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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) 
• Current technology can only reliably ‘read’ 
a short stretch – typically hundreds of base 
pairs
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 
• There are competing methods of assembly 
• The competing – public and private – 
sequencing teams used competing assembly 
methods
Assembly: 
• Putting sequenced fragments of DNA 
into their correct chromosomal 
positions
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 
• Contiguous sequence of DNA created 
by assembling overlapping sequenced 
fragments of a chromosome (whether 
natural or artificial, as in BACs)
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 
• Successively sequencing DNA from 
adjacent stretches of chromosome
Draft sequence 
• Sequence with lower accuracy than a 
finished sequence; some segments are 
missing or in the wrong order or 
orientation
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
Exon 
• Region of a gene’s DNA that encodes 
a portion of its protein; exons are 
interspersed with noncoding introns
Genome 
• The entire chromosomal genetic 
material of an organism
Intron 
• Region of a gene’s DNA that is not 
translated into a protein
Kilobase (kb) 
• Unit of DNA equal to 1000 bases
Locus 
• Chromosomal location of a gene or 
other piece of DNA
Megabase (mb) 
• Unit of DNA equal to 1 million bases
PCR 
• Polymerase chain reaction: a technique 
for amplifying a piece of DNA quickly 
and cheaply
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 
• Loop of bacterial DNA that replicates 
independently of the chromosomes; 
artificial plasmids can be inserted into 
bacteria to amplify DNA for 
sequencing
Regulatory region 
• A segment of DNA that controls 
whether a gene will be expressed and 
to what degree
Repetitive DNA 
• Sequences of varying lenths that occur 
in multiple copies in the genome; it 
represents much of the genome
Restriction enzyme 
• An enzyme that cuts DNA at specific 
sequences of base pairs
RFLP 
• Restriction fragment length 
polymorphism: genetic variation in the 
length of DNA fragments produced by 
restriction enzymes; useful as markers 
on maps
Scaffold 
• A series of contigs that are in the right 
order but are not necessarily connected 
in one continuous stretch of sequence
Shotgun sequencing 
• Breaking DNA into many small pieces, 
sequencing the pieces, and assembling 
the fragments
STS 
• Sequence tagged site: a unique stretch 
of DNA whose location is known; 
serves as a landmark for mapping and 
assembly
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 
• 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 
• 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
Physical map
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’
Hierarchical v. whole-genome
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 e. 
– Unknown orientation. Don’t know whether to 
use read or its Watson-Crick complement.
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 
• 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)
Gaps 
• Read length LR hasn’t changed much 
• w = LR /G gets steadily smaller 
• Gaps ~ Re- wR (Waterman & Lander)
How deep must coverage be?
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
Genomic results
HGSC v Celera results
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? 
• “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 
• 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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sequencing of genome

  • 2. Definition • Determining the identity and order of nucleotides in the genetic material – usually DNA, sometimes RNA, of an organism
  • 3. 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
  • 4. 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
  • 5. A human drama • There are competing methods of assembly • The competing – public and private – sequencing teams used competing assembly methods
  • 6. Assembly: • Putting sequenced fragments of DNA into their correct chromosomal positions
  • 7. 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.
  • 8. Contig • Contiguous sequence of DNA created by assembling overlapping sequenced fragments of a chromosome (whether natural or artificial, as in BACs)
  • 9. Cosmid • DNA from a bacterial virus spliced with a small fragment of a genome (45 kb or less) to be amplified and sequenced
  • 10. Directed sequencing • Successively sequencing DNA from adjacent stretches of chromosome
  • 11. Draft sequence • Sequence with lower accuracy than a finished sequence; some segments are missing or in the wrong order or orientation
  • 12. 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
  • 13. Exon • Region of a gene’s DNA that encodes a portion of its protein; exons are interspersed with noncoding introns
  • 14. Genome • The entire chromosomal genetic material of an organism
  • 15. Intron • Region of a gene’s DNA that is not translated into a protein
  • 16. Kilobase (kb) • Unit of DNA equal to 1000 bases
  • 17. Locus • Chromosomal location of a gene or other piece of DNA
  • 18. Megabase (mb) • Unit of DNA equal to 1 million bases
  • 19. PCR • Polymerase chain reaction: a technique for amplifying a piece of DNA quickly and cheaply
  • 20. 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
  • 21. Plasmid • Loop of bacterial DNA that replicates independently of the chromosomes; artificial plasmids can be inserted into bacteria to amplify DNA for sequencing
  • 22. Regulatory region • A segment of DNA that controls whether a gene will be expressed and to what degree
  • 23. Repetitive DNA • Sequences of varying lenths that occur in multiple copies in the genome; it represents much of the genome
  • 24. Restriction enzyme • An enzyme that cuts DNA at specific sequences of base pairs
  • 25. RFLP • Restriction fragment length polymorphism: genetic variation in the length of DNA fragments produced by restriction enzymes; useful as markers on maps
  • 26. Scaffold • A series of contigs that are in the right order but are not necessarily connected in one continuous stretch of sequence
  • 27. Shotgun sequencing • Breaking DNA into many small pieces, sequencing the pieces, and assembling the fragments
  • 28. STS • Sequence tagged site: a unique stretch of DNA whose location is known; serves as a landmark for mapping and assembly
  • 29. 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
  • 30. 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
  • 31. 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
  • 33. 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’
  • 35. 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 e. – Unknown orientation. Don’t know whether to use read or its Watson-Crick complement.
  • 36. 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)
  • 37. 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)
  • 38. Gaps • Read length LR hasn’t changed much • w = LR /G gets steadily smaller • Gaps ~ Re- wR (Waterman & Lander)
  • 39. How deep must coverage be?
  • 40. 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
  • 42. HGSC v Celera results
  • 43. 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
  • 44. 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
  • 45. 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.”