Quiz next week

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Tutorial 2
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Quiz next week

• Cover everything youve seen in the course so far
• Combination of True/False, definition, short
  answer, or some similar question from the problem
  set

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How to design a PCR primer?

• Primer length and sequence are of critical
  importance in designing the parameters of a
  successful amplification
• A simple formula for calculating the Tm
• Tm 4(G C) 2(A T)
• When designing a PCR primer, Tm is not the only
  thing, should also consider the GC content, any
  secondary structure or hairpin loop

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Example
Design PCR primer to amplify IFI16 (interferon,
gamma-inducible protein 16)
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NCBI
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Synonymous Vs Nonsynonymous

• When studying the evolutionary divergences of DNA
  sequence
• Synonymous silent
• Nonsynonymous amino acid altering
• The rates of these nucleotide substitution maybe
  used as a molecular clock for dating the
  evolutionary time of closely related species

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Calculating Synonymous sites (s) and
nonsynonymous sites (n)

• Each codon has 3 nucleotides, denote by fi (I
  1,2,3)
• Where s and n for a codon are given by
• s ?3i1fi and n (3-s)
• Ex. TTA (Leu) f11/3 (T?C)
• f20
• f31/3 (A?G)
• Thus, s 2/3 and n 7/3
• For DNA sequence of r codons, it will be
• s ?ri1si and n (3r-s),
• where si is the value of s for the ith codon

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Calculation of s and n for 2 nucleotide
differences between 2 codons

• Ex. GTT (Val) and GTA (Val)
• 1 synonymous difference
• Denote sd and nd the number of synonymous and
  nonsynonymous differences per codon, respectively
• sd 1
• nd 0

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Cont

• Ex. TTT and GTA, 2 pathways to get there
• Pathway 1 TTT(Phe)?GTT(Val)?GTA(Val)
• Pathway 2 TTT(Phe)?TTA(Leu)?GTA(Val)
• Pathway 1 involve 1 synonymous and 1
  nonsynonymous substitution
• Pathway 2 involve 2 nonsynonymous substitution
• sd 1 synonymous substitution / 2 change state
  0.5
• nd 3 nonsysnonymous substitution / 2 change
  state 1.5
• D in the problem set proportion of synonymous
  or nonsynonymous differences, therefore, for this
  nonsynonymous site, the Dn would be
• 1 / 1.5 0.667
• Note that sd nd is equal to the total number of
  nucleotide differences between the two DNA
  sequences compared

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Sequence Alignment

• Every alignment will have a scoring system
• Base change cost 1
• Gap cost 2
• Gap extension cost 1
• Ex. ACT GTT GCC
• AG - C - - GCT
• Score of this alignment would be
• 3 2x2 1 8
• In this case, a higher score means a worst
  alignment

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MLST - Methods

• Isolate multiple strains of species of interest
• PCR 500bp regions of 4-20 housekeeping genes
  (loci)
• Sequence PCR products
• Assign allele numbers to each locus
• Arbitrary, each represents a different sequence

1
2
3
1
2
1
1
1
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MLST - Methods

• Collate the information into a table
• Row isolate
• Column loci
• Fill in allele numbers

Locus A Locus B Locus C
Isolate 1 1 1 1
Isolate 2 2 2 1
Isolate 3 3 1 2
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MLST of a Halorubrum Population

• 36 isolates
• 4 housekeeping genes
• atpB
• ef-2
• radA
• secY
• 500bp PCR product
• Allelic profiles vary
• Few identical pairs
• All loci polymorphic
• 8-15 alleles

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Insights from the MLST Data - 1
How genetically diverse is the saltern Archaeal
population?

• Genetic diversity H 1-Sxi2
• Overall genetic diversity 0.69
• Varied between ponds of different salinity
• 0.57 in 23 saline pond
• 0.83 in 36 saline pond
• Higher than E. coli diversity of 0.47
• gt5x higher than eukaryotic diversity

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Insights from the MLST Data - 2
Is recombination occurring in the Archaea?

• Linkage disequilibrium calculator mlst.net
• LD Alleles are linked and are transferred
  together during recombination
• LE Alleles are not linked and recombination
  scatters them randomly
• Halorubrum population is near linkage equilibrium
• Suggests recombination is occurring

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Tetraodon Nigroviridis
2X?
Nature Reviews Genetics 3 838-849 (2002)
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Phylogenetic tree

• Phylogenetics is the field of systematics that
  focuses on evolutionary relationship between
  organisms or genes/proteins (phylogeny)

A node
Human Mouse Fly
A clade

• clade -- A monophyletic taxon
• taxon -- Any named group of organisms, not
  necessarily a clade.

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A phylogenetic tree
A node
ABC is less than DBC So the mouse Sequence
is more related to fly than the human sequence is
to fly in this example
Human Mouse Fly
D A C
A clade
B
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Tetraodon gene evolution

• Fourfold degenerate (4D) site substitution - a
  mesure of neutral nucleotide mutations
• 4D site 3rd base of codon free to change with
  no FX on AA
• of AA changes at these sites neutral
  mutations
• Fish proteins have diverged faster vs. mammalian
  homologues

Figure 3
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Brief generalization of the papers

• Comparative genomics help identifying region of
  DNA that are shared between two different species
  and allows the transfer of information between
  both species in the common region.
• It can also detect regions that have gone through
  chromosomes rearrangement occurring in many
  different diseases. This information can be of
  different type.
• 1) Using one of the species it is possible to
  transfer annotation information that were not
  known in the other species,
• 2) identify region that are under selective
  pressure,
• 3) It is also possible to compare for examples
  regions that have gone through chromosomes
  rearrangement with annotation genes map to
  identify genes responsible for a particular
  disease

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Homologs

• Have common origins but may or may not have
  common activity
• Orthologs Homologs produced by speciation. They
  tend to have similar function
• Paralogs Homologs produced by gene duplication.
  They tend to have differing function
• Xenologs Homologs resulting from horizontal
  gene transfer between two organism

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BLAST

• Basic Local Alignment Search Tool
• Developed in 1990 and 1997 (S. Altschul)
• A heuristic method (Fast alignment method) for
  performing local alignments through searches of
  high scoring segment pairs (HSPs)
• 1st to use statistics to predict significance of
  initial matches - saves on false leads
• Offers both sensitivity and speed

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BLAST

• Looks for clusters of nearby or locally dense
  similar or homologous k-tuples
• Uses look-up tables to shorten search time
• Uses larger word size than FASTA to accelerate
  the search process
• Can generate domain friendly local alignments
• Fastest and most frequently used sequence
  alignment tool BECAME THE STANDARD

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Connecting HSPs
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Extreme Value Distribution

• Kmne-lS is called Expect or E-value
• In BLAST, default E cutoff 10 so P 0.99995
• If E is small then P is small
• Why does BLAST report an E-value instead of a p
  value?
• E-values of 5 and 10 are easier to understand
  than P-values of 0.993 and 0.99995.
• However, note that when E lt 0.01, P-values and
  E-value are nearly identical.

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Expect value

• Kmne-lS Expect or E-value
• What parameters does it depend on?
• - l and K are two parameters natural scales for
  search space size and scoring system,
  respectively
• l lnq/p and K (q-p)2/q
• p probability of match (i.e. 0.05)
• q probability of not match (i.e. 0.95)
• Then l 2.94 and K 0.85
• p and q calculated from a random sequence model
  (Altschul, S.F. Gish, W. (1996) "Local
  alignment statistics." Meth. Enzymol.
  266460-480.) based on given subst. matrix and
  gap costs
• - m length of sequence
• - n length of database
• - S score for given HSP

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Expect value

• Expect value an intuitive value but
• Expect value changes as database changes
• Expect value becomes zero quickly
• Alternative bit score
• S' (bits) lambda S (raw) - ln K / ln 2
• Independent of scoring system used - normalized
• Larger value for more similar sequences,
  therefore useful in analyses of very similar
  sequences

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Similarity by chance the impact of sequence
complexity
MCDEFGHIKLAN. High Complexity
ACTGTCACTGAT. Mid Complexity
NNNNTTTTTNNN. Low Complexity
Low complexity sequences are more likely to
appear similar by chance