Using DNA sequences to identify target organisms Obtain sequence Align sequences, number of parsimony informative sites Gap handling Picking sequences.

Using DNA sequences to identify target organisms

• 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

2

Sequencing reaction (a)

Sequencing reaction requires:

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

3

Sequencing reaction (b)

1. Annealing

2. Elongation

3. Incorporation of ddNTP and stop of the elongation

ddATP FAM

ddCTP HEX

5’

5’

5’

5’

4

5

Alignment of the 2 sequences obtained using the Forward and the Reverse primers on the same PCR amplification product

6

Alignment of several sequences showing a T/C substitution (homozygote)

Good chromatogram!

Bad chromatogram…

Pull-up (too much signal) Loss of fidelity leads to slips, skips and mixed signals

Reverse reaction suffers same problems in opposite direction

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

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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

Using DNA sequences

• 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

• Blonde

• Blue-eyed

• Hairy

• 6 feet tall

• 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.

20

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

• SNPsSingle Nucleotide Polymorphisms

substitution of a nucleotide4 alleles: Adenine, Guanine, Cysteine, Thymine

Insertion/deletion of a nucleotide2 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 microsatellitesSNPs 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

MicrosatellitesSNPs in non-coding regions of genesAnonymous SNPs in the genome

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PCR amplification (a)

PCR amplification requires:

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. Elongation

5’5’

5’

3’3’

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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

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html

Tobin & Dusheck, Asking About Life, 2nd ed. Copyright 2001, Harcourt, Inc.

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Microsatellites Short tandem repeats

ACT ACT ACTACT ACT

ACT ACT ACTACT

DNA

DNA

Microsatellites are located in non-coding regions

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Fluorescent genotyping of microsatellites

1. PCR amplification using 1 primer fluorescently labeled2. PCR amplification product mixed with a size marker3. PCR fragments separated by capillary electrophoresis

ACT ACT ACT ACTACT

5’ 5’

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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 1homozygote for allele 2 heterozygote

31Tetra repeat: allele 1 486 bp

32Tetra repeat: allele 2 490 bp

33

Sequencing reaction (a)

Sequencing reaction requires:

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

34

Sequencing reaction (b)

1. Annealing

2. Elongation

3. Incorporation of ddNTP and stop of the elongation

ddATP FAM

ddCTP HEX

5’

5’

5’

5’

35

36

Alignment of the 2 sequences obtained using the Forward and the Reverse primers on the same PCR amplification product

37

Alignment of several sequences showing a T/C substitution (homozygote)

38

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

PCR amplification of the region containing the restriction sites

Electrophoresis to identify presence or absence of bands

Size marker

Sample 1 Sample 2

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PCR-RFLPFluorescent electrophoresis

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P. ramorumCoxI-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-SSCPSingle 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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• 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

resolution is limited!)436

281160303

468 485

T-RFLPTerminal Restriction Fragment Length Polymorphisms

44

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

48

RAPDRandom 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’

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

3’3’ 5’5’5’

49

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

GAATTCCTTAAG

TTAAAATT

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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)

NN N N

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Selective amplification using primers complementary to the adaptor (+1 bp) + 2 bp

NNNNNN NNN NNN

53

AFLP genotyping

PCR amplification using primers corresponding to the new sequenceIf there are 2 new priming sites within 400 – 1600 bp there is amplification

The result is: Presence or absence of amplification1 or 0Dominant 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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AFLPFluorescent electrophoresis