Transcript Lecture #7 (cont.) ppt

Using DNA sequences to
identify target organisms
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Obtain sequence
Align sequences, number of parsimony informative sites
Gap handling
Picking sequences (order)
Analyze sequences
(similarity/parsimony/exhaustive/bayesian
• Analyze output; CI, HI Bootstrap/decay indices
Sequencing reaction (a)
Sequencing reaction requires:
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PCR amplification product as template
1 oligonucleotide - Primer
Nucleotides dATP, dCTP, dGTP, dTTP
Taq polymerase
Modified nucleotides ddATP, ddCTP, ddGTP,
ddTTP
– ddNTPs are incorporated into the polynucleotide
chain and block further elongation
– ddNTPs are fluorescently labeled, each with a
different fluorocrome
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Sequencing reaction (b)
5’
1.
Annealing
2.
Elongation
3.
Incorporation of ddNTP
and stop of the elongation5’
5’
5’
ddCTP HEX
ddATP FAM
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4
Alignment of the 2 sequences obtained using the
Forward and the Reverse primers on the same
PCR amplification product
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Alignment of several sequences showing a T/C
substitution (homozygote)
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Good chromatogram!
Bad chromatogram…
Reverse reaction suffers same problems in opposite direction
Pull-up (too much signal)
Loss of fidelity leads to slips,
skips and mixed signals
Alignments (Se-Al)
Using DNA sequences
• Bootstrap: the presence of a branch separating two
groups of microbial strains could be real or simply one
of the possible ways we could visualize microbial
populations. Bootstrap tests whether the branch is real.
It does so by trying to see through iterations if a similar
branch can come out by chance for a given dataset
• BS value over 65 ok over 80 good, under 60 bad
Statistical support
Re-sampling (~ 10000 times)
Bootstrap analysis
The original loci are randomly re-sampled
with replacement
Jacknife analysis
From the original data 1 locus is randomly
removed
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Using DNA sequences
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Testing alternative trees: kashino hasegawa
Molecular clock
Outgroup
Spatial correlation (Mantel)
• Networks and coalescence approaches
Genotype
• A unique individual as defined by an array
of genetic markers. (the more markers you
have the less mistaken identity you will
have.
blonde
• Blonde
• Blue-eyed
• Blonde
• Blue-eyed
• Hairy
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Blonde
Blue-eyed
Hairy
6 feet tall
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Blonde
Blue-eyed
Hairy
6 feet tall
Missing two molars
In the case of microbes it will
probably be something like
• Genotype A= 01010101
• Genotype B= 00110101
• Genotype C= 00010101
Dominant vs. co-dominant
markers
• Flowers are red or white or yellow, DNA
sequence is agg, agt, agc; DNA fragment
is 10, 12 0r 14 bp long (CO-DOMINANT,
we know what alternative alleles are)
• Flowers are red or non-red, DNA is agg or
not, size is 10bp or not. We only see the
dominant allele and we express it in binary
code 1(present), 0(absent)
Limitations of co-dominant
markers
• Not all non-red flowers are the same, but we assume
they are (non red flowers can be orange or yellow)
• If at one locus we have a dominant A allele and a
recessive a allele, using a codominant marker we would
say AA=Aa but not aa. We know in reality AA and Aa
are quite different.
Study the genetic structure of a population
in an area
 Number of different genotypes
 Determine gene flow between two
population
 Determine if there is an ongoing invasion
 Duration of infestation
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Some Considerations in
Choosing a Genotyping Method
• Level of taxonomic resolution desired
(Populations? Species? Phyla?)
• Level of genotypic resolution desired
– Dominant vs. codominant markers
– Fine (e.g., nucleotide-level) data vs. coarse
(e.g., fragment size) genomic scale
• Previous sequence knowledge
• Cost and labor constraints
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Genetic Markers
• SNPs
Single Nucleotide Polymorphisms
 substitution of a nucleotide
4 alleles: Adenine, Guanine, Cysteine, Thymine
 Insertion/deletion of a nucleotide
2 alleles: presence or absence of the nucleotide
 Approximately every 200 – 300 bp
 Different degrees of variability
• Microsatellites
 variation in number of short tandem repeats
 Unknown number of alleles
 High variability
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Choice of genetic marker (a)
Comparison of individuals of the same
species but isolated requires markers with
low level of variability
No microsatellites
SNPs in genes necessary for the survival
of the cell
• ATPase (cellular energy)
• Cyt b (cytochrome b)
• Cox1 (cytochrome c oxidase subunit 1)
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Choice of genetic marker (b)
• Comparison of individuals closely related
requires markers with high level of variability
 Microsatellites
 SNPs in non-coding regions of genes
 Anonymous SNPs in the genome
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PCR amplification (a)
PCR amplification requires:
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DNA template
2 oligonucleotides - Primers
Nucleotides dATP, dCTP, dGTP, dTTP
Taq polymerase
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PCR reaction (b)
1.
Double strand denaturation
2.
Annealing of the primers
3’
5’
3.
5’
3’
Elongation
5’
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Restriction Enzymes
• Found in bacteria
• Cut DNA within the molecule (endonuclease)
• Cut at sequences that are specific for each enzyme
(restriction sites)
• Leave either blunt or sticky ends, depending upon the
specific enzyme
Tobin & Dusheck, Asking About Life, 2nd ed. Copyright 2001, Harcourt, Inc.
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http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html
Microsatellites
Short tandem repeats
DNA
ACT
ACT
ACT
ACT
ACT
DNA
ACT
ACT ACT
ACT
Microsatellites are located in non-coding regions
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Fluorescent genotyping of
microsatellites
5’
5’
ACT
1.
2.
3.
ACT
ACT
ACT
ACT
PCR amplification using 1 primer fluorescently labeled
PCR amplification product mixed with a size marker
PCR fragments separated by capillary electrophoresis
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Size of the amplification product is variable and
corresponds to the length of the flanking
sequences plus a multiple of the size of the
repeat
Co-dominant:
 homozygote for allele 1
 homozygote for allele 2
 heterozygote
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Tetra repeat: allele 1 486 bp
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Tetra repeat: allele 2 490 bp
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PCR-RFLP
Restriction Fragment Length Polymorphism
• Restriction enzymes cut the DNA at specific sequences
• DNA fragment containing a restriction sequence (EcoRI)
AGGTGAATCCAAAATTTT
• DNA fragment after restriction digestion
AGGTG
AATTCAAATTT
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Scoring PCR-RFLP
Sample 1 Sample 2
 PCR amplification of the
region containing the
restriction sites
Size marker
 Electrophoresis to identify
presence or absence of bands
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PCR-RFLP
Fluorescent electrophoresis
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P. ramorum
CoxI-PCR-RFLP
 PCR amplification of a 972 bp portion of the CoxI gene
 Restriction digestion with Apo I
 EU isolates (mating type A1) have a C at position 377 of the
amplicon
Apo I cuts
 US isolates (mating type A2) have a T at position 377 of the
amplicon
Apo I does not cut
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PCR-SSCP
Single Strand Conformation Polymorphisms
• Denatured DNA (single strand) can be
differentiate using electrophoresis on the
basis of a single nucleotide difference
 PCR amplification of region containing the
polymorphism
 Denaturation
 Gel electrophoresis
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T-RFLP
Terminal Restriction Fragment Length Polymorphisms
436
468
281
485
303
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• PCR amplification of a selected gene,
with one primer labeled with a
fluorophore.
• Digestion of DNA with a restriction
enzyme; number and length of the
resulting fragments is determined by the
presence/absence of appropriate
restriction sites (i.e., depends upon the
underlying DNA sequence
• Because the fluorophore is bound to
the 5’ end of the PCR product, only the
fragment that occurs 5’ to the restriction
site will appear when run on an
automated DNA sequencer
• Size of the fragment may be specific to
a certain genotype (though
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resolution is limited!)
T-RFLP Analysis I:
Hierarchical Clustering
Grouping by overall similarity (distance) calculated between plots
or communities -- e.g., Jaccard’s index: J=M/(M+N), where M
= #matches and N= #mismatches; followed by clustering (e.g.,
UPGMA)
Figure: Plots clustered by bacterial community composition. Groupings
do not correspond to carbon dioxide enrichment treatment
(Osmundson, Naeem et al., in prep.)
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T-RFLP Analysis II:
MRPP & Indicator Species
Analysis
Multiresponse permutation procedure
(MRPP):
Do a priori groups (in this example, based
on carbon dioxide treatment) differ
significantly in their biotic (in this example,
microbial) communities?
Indicator Species Analysis:
Are there species that discriminate
between groups?
(Osmundson, Naeem et al., in prep)
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T-RFLP Analysis III:
NMS (Nonmetric Multidimensional
Scaling)
Ordination based on community presence/absence matrix
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Random Genomic Markers
 DNA sequence of suitable SNPs is not available
 Relatively inexpensive
 Scan the entire genome producing information on
several variations in the same reaction
 RAPD Random Amplification of Polymorphic DNA
 AFLP Amplified Fragment Length Polymorphism
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RAPD
Random Amplification of Polymorphic DNA
 Amplification of genomic DNA included between 2 identical short sequences
(random)
 Genomic DNA is amplified with 1 pair of identical (complementary) primers
(generally 10 bp and GC rich)
example: 5’ AATCGGTACA 3’ and 5’ TGTACCGATT 3’
5’
5’
3’
3’
5’
 Amplification using a low annealing temperature (increased amplification for
sequences not exactly complementary to the primer sequence)
 The primers amplify or not depending on the presence or absence of the
short sequence used to design the primers
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Scoring RAPD
 Presence (1) or absence (0) of amplification
product = Dominant marker
Mismatches between primer and template might
also result in decreased amount of PCR product
Nucleotide substitution at 3’ end of the primer
 no annealing = no amplification
Nucleotide substitution at 5’ end of the primer
 < annealing = < amplification
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AFLP
Amplified Fragment Length Polymorphisms
(Vos et al., 1995)
 Genomic DNA digested with 2 restriction
enzymes:
– EcoRI (6 bp restriction site)
cuts infrequently
– MseI(4 bp restriction site)
cuts frequently
GAATTC
CTTAAG
TTAA
AATT
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 Fragments of DNA resulting from restriction digestion are
ligated with end-specific adaptors (a different one for
each enzyme) to create a new PCR priming site
 Pre selective PCR amplification is done using primers
complementary to the adaptor + 1 bp (chosen by the
user)
N
N
N
N
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 Selective amplification using primers complementary to
the adaptor (+1 bp) + 2 bp
NNN
NNN
NNN NNN
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AFLP genotyping
 PCR amplification using primers corresponding to the
new sequence
If there are 2 new priming sites within 400 – 1600 bp
there is amplification
 The result is: Presence or absence of amplification
1 or 0
Dominant marker: does not distinguish between
heterozygote and homozygote
 Due mostly to SNPs but also to deletions/insertions
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AFLP OVERVIEW
(VOS ET AL., 1995)
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AFLP
Fluorescent electrophoresis
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