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’

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

10 Statistical support  Re-sampling (~ 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= Genotype B= Genotype C=

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

21 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

22 Genetic Markers SNPsSingle 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

23 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)

24 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

25 PCR amplification (a) PCR amplification requires:  DNA template  2 oligonucleotides - Primers  Nucleotides dATP, dCTP, dGTP, dTTP  Taq polymerase

26 PCR reaction (b) 1.Double strand denaturation 2.Annealing of the primers 3.Elongation 5’ 3’

27 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.

28 Microsatellites Short tandem repeats ACT DNA Microsatellites are located in non-coding regions

29 Fluorescent genotyping of microsatellites 1.PCR amplification using 1 primer fluorescently labeled 2.PCR amplification product mixed with a size marker 3.PCR fragments separated by capillary electrophoresis ACT 5’

30  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

31 Tetra repeat: allele bp

32 Tetra repeat: allele bp

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34 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

35 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

36 PCR-RFLP Fluorescent electrophoresis

37 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

38 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

39 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!) T-RFLP Terminal Restriction Fragment Length Polymorphisms

40 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.)

41 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)

42 T-RFLP Analysis III: NMS (Nonmetric Multidimensional Scaling) Ordination based on community presence/absence matrix

43 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

44 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’ 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’ 5’

45 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

46 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

47  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) NNN N

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

49 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

50 AFLP OVERVIEW (VOS ET AL., 1995)

51 AFLP Fluorescent electrophoresis