7.91 / 7.36 / BE.490
Lecture #1
Feb. 24, 2004
Genome Sequencing
&
DNA Sequence Analysis
Chris Burge
What is a Genome?
A genome is NOT a bag of proteins
What s in the Human Genome?
Outline of Unit II:
DNA/RNA Sequence Analysis
Reading*
2/24 Genome Sequencing & DNA Sequence Analysis M Ch. 3
2/26 DNA Sequence Comparison & Alignment M Ch. 7
3/2 DNA Motif Modeling & Discovery M Ch. 4
3/4 Markov and Hidden Markov Models for DNA M Ch. 4
3/9 DNA Sequence Evolution M Ch. 6
3/11 RNA Structure Prediction & Applications M Ch. 5
3/16 Literature Discussion TBA
* M = Mount,  Bioinformatics: Sequence and Genome Analysis
Feedback to Instructor
Examples from past years:
" Comic font looks stupid
" Burge uses too much genomics jargon
" Better synergy between Yaffe/Burge sections
" Asks questions to the class, student answers,
but I didn t hear/understand the answer&
DNA vs Protein Sequence Analysis
Protein Sequence Analysis DNA Sequence Analysis
- emphasis on chemistry - emphasis on regulation
- protein structure - RNA structure
- selection is everywhere - signal vs noise (statistics)
- multiple alignment - motif finding
- comparative proteomics - comparative genomics
- data: O(10^8) aa - data: O(10^10) nt
Read your probability/statistics primer!
Genome Sequencing
& DNA Sequence Analysis
" The Language of Genomics
" Shotgun Sequencing
- Progress: genomes, transcriptomes, etc.
" DNA Sequence Alignment I
- How to choose a mismatch penalty
" Comparative Genomics Examples
- PipMaker, Phylogenetic Shadowing
Recent Media Attention
Genomespeak
Bork, Peer, and Richard Copley. " Genome Speak."
Nature 409 (15 February 2001): 815.
Learn to speak genomic
In the following article, note the use of the following genomic terms:
euchromatic, whole-genome shotgun sequencing, sequence reads,
5.11-fold coverage, plasmid clones, whole-genome assembly,
regional chromosome assembly.
Venter, JC, MD Adams, EW Myers, PW Li, RJ Mural, GG Sutton, HO Smith, & "The
Sequence of The Human Genome." Science 291, no. 5507 (16 February 2001): 1304-51.
Types of Nucleotides
" ribonucleotides
" deoxyribonucleotides
" dideoxyribonucleotides
DNA Sequencing
Adapted from Fig. 4.2 of  Genomes by T. A. Brown, John Wiley & Sons, NY, 1999
Shotgun Sequencing a BAC or a Genome
200 kb (NIH)
3 Gb (Celera)
Sonicate, Subclone
Subclones
Sequence, Assemble
What
would
cause
problems
with
assembly?
Shotgun Contigs
Shotgun Coverage
(Poisson distribution)
Sequence N reads, 500 bp each, from a 200kb BAC
Coverage/read p = 500/200,000 = 0.0025
Total coverage C = Np
Y = no. of reads covering the point x
P(Y=k) = (N!/(N-k)!k!) pk(1-p)N-k
H" e-cck / k!
.
x
P(Y=0)= e-c Examples: e-2 H" 0.14 e-4 H" 0.02
What could cause reality to differ from theory?
Clickable Genomes
Eukaryotes Protists Eubacteria
S. cerevisiae Plasmodium E. coli
S. pombe Giardia B. subtilis
C. elegans & S. aureus
Drosophila (several) &
Anopheles (>100)
Ciona Archaea
Arabidopsis Methanococcus
Human Sulfolobus Phages/Viruses
Mouse & Lots
Tetraodon (total of ~16)
Fugu Organelles
Zebrafish Lots
Neurospora
Aspergillus
&
Large-scale Transcript Sequencing
Please see the following example article that uses large-scale
transcript sequencing.
Okazaki, Y, M Furuno, T Kasukawa, J Adachi, H Bono, S Kondo, & "Analysis of The
Mouse Transcriptome Based On Functional Annotation of 60,770 Full-length cDNAs."
Nature 420, no. 6915 (5 December 2002): 563-73.
EST Sequencing
dbEST release 022004 No. of public entries: 20,039,613
Summary by Organism - as of February 20, 2004
Homo sapiens (human) 5,472,005
Mus musculus + domesticus (mouse) 4,055,481
Rattus sp. (rat) 583,841
Triticum aestivum (wheat) 549,926
Ciona intestinalis 492,511
Gallus gallus (chicken) 460,385
Danio rerio (zebrafish) 450,652
Zea mays (maize) 391,417
Xenopus laevis (African clawed frog) 359,901
Hordeum vulgare + subsp. vulgare (barley) 352,924
Source: NCBI - http://ncbi.nlm.nih.gov
-omes and -omics *
Glycome ???
Ribonome?
Proteome
Mass spec, Y2H, ?
Variome
SNPs, haplotypes
Transcriptome
ESTs, cDNAs, microarrays
Genome
Genome sequences
*Warning: some of the words on this slide may not be in Webster s dictionary
DNA Sequence Alignment I
How does DNA alignment differ from protein alignment?
Query:
1 ttgacctagatgagatgtcgttcacttttactgagctacagaaaa 45
|||| |||||||||||| | |||||||||||||||||||||||||
Subject:
403 ttgatctagatgagatgccattcacttttactgagctacagaaaa 447
Use BLASTN instead of BLASTP
Nucleotide-
nucleotide
BLAST Web
Server
(BLASTN)
DNA Sequence Alignment II
Translating searches:
translate in all possible reading frames
search peptides against protein database (BLASTP)
ttgacctagatgagatgtcgttcactttactgagctacagaaaa
ttg|acc|tag|atg|aga|tgt|cgt|tca|ctt|tta|ctg|agc|tac|aga|aaa
M
L T x R C R S L L L S Y R K
t|tga|cct|aga|tga|gat|gtc|gtt|cac|ttt|tac|tga|gct|aca|gaa|aa
x P R x D V V H F Y x S T E
tt|gac|cta|gat|gag|atg|tcg|ttc|act|ttt|act|gag|cta|cag|aaa|a
D L D E M S F T F T E L Q K
Also consider reading frames on complementary DNA strand
DNA Sequence Alignment III
Common flavors of BLAST:
Program Query Database
aa aa
BLASTP
nt nt
BLASTN
nt (Ò! aa) aa
BLASTX
aa nt (Ò! aa)
TBLASTN
nt (Ò! aa) nt (Ò! aa)
TBLASTX
aa (aa msa) aa
PsiBLAST
Which would be best for searching ESTs against a genome?
DNA Sequence Alignment IV
Which alignments are significant?
Q:
1 ttgacctagatgagatgtcgttcacttttactgagctacagaaaa 45
|||| |||||||||||| | |||||||||||||||||||||||||
S:
403 ttgatctagatgagatgccattcacttttactgagctacagaaaa 447
Identify high scoring segments whose score S exceeds
a cutoff x using dynamic programming.
Scores follow an extreme value distribution:
P(S > x) = 1 - exp[-Kmn e-x]
For sequences of length m, n where K,  depend on the score
matrix and the composition of the sequences being compared
(Same theory as for protein sequence alignments)
Notes (cont)
From M. Yaffe
Lecture #2
Probability values for the extreme value distribution (A) and the
normal distribution (B). The area under each curve is 1.
A.
B.
0.4
" The random sequence alignment scores
0.4
would give rise to an  extreme value
0.2
distribution  like a skewed gaussian.
Yev
Yn
" Called Gumbel extreme value
distribution
-4
4
-2 -1 0 1 2 3 4 5
X X
For a normal distribution with a mean m and a variance Ã, the height of the
curve is described by Y=1/(Ã"2Ä„) exp[-(x-m)2/2Ã2]
For an extreme value distribution, the height of the curve is described by
Y=exp[-x-e-x] & and P(S>x) = 1-exp[-e-(x-u)] where u=(ln Kmn)/
Can show that mean extreme score is ~ log2(nm), and the probability of
getting a score that exceeds some number of  standard deviations x is:
P(S>x)~ Kmne-x. ***K and  are tabulated for different matrices ****
For the less statistically inclined: E~ Kmne -S
DNA Sequence Alignment V
How is  related to the score matrix?
 is the unique positive solution to the equation*:
" pipjesij = 1
i,j
pi = frequency of nt i, sij = score for aligning an i,j pair
What kind of an equation is this? (transcendental)
What would happen to  ð if we doubled all the scores? (reduced by half)
What does this tell us about the nature of ? (scaling factor)
*Karlin & Altschul, 1990
DNA Sequence Alignment VI
What scoring matrix to use for DNA?
Usually use simple match-mismatch matrices:
i j: A C G T
A 1 m m m
C m 1 m m
G m m 1 m
si,j :
T m m m 1
m =  mismatch penalty (must be negative)
DNA Sequence Alignment VII
How to choose the mismatch penalty?
Use theory of High Scoring Segment composition*
High scoring alignments will have composition:
qij = pipjesij
where qij = frequency of i,j pairs ( target frequencies )
pi, pj = freq of i, j bases in sequences being compared
What would happen to the target frequencies if we
doubled all of the scores?
*Karlin & Altschul, 1990