Download DNA Sequencing (Batzoglou

DNA Sequencing
DNA sequencing
How we obtain the sequence of nucleotides of a species
…ACGTGACTGAGGACCGTG
CGACTGAGACTGACTGGGT
CTAGCTAGACTACGTTTTA
TATATATATACGTCGTCGT
ACTGATGACTAGATTACAG
ACTGATTTAGATACCTGAC
TGATTTTAAAAAAATATT…
CS262 Lecture 9, Win06, Batzoglou
Which representative of the species?
Which human?
Answer one:
Answer two: it doesn’t 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
CS262 Lecture 9, Win06, Batzoglou
CS262 Lecture 9, Win06, Batzoglou
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
CS262 Lecture 9, Win06, Batzoglou
Why humans are so similar
Out of Africa
A small population that interbred
reduced the genetic variation
Out of Africa ~ 40,000 years ago
CS262 Lecture 9, Win06, Batzoglou
Migration of human variation
http://info.med.yale.edu/genetics/kkidd/point.html
CS262 Lecture 9, Win06, Batzoglou
Migration of human variation
http://info.med.yale.edu/genetics/kkidd/point.html
CS262 Lecture 9, Win06, Batzoglou
Migration of human variation
http://info.med.yale.edu/genetics/kkidd/point.html
CS262 Lecture 9, Win06, Batzoglou
Human variation in Y chromosome
CS262 Lecture 9, Win06, Batzoglou
CS262 Lecture 9, Win06, Batzoglou
CS262 Lecture 9, Win06, Batzoglou
DNA Sequencing – Overview
•
Gel electrophoresis
1975
 Predominant, old technology by F. Sanger
•
Whole genome strategies
 Physical mapping
 Walking
 Shotgun sequencing
•
Computational fragment assembly
•
The future—new sequencing technologies
 Pyrosequencing, single molecule methods, …
 Assembly techniques
•
Future variants of sequencing
 Resequencing of humans
 Microbial and environmental sequencing
 Cancer genome sequencing
2015
CS262 Lecture 9, Win06, Batzoglou
DNA Sequencing
Goal:
Find the complete sequence of A, C, G, T’s 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
CS262 Lecture 9, Win06, Batzoglou
DNA Sequencing – vectors
DNA
Shake
DNA fragments
Vector
Circular genome
(bacterium, plasmid)
CS262 Lecture 9, Win06, Batzoglou
+
=
Known
location
(restriction
site)
Different types of vectors
VECTOR
Size of insert
Plasmid
2,000-10,000
Can control the size
Cosmid
40,000
BAC (Bacterial Artificial
Chromosome)
70,000-300,000
YAC (Yeast Artificial
Chromosome)
> 300,000
Not used much
recently
CS262 Lecture 9, Win06, Batzoglou
DNA Sequencing – gel electrophoresis
1.
Start at primer (restriction
site)
2.
Grow DNA chain
3.
Include dideoxynucleoside
(modified a, c, g, t)
4.
Stops reaction at all
possible points
5.
Separate products with
length, using gel
electrophoresis
CS262 Lecture 9, Win06, Batzoglou
Electrophoresis diagrams
CS262 Lecture 9, Win06, Batzoglou
Challenging to read answer
CS262 Lecture 9, Win06, Batzoglou
Challenging to read answer
CS262 Lecture 9, Win06, Batzoglou
Challenging to read answer
CS262 Lecture 9, Win06, Batzoglou
Reading an electropherogram
1.
2.
3.
4.
Filtering
Smoothening
Correction for length compressions
A method for calling the letters – PHRED
PHRED – PHil’s Read EDitor (by Phil Green)
Several better methods exist, but labs are reluctant to change
CS262 Lecture 9, Win06, Batzoglou
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
CS262 Lecture 9, Win06, Batzoglou
Method to sequence longer regions
genomic segment
cut many times at
random (Shotgun)
Get one or two reads from
each segment
~500 bp
CS262 Lecture 9, Win06, Batzoglou
~500 bp
Reconstructing the Sequence
(Fragment Assembly)
reads
Cover region with ~7-fold redundancy (7X)
Overlap reads and extend to reconstruct the original genomic
region
CS262 Lecture 9, Win06, Batzoglou
Definition of Coverage
C
Length of genomic segment:
Number of reads:
Length of each read:
L
n
l
Definition:
C=nl/L
Coverage
How much coverage is enough?
Lander-Waterman model:
Assuming uniform distribution of reads, C=10 results in 1
gapped region /1,000,000 nucleotides
CS262 Lecture 9, Win06, Batzoglou
Repeats
Bacterial genomes:
Mammals:
5%
50%
Repeat types:
•
Low-Complexity DNA (e.g. ATATATATACATA…)
•
Microsatellite repeats
•
Transposons
 SINE
(a1…ak)N where k ~ 3-6
(e.g. CAGCAGTAGCAGCACCAG)
(Short Interspersed Nuclear Elements)
e.g., ALU: ~300-long, 106 copies
 LINE
 LTR retroposons
(Long Interspersed Nuclear Elements)
~4000-long, 200,000 copies
(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
CS262 Lecture 9, Win06, Batzoglou
Sequencing and Fragment Assembly
AGTAGCACAGA
CTACGACGAGA
CGATCGTGCGA
GCGACGGCGTA
GTGTGCTGTAC
TGTCGTGTGTG
TGTACTCTCCT
3x109 nucleotides
50% of human DNA is composed of repeats
Error!
Glued together two distant regions
CS262 Lecture 9, Win06, Batzoglou
What can we do about repeats?
Two main approaches:
• Cluster the reads
•
Link the reads
CS262 Lecture 9, Win06, Batzoglou
What can we do about repeats?
Two main approaches:
• Cluster the reads
•
Link the reads
CS262 Lecture 9, Win06, Batzoglou
What can we do about repeats?
Two main approaches:
• Cluster the reads
•
Link the reads
CS262 Lecture 9, Win06, Batzoglou
Sequencing and Fragment Assembly
AGTAGCACAGA
CTACGACGAGA
CGATCGTGCGA
GCGACGGCGTA
GTGTGCTGTAC
TGTCGTGTGTG
TGTACTCTCCT
3x109 nucleotides
A
R
B
ARB, CRD
or
C
CS262 Lecture 9, Win06, Batzoglou
R
D
ARD, CRB ?
Sequencing and Fragment Assembly
AGTAGCACAGA
CTACGACGAGA
CGATCGTGCGA
GCGACGGCGTA
GTGTGCTGTAC
TGTCGTGTGTG
TGTACTCTCCT
3x109 nucleotides
CS262 Lecture 9, Win06, Batzoglou
Strategies for whole-genome sequencing
1.
Hierarchical – Clone-by-clone
i.
ii.
iii.
Break genome into many long pieces
Map each long piece onto the genome
Sequence each piece with shotgun
Example: Yeast, Worm, Human, Rat
2.
Online version of (1) – Walking
i.
ii.
iii.
Break genome into many long pieces
Start sequencing each piece with shotgun
Construct map as you go
Example: Rice genome
3.
Whole genome shotgun
One large shotgun pass on the whole genome
Example: Drosophila, Human (Celera),
Neurospora, Mouse, Rat, Dog
CS262 Lecture 9, Win06, Batzoglou
Hierarchical Sequencing
Hierarchical Sequencing Strategy
a BAC clone
genome
1.
2.
3.
4.
5.
6.
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
CS262 Lecture 9, Win06, Batzoglou
map
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
CS262 Lecture 9, Win06, Batzoglou
1.
Hybridization
p1
Short words, the probes, attach to complementary words
1.
2.
3.
4.
Construct many probes
Treat each BAC with all probes
Record which ones attach to it
Same words attaching to BACS X, Y  overlap
CS262 Lecture 9, Win06, Batzoglou
pn
Hybridization – Computational Challenge
(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 > n)
CS262 Lecture 9, Win06, Batzoglou
Cj1Cj2 ……………….Cjn
Matrix:
m probes  n clones
C1 C2 ……………….Cn
p1 p2 …………………….pm
0 0 1 …………………..1
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
0 0 0 0 0 0………1 1 1 0
0 0 0 0 0 0………0 1 1 1
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
0 0 0 0 0 0………1 1 1 0
0 0 0 0 0 0………0 1 1 1
Cj1Cj2 ……………….Cjn
Cj1Cj2 ……………….Cjn
Hybridization – Computational Challenge
If we put the matrix in consecutive-ones form,
then we can deduce the order of the clones
& which pairs of clones overlap
CS262 Lecture 9, Win06, Batzoglou
Hybridization – Computational 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
CS262 Lecture 9, Win06, Batzoglou
p1 p2 …………………….pm
C1 C2 ……………….Cn
Additional challenge:
0 0 1 …………………..1
1 1 0 …………………..0
1 0 1…………………...0
2.
Digestion
Restriction enzymes cut DNA where specific words
appear
1. Cut each clone separately with an enzyme
2. Run fragments on a gel and measure length
3. Clones Ca, Cb have fragments of length { li, lj, lk } 
overlap
Double digestion:
Cut with enzyme A, enzyme B, then enzymes A + B
CS262 Lecture 9, Win06, Batzoglou
Online Clone-by-clone
The Walking Method
The Walking Method
1.
Build a very redundant library of BACs with sequenced cloneends (cheap to build)
2.
Sequence some “seed” clones
3.
“Walk” from seeds using clone-ends to pick library clones that
extend left & right
CS262 Lecture 9, Win06, Batzoglou
Walking: An Example
CS262 Lecture 9, Win06, Batzoglou
Walking off a Single Seed
• Low redundant sequencing
• Many sequential steps
CS262 Lecture 9, Win06, Batzoglou
Walking off a single clone is impractical
Cycle time to process one clone: 1-2 months
1.
2.
3.
4.
5.
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 !
CS262 Lecture 9, Win06, Batzoglou
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 <20% redundant sequencing
CS262 Lecture 9, Win06, Batzoglou
Whole-Genome Shotgun Sequencing
Whole Genome Shotgun
Sequencing
genome
cut many times at
random
plasmids (2 – 10 Kbp)
known dist
cosmids (40 Kbp)
~500 bp
CS262 Lecture 9, Win06, Batzoglou
forward-reverse paired
reads
~500 bp