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Sequencing a genome. Definition. Determining the identity and order of nucleotides in the genetic material – usually DNA, sometimes RNA, of an organism. Basic problem. Genomes are large (typically millions or billions of base pairs)

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definition
Definition
  • Determining the identity and order of nucleotides in the genetic material – usually DNA, sometimes RNA, of an organism
basic problem
Basic problem
  • Genomes are large (typically millions or billions of base pairs)
  • Current technology can only reliably ‘read’ a short stretch – typically hundreds of base pairs
elements of a solution
Elements of a solution
  • Automation – over the past decade, the amount of hand-labor in the ‘reads’ has been steadily and dramatically reduced
  • Assembly of the reads into sequences is an algorithmic and computational problem
a human drama
A human drama
  • There are competing methods of assembly
  • The competing – public and private – sequencing teams used competing assembly methods
assembly
Assembly:
  • Putting sequenced fragments of DNA into their correct chromosomal positions
slide7
BAC
  • Bacterial artificial chromosome: bacterial DNA spliced with a medium-sized fragment of a genome (100 to 300 kb) to be amplified in bacteria and sequenced.
contig
Contig
  • Contiguous sequence of DNA created by assembling overlapping sequenced fragments of a chromosome (whether natural or artificial, as in BACs)
cosmid
Cosmid
  • DNA from a bacterial virus spliced with a small fragment of a genome (45 kb or less) to be amplified and sequenced
directed sequencing
Directed sequencing
  • Successively sequencing DNA from adjacent stretches of chromosome
draft sequence
Draft sequence
  • Sequence with lower accuracy than a finished sequence; some segments are missing or in the wrong order or orientation
slide12
EST
  • Expressed sequence tag: a unique stretch of DNA within a coding region of a gene; useful for identifying full-length genes and as a landmark for mapping
slide13
Exon
  • Region of a gene’s DNA that encodes a portion of its protein; exons are interspersed with noncoding introns
genome
Genome
  • The entire chromosomal genetic material of an organism
intron
Intron
  • Region of a gene’s DNA that is not translated into a protein
kilobase kb
Kilobase (kb)
  • Unit of DNA equal to 1000 bases
locus
Locus
  • Chromosomal location of a gene or other piece of DNA
megabase mb
Megabase (mb)
  • Unit of DNA equal to 1 million bases
slide19
PCR
  • Polymerase chain reaction: a technique for amplifying a piece of DNA quickly and cheaply
physical map
Physical map
  • A map of the locations of identifiable markers spaced along the chromosomes; a physical map may also be a set of overlapping clones
plasmid
Plasmid
  • Loop of bacterial DNA that replicates independently of the chromosomes; artificial plasmids can be inserted into bacteria to amplify DNA for sequencing
regulatory region
Regulatory region
  • A segment of DNA that controls whether a gene will be expressed and to what degree
repetitive dna
Repetitive DNA
  • Sequences of varying lenths that occur in multiple copies in the genome; it represents much of the genome
restriction enzyme
Restriction enzyme
  • An enzyme that cuts DNA at specific sequences of base pairs
slide25
RFLP
  • Restriction fragment length polymorphism: genetic variation in the length of DNA fragments produced by restriction enzymes; useful as markers on maps
scaffold
Scaffold
  • A series of contigs that are in the right order but are not necessarily connected in one continuous stretch of sequence
shotgun sequencing
Shotgun sequencing
  • Breaking DNA into many small pieces, sequencing the pieces, and assembling the fragments
slide28
STS
  • Sequence tagged site: a unique stretch of DNA whose location is known; serves as a landmark for mapping and assembly
slide29
YAC
  • Yeast artificial chromosome: yeast DNA spliced with a large fragment of a genome (up to 1 mb) to be amplified in yeast cells and sequenced
readings
Readings
  • Myers, “Whole Genome DNA Sequencing,” http://www.cs.arizona.edu/people/gene/PAPERS/whole.IEEE.pdf
  • Venter, et al, “The Sequence of the Human Genome,” Science, 16 Feb 2001, Vol. 291 No 5507, 1304 (parts 1 & 2)
  • Waterston, Lander, Sulston, “On the sequencing of the human genome,” PNAS, March 19, 2002, Vol 99, no 6, 3712-3716
  • Myers, et.al., “On the sequencing and assembly of the human genome,” www.pnas.org/cgi/doi/10.1073/pnas.092136699
hierarchical sequencing
Hierarchical sequencing
  • Create a high-level physical map, using ESTs and STSs
  • Shred genome into overlapping clones
  • Multiply clones in BACs
  • ‘shotgun’ each clone
  • Read each ‘shotgunned’ fragment
  • Assemble the fragments
whole genome sequencing wgs
Whole genome sequencing (WGS)
  • Make multiple copies of the target
  • Randomly ‘shotgun’ each target, discarding very big and very small pieces
  • Read each fragment
  • Reassemble the ‘reads’
the fragment assembly problem
The fragment assembly problem
  • Aim: infer the target from the reads
  • Difficulties –
    • Incomplete coverage. Leaves contigs separated by gaps of unknown size.
    • Sequencing errors. Rate increases with length of read. Less than some .
    • Unknown orientation. Don’t know whether to use read or its Watson-Crick complement.
scaling and computational complexity
Scaling and computational complexity
  • Increasing size of target G.
    • 1990 – 40kb (one cosmid)
    • 1995 – 1.8 mb (H. Influenza)
    • 2001 – 3,200 mb (H. sapiens)
the repeat problem
The repeat problem
  • Repeats
    • Bigger G means more repeats
    • Complex organisms have more repetitive elements
    • Small repeats may appear multiple times in a read
    • Long repeats may be bigger than reads (no unique region)
slide38
Gaps
  • Read length LR hasn’t changed much
  •  = LR /G gets steadily smaller
  • Gaps ~ Re- R (Waterman & Lander)
double barreled shotgun sequencing
Double-barreled shotgun sequencing
  • Choose longer fragments (say, 2 x LR)
  • Read both ends
  • Such fragments probably span gaps
  • This gives an approximate size of the gap
  • This links contigs into scaffolds
to do or not to do
To do or not to do?
  • “The idea is gathering momentum. I shiver at the thought.” – David Baltimore, 1986
  • “If there is anything worth doing twice, it’s the human genome.” – David Haussler, 2000
public or private
Public or private?
  • “This information is so important that it cannot be proprietary.” – C Thomas Caskey, 1987
  • “If a company behaves in what scientists believe is a socially responsible manner, they can’t make a profit.” – Robert Cook-Deegan, 1987
hw for feb 17
HW for Feb 17
  • Comment on these assertions (500-1000 words):
    • WLS – “Our analysis indicates that the Celera paper provides neither a meaningful test of the WGS approach nor an independent sequence of the human genome.”
    • Venter – “This conclusion is based on incorrect assumptions and flawed reasoning.”
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