 1...0
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2. Digestion

• Restriction enzymes cut DNA where specific words
  appear
• Cut each clone separately with an enzyme
• Run fragments on a gel and measure length
• Clones Ca, Cb have fragments of length li, lj,
  lk ? overlap
• Double digestion
• Cut with enzyme A, enzyme B, then enzymes A B

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Online Clone-by-cloneThe Walking Method
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The Walking Method

• Build a very redundant library of BACs with
  sequenced clone-ends (cheap to build)
• Sequence some seed clones
• Walk from seeds using clone-ends to pick
  library clones that extend left right

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Walking An Example
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Advantages Disadvantages of Hierarchical
Sequencing

• Hierarchical Sequencing
• ADV. Easy assembly
• DIS. Build library physical map
• redundant sequencing
• Whole Genome Shotgun (WGS)
• ADV. No mapping, no redundant sequencing
• DIS. Difficult to assemble and resolve repeats
• The Walking method motivation
• Sequence the genome clone-by-clone without a
  physical map
• The only costs involved are
• Library of end-sequenced clones (cheap)
• Sequencing

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Walking off a Single Seed

• Low redundant sequencing
• Many sequential steps

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Walking off a single clone is impractical

• Cycle time to process one clone 1-2 months
• Grow clone
• Prepare Shear DNA
• Prepare shotgun library perform shotgun
• Assemble in a computer
• Close remaining gaps
• A mammalian genome would need 15,000 walking
  steps !

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Walking off several seeds in parallel
Efficient
Inefficient

• Few sequential steps
• Additional redundant sequencing
• In general, can sequence a genome in 5 walking
  steps,
• with lt20 redundant sequencing

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Using Two Libraries
Most inefficiency comes from closing a small gap
with a much larger clone
Solution Use a second library of small clones
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Whole-Genome Shotgun Sequencing
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Whole Genome Shotgun Sequencing
genome
plasmids (2 10 Kbp)
forward-reverse paired reads
known dist
cosmids (40 Kbp)
500 bp
500 bp