MICROSATELLITE MARKERS FOR LIVESTOCK
GENETIC DIVERSITY ANALYSES
Karan Veer Singh
National Bureau of Animal Genetic Resourc...
LIVESTOCK DIVERSITY
About 40 species of domestic animals and poultry contribute to meeting the
About 40 species of domesti...
LIVESTOCK DIVERSITY IN INDIA
Species

No. of recognized
breeds

Buffalo

13

Cattle

37

Sheep

39

Goat

23

Camel

8

Ho...
REASONS FOR DECLINE IN DOMESTIC ANIMAL BIODIVERSITY
•

Conservation of indigenous breeds has received little
attention in ...
LIVESTOCK GENETIC ANALYSIS
Livestock Breed analysis/characterization requires
knowledge of genetic variation.
Genetic vari...
MOLECULAR/DNA MARKERS
Any DNA fragment or gene coding for a trait which is free of
environmental effect and does not inter...
MICROSATELLITE/SSR MARKERS
• Litt and Luty 1989 (Am. J. Hum. Gen.)
• Litt and Luty 1989 (Am. J. Hum. Gen.)
• Sequences of ...
REPEAT STRUCTURE OF MICROSATELLITES
Mononucleotide - (A)11
AAAAAAAAAAA
Dinucleotide - (GT)6
GTGTGTGTGTGT
Trinucleotide - (...
TYPES OF MICROSATELLITES BASED ON THE
NATURE OF REPEATS
POLYMORPHISM
AATG

7 repeats
8 repeats
the repeat region is variable between samples while the
flanking regions where PCR ...
EVOLUTION OF MICROSATELLITES
•• Mutation
Mutation
•• It is estimated that microsatellites mutate 100 to
It is estimated th...
How do microsatellites mutate?
• Microsatellites alleles change rather quickly over time
 E. coli – 10-2 events per locus...
MICROSATELLITES - TOOLS OF CHOICE
 Low quantities of template DNA required (10-100
ng)
 High genomic abundance
 Random ...
Stutter Bands in SSR
 Often there are minor bands in addition to the major bands. These
minor bands are called stutter ba...
Homology vs. Homoplasy
•

.

Homology is any similarity
between characters that is due
to their shared ancestry

•

Homopl...
HOW DO WE DEVELOP MICROSATELLITE
PRIMERS?


















DNA Extraction
DNA Extraction
Digestion of gen...
WHAT ARE MICROSATELLITES FOR?
• Microsatellites are “junk” DNA.
In humans, 90% of
microsatellites are found in noncoding r...
APPLICATIONS
• Forensics and parentage analysis
• Disease diagnosis
• Diversity analysis
• Population Studies
• Conservati...
• Forensics
Because microsatellites are so variable, by
studying several at one time (and getting a
DNA fingerprint), indi...
PARENTAGE VERIFICATION

Exclusion of false parents with a probability of
as high as 99.999 % against 40 – 60 % from
bioche...
Disease Diagnosis – Huntington’s disease

Huntington's disease is caused by a genetic defect on chromosome 4. The defect c...
DIVERSITY ANALYSIS

• Observed heterozygosity (Ho) and gene
diversity or expected heterozygosity (H e) are
measures of gen...
INTRASPECIFIC (WITHIN SPECIES)
Genetic variability between & within breeds- through genetic
distancing and heterozygosity ...
Ja
la

wa
d

Na

er i

i

Ma
l pu
ra
Ra
mp
urB
us
ha
ir

Kh

Pu
ga
l

Pa
tan

ri

na
gr i

ra

Na
li

f ar

Ma
g
za

la

i...
DENDROGRAM BASED ON NEI’S STANDARD GENETIC
DISTANCE (Ds)
Radiation tree using individual animals as taxonomic units constructed with a distance
matrix with simple allele sharing s...
Average membership coefficient (q) for each
given breed for k=15 clustering result
INTER-SPECIFIC LEVEL (BETWEEN CLOSELY RELATED
SPECIES)
•

To study relatedness– through Phylogenetics

•

Reconstruction o...
CONSERVATION BIOLOGY



In order to plan a conservation management strategy, it is
In order to plan a conservation manag...
Which breeds should be prioritized for economically viable
conservation plans?
The marginal diversity reflects the change ...
IMPLICATIONS
 The overall magnitude of genetic diversity within each livestock
species
 The genetic relationships, expre...
ANALYSIS OF MICROSATELLITE DATA
Three main steps are involved in the statistical
analysis of molecular data in diversity s...
Data collection
• Sample collection
• DNA isolation
• PCR amplification
• Checking of PCR products
• Resolution and Visual...
Sampling Procedure
• Any of the biological materials like fresh blood, tissue, hair,
bone etc. may potentially be used for...
DNA Extraction
•The collected blood samples in vacutainer tubes
containing anticoagulant such as EDTA are transported to
t...
8
8-1
8-4
The number of repeats can be determined by separating
microsatellites by size using electrophoresis.

Gel Electrophoresis
...
DETECTION
1. Radioactive (P33) end-labelling
2. Silver staining
6% urea PAGE showing microsatellite polymorphism

A

A
B C

B C

D

F

1 DD
2 BB
3 CC
4 CF
5 AC

E

D F...
BM6526

Entry of band/allele information into the computer. It can be
done manually or it can be read from gel directly by...
Multiplex PCR
(Parallel Sample Processing)


Compatible primers are the key
to successful multiplex PCR



10 or more ST...
GENOTYPING
Each individual can be genotyped manually by scoring the
band (alleles) as two digits or as their interger size...
Statistical Parameters for estimation of the
Variability
••
••
••
••
••
••

Heterozygosity
Heterozygosity
Polymorphism Inf...
Statistical Analysis of Data
Allele number
Allele number
Alleles are a set of alternative forms of the same gene
Alleles a...
Allele Frequency
The frequency of an allele ‘A’ is the number of
The frequency of an allele ‘A’ is the number of
‘A’ allel...
Heterozygosity
Heterozygosity is the state of possessing different alleles at a given locus in
regard to a given character...
Polymorphism Information Content (PIC)
The polymorphism information content is another
important measure of DNA polymorphi...
Genetic Distancing
• Genetic distance expresses the genetic differences between two
populations as a single number.
• It i...
Methods of genetic distancing
• Nei's

(1972) standard genetic distance

• Average
• Delta

square distance (Goldstein et ...
Molecular data processing
1.GenAlex
2.POPGENE
3.GDA
(Genetic
Analysis)
4.GENEPOP
5.Phylip
6.Microsat
7.TreeView
8.FSTAT
9....
SNPs vs STR
• Each SNP is less informative
- Because only has two alleles
• Need to genotype more SNPs to equal distinctiv...
MICROSATELITE Markers for LIVESTOCK Genetic DIVERSITY ANALYSES
MICROSATELITE Markers for LIVESTOCK Genetic DIVERSITY ANALYSES
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MICROSATELITE Markers for LIVESTOCK Genetic DIVERSITY ANALYSES

  1. 1. MICROSATELLITE MARKERS FOR LIVESTOCK GENETIC DIVERSITY ANALYSES Karan Veer Singh National Bureau of Animal Genetic Resources Karnal-132001
  2. 2. LIVESTOCK DIVERSITY About 40 species of domestic animals and poultry contribute to meeting the About 40 species of domestic animals and poultry contribute to meeting the needs of humankind, providing meat, fibre, milk, eggs, draught animal power, needs of humankind, providing meat, fibre, milk, eggs, draught animal power, skins, and manure, and are an essential component of many mixed farming skins, and manure, and are an essential component of many mixed farming systems. systems. Within these species, more than 8000 breeds and strains (FAO, 2011) constitute Within these species, more than 8000 breeds and strains (FAO, 2011) constitute the animal genetic resources (AnGR) that are of crucial significance for food and the animal genetic resources (AnGR) that are of crucial significance for food and agriculture. agriculture. According to the report on the Status and trends of animal genetic resources –– According to the report on the Status and trends of animal genetic resources 2010 (FAO, 2011), approximately 88 percent of reported livestock breeds have 2010 (FAO, 2011), approximately percent of reported livestock breeds have become extinct and an additional 21 percent are considered to be at risk of become extinct and an additional 21 percent are considered to be at risk of extinction. Moreover, the situation is presently unknown for 35 percent of extinction. Moreover, the situation is presently unknown for 35 percent of breeds, most of which are reared in developing countries. breeds, most of which are reared in developing countries. FAO. 2011. Status and trends of animal genetic resources – –2010. Commission on Genetic Resources for Food and Agriculture, Thirteenth FAO. 2011. Status and trends of animal genetic resources 2010. Commission on Genetic Resources for Food and Agriculture, Thirteenth Regular Session, Rome, 18–22 July 2011, (CGRFA-13/11/Inf.17). Rome (available at http://www.fao.org/docrep/meeting/022/am649e.pdf). Regular Session, Rome, 18–22 July 2011, (CGRFA-13/11/Inf.17). Rome (available at http://www.fao.org/docrep/meeting/022/am649e.pdf).
  3. 3. LIVESTOCK DIVERSITY IN INDIA Species No. of recognized breeds Buffalo 13 Cattle 37 Sheep 39 Goat 23 Camel 8 Horse/Pony 6 Poultry 15 Pig 2 Donkey 1 Yak, Mithun, ducks, geese and other non descript populations It is estimated that 50% of indigenous goats, 27% of indigenous sheep, 20% of indigenous cattle and 26% poultry breeds in India are threatened.
  4. 4. REASONS FOR DECLINE IN DOMESTIC ANIMAL BIODIVERSITY • Conservation of indigenous breeds has received little attention in the country. • No serious efforts are made for conservation of the breeds at risk. • Lack of basic descriptive information on animal genetic resources. • Replacement of Indigenous breeds by exotic or crossbreds. • Shifting of traditional farming to commercial farming.
  5. 5. LIVESTOCK GENETIC ANALYSIS Livestock Breed analysis/characterization requires knowledge of genetic variation. Genetic variation be effectively measured within and between populations. Various types of markers are available to assess such genetic variations/polymorphism.
  6. 6. MOLECULAR/DNA MARKERS Any DNA fragment or gene coding for a trait which is free of environmental effect and does not interact with other genes or alleles, is called a DNA marker.Viz. RAPD, SSR, RFLP, AFLP etc. Typical characteristics • Not affected by environment or the developmental stage • Not tissue /organ/sex specific • More efficient than protein or biochemical polymorphism • More informative • Explore complete genome and show Mendelian inheritance
  7. 7. MICROSATELLITE/SSR MARKERS • Litt and Luty 1989 (Am. J. Hum. Gen.) • Litt and Luty 1989 (Am. J. Hum. Gen.) • Sequences of DNA consisting of repeats of 2-6 base pair motifs, • Sequences of DNA consisting of repeats of 2-6 base pair motifs, almost any combination possible (e.g. CA, GA, GGGAA). almost any combination possible (e.g. CA, GA, GGGAA). Polymorphisms are based on number of repeat units and are Polymorphisms are based on number of repeat units and are hypervariable (have many alleles) hypervariable (have many alleles) SYNONYMS Microsatellites are also known as • simple sequence repeats (SSR), • short tandem repeats (STR)
  8. 8. REPEAT STRUCTURE OF MICROSATELLITES Mononucleotide - (A)11 AAAAAAAAAAA Dinucleotide - (GT)6 GTGTGTGTGTGT Trinucleotide - (CTG)4 CTGCTGCTGCTG Tetranucleotide - (ACTC)4 ACTCACTCACTCACTC
  9. 9. TYPES OF MICROSATELLITES BASED ON THE NATURE OF REPEATS
  10. 10. POLYMORPHISM AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another
  11. 11. EVOLUTION OF MICROSATELLITES •• Mutation Mutation •• It is estimated that microsatellites mutate 100 to It is estimated that microsatellites mutate 100 to 10,000 times as fast as base pair substitutions. 10,000 times as fast as base pair substitutions. How do microsatellites evolve? Unequal crossing-over during meiosis Replication Slippage
  12. 12. How do microsatellites mutate? • Microsatellites alleles change rather quickly over time  E. coli – 10-2 events per locus per replication  Drosophila – 6 X 10-6 events per locus per generation  Human – 10-3 events per locus per generation DNA polymerase slippage Unequal crossing over
  13. 13. MICROSATELLITES - TOOLS OF CHOICE  Low quantities of template DNA required (10-100 ng)  High genomic abundance  Random distribution throughout the genome  High level of polymorphism  Band profiles can be interpreted in terms of loci and alleles  Codominance of alleles  Allele sizes can be determined with an accuracy of 1 bp, allowing accurate comparison across different gels  High reproducibility  Different STRs may be multiplexed in PCR or on gel  Wide range of applications  Amenable to automation
  14. 14. Stutter Bands in SSR  Often there are minor bands in addition to the major bands. These minor bands are called stutter bands (shadow bands) and they usually differ (smaller in size) from the major bands by a few nucleotides.
  15. 15. Homology vs. Homoplasy • . Homology is any similarity between characters that is due to their shared ancestry • Homoplasy occurs when characters are similar, but are not derived from a common ancestor.
  16. 16. HOW DO WE DEVELOP MICROSATELLITE PRIMERS?                 DNA Extraction DNA Extraction Digestion of genomic DNA with Restriction Enzymes Digestion of genomic DNA with Restriction Enzymes Cloning the resulting fragments into suitable cloning vectors to form Cloning the resulting fragments into suitable cloning vectors to form genomic library genomic library Plating these cloning vectors on nylon membrane Plating these cloning vectors on nylon membrane Probe the membrane with labeled oligonucleotides of desirable repeats Probe the membrane with labeled oligonucleotides of desirable repeats Culture the positive clones Culture the positive clones Cut the insert out and run on agarose gel Cut the insert out and run on agarose gel Sequence the positive clones and design the appropriate primers from Sequence the positive clones and design the appropriate primers from flanking regions flanking regions
  17. 17. WHAT ARE MICROSATELLITES FOR? • Microsatellites are “junk” DNA. In humans, 90% of microsatellites are found in noncoding regions of the genome. • Microsatellites may provide a source of genetic variation. In bacteria, variation in microsatellites alleles in coding regions is thought to be adaptive in different environments. • Microsatellites may help regulate gene expression.
  18. 18. APPLICATIONS • Forensics and parentage analysis • Disease diagnosis • Diversity analysis • Population Studies • Conservation Biology
  19. 19. • Forensics Because microsatellites are so variable, by studying several at one time (and getting a DNA fingerprint), individuals can be identified. • Paternity studies Because individuals receive one allele from their mother and one from their father, paternity (or maternity) can be determined
  20. 20. PARENTAGE VERIFICATION Exclusion of false parents with a probability of as high as 99.999 % against 40 – 60 % from biochemical markers
  21. 21. Disease Diagnosis – Huntington’s disease Huntington's disease is caused by a genetic defect on chromosome 4. The defect causes a part of DNA, called a CAG repeat, to occur many more times than it is supposed to. Normally, this section of DNA is repeated 10 to 28 times. But in persons with Huntington's disease, it is repeated 36 to 120 times.
  22. 22. DIVERSITY ANALYSIS • Observed heterozygosity (Ho) and gene diversity or expected heterozygosity (H e) are measures of genetic diversity within a population. • Allelic polymorphisms in a population.
  23. 23. INTRASPECIFIC (WITHIN SPECIES) Genetic variability between & within breeds- through genetic distancing and heterozygosity to look into the effects of • Bottlenecks suffered by a breed • Inbreeding depressions due to declining population Relationship among breeds • Helps in finding the most diverse groups • Helps to decide about the conservation programs
  24. 24. Ja la wa d Na er i i Ma l pu ra Ra mp urB us ha ir Kh Pu ga l Pa tan ri na gr i ra Na li f ar Ma g za la i al m eri Ch ok So na d Ja is Mu z un Ga r ol e pu j am t an ag Ga n al i Ma rw ari Ch ho gy an i 0 Ma d De cc Mean 12 8 0.600 6 0.500 0.400 4 No. Private Alleles Heterozygosity Allelic Patterns across Populations 0.900 10 0.800 0.700 0.300 2 0.200 0.100 0.000 Populations He
  25. 25. DENDROGRAM BASED ON NEI’S STANDARD GENETIC DISTANCE (Ds)
  26. 26. Radiation tree using individual animals as taxonomic units constructed with a distance matrix with simple allele sharing statistics.
  27. 27. Average membership coefficient (q) for each given breed for k=15 clustering result
  28. 28. INTER-SPECIFIC LEVEL (BETWEEN CLOSELY RELATED SPECIES) • To study relatedness– through Phylogenetics • Reconstruction of the evolutionary relationships among the organisms To study cross-species homologies for both coding and non-coding sequences for construction of comparative maps •
  29. 29. CONSERVATION BIOLOGY   In order to plan a conservation management strategy, it is In order to plan a conservation management strategy, it is necessary to define, record and assess the genetic resources at necessary to define, record and assess the genetic resources at risk. risk.   Full description or characterization of animal genetic resources Full description or characterization of animal genetic resources is essential at the level of comparative molecular description for is essential at the level of comparative molecular description for which microsatellite markers can be used to establish which which microsatellite markers can be used to establish which breed harbor significant genetic diversity in order to better breed harbor significant genetic diversity in order to better target conservation action. target conservation action.
  30. 30. Which breeds should be prioritized for economically viable conservation plans? The marginal diversity reflects the change of diversity in the whole population in case of an increase in the extinction probability of one breed. Weitzman Diversity Deccani, 1.85 Madgyal, 6.35 Rampur Bushair, 3.65 Chokla, 7.53 Magra, 1.85 Nali, 1.83 Marwari, 3.35 Garole, 11.3 Jaisalmeri, 2.35 Pugal, 2.65 Chhotanagpuri, 6.35 Patanwadi, 4.28 Ganjam, 7.03 Jalauni, 2.88 Muzzafarnagri, 4.98 Sonadi, 9.68 Malpura, 2.1 Kheri, 2.5
  31. 31. IMPLICATIONS  The overall magnitude of genetic diversity within each livestock species  The genetic relationships, expressed as genetic distances among breeds, within each species.  allow for interpretation of gene flow in animal populations, which might be related to human migrations  possibly give some indication of levels of inbreeding in each breed  enhance the global information system on domestic animal diversity, and consequently the development of more effective and efficient conservation programmes  alert national governments of the need to better characterize and conserve the indigenous animal genetic resources, and guide in the establishment of sound policies and sustainable agriculture.
  32. 32. ANALYSIS OF MICROSATELLITE DATA Three main steps are involved in the statistical analysis of molecular data in diversity studies: • Data collection • Data analysis • Interpretation of the data http://www.fao.org/docrep/014/i2413e/i2413e00.htm
  33. 33. Data collection • Sample collection • DNA isolation • PCR amplification • Checking of PCR products • Resolution and Visualization of different alleles by PAGE, silver staining, autoradiography or by automated sequencer
  34. 34. Sampling Procedure • Any of the biological materials like fresh blood, tissue, hair, bone etc. may potentially be used for DNA analysis. •Sample should be collected from unrelated animals by visiting the breeding tract of the breed in question and not more than 10 % of any one herd or village population should be sampled. Whenever possible, pedigree records should be consulted for identifying unrelated individuals. • To achieve clearer differentiation among closely related populations/ breeds, it is recommended that per breed 50 unrelated animals (preferably 25 each of both the sexes) should be assayed .
  35. 35. DNA Extraction •The collected blood samples in vacutainer tubes containing anticoagulant such as EDTA are transported to the laboratory under chilled condition for further processing. •Genomic DNA from total blood is then isolated using proteinase-K digestion followed by standard phenol/ chloroform extraction. •Both the quality as well as quantity of isolated genomic DNA is assessed and subsequently stored at –200C/40C for further analysis with microsatellite markers.
  36. 36. 8 8-1 8-4
  37. 37. The number of repeats can be determined by separating microsatellites by size using electrophoresis. Gel Electrophoresis Capillary Electrophoresis
  38. 38. DETECTION 1. Radioactive (P33) end-labelling
  39. 39. 2. Silver staining 6% urea PAGE showing microsatellite polymorphism A A B C B C D F 1 DD 2 BB 3 CC 4 CF 5 AC E D F E
  40. 40. BM6526 Entry of band/allele information into the computer. It can be done manually or it can be read from gel directly by a computer installed with software.
  41. 41. Multiplex PCR (Parallel Sample Processing)  Compatible primers are the key to successful multiplex PCR  10 or more STR loci can be simultaneously amplified Challenges to Multiplexing –primer design to find compatible primers (no program exists) –reaction optimization is highly empirical often taking months Advantages of Multiplex PCR –Increases information obtained per unit time (increases power of discrimination) –Reduces labor to obtain results –Reduces template required (smaller sample consumed)
  42. 42. GENOTYPING Each individual can be genotyped manually by scoring the band (alleles) as two digits or as their interger size in base pair in which case heterozygous individuals yield two bands and those that are homozygous yield one band. A. Because humans are diploid organisms, each individual has two alleles per locus. B. Individuals could be: 1. Homozygous—two copies of the same overall length 2. Heterozygous—two copies of different overall length. A. Many alleles exist in a population with the maximum number of alleles being two times the number of people in the population.
  43. 43. Statistical Parameters for estimation of the Variability •• •• •• •• •• •• Heterozygosity Heterozygosity Polymorphism Information Content (PIC) Polymorphism Information Content (PIC) Genetic Distances Genetic Distances Divergence times Divergence times Probability of individual identification Probability of individual identification Probability of exclusion of false parents Probability of exclusion of false parents
  44. 44. Statistical Analysis of Data Allele number Allele number Alleles are a set of alternative forms of the same gene Alleles are a set of alternative forms of the same gene occupying the same relative position or locus on homologous occupying the same relative position or locus on homologous chromosomes. chromosomes. Allele number is the total number of alleles for a given Allele number is the total number of alleles for a given marker // locus in a population, which is counted with a nonmarker locus in a population, which is counted with a nonzero frequency. zero frequency. The allele number for each locus can be determined The allele number for each locus can be determined manually from the silver stained gels/autoradiograms. manually from the silver stained gels/autoradiograms.
  45. 45. Allele Frequency The frequency of an allele ‘A’ is the number of The frequency of an allele ‘A’ is the number of ‘A’ alleles in the population divided by the total ‘A’ alleles in the population divided by the total number of alleles/genes. number of alleles/genes. It gives an indication of the most or least It gives an indication of the most or least prevalent alleles in the population. prevalent alleles in the population. The allele frequency is affected over time by The allele frequency is affected over time by forces such as genetic drift, mutation and migration. forces such as genetic drift, mutation and migration.
  46. 46. Heterozygosity Heterozygosity is the state of possessing different alleles at a given locus in regard to a given character. It is a measure of heterozygotes or genic variation in a population. The population heterozygosity at a locus is given by the formula: H = 1 – Σ Pi2 where ∑ stands for summation over all alleles (Nei, 1978) and Pi is the frequency of the ith allele at a locus in a population. The average heterozygosity per locus (H) is defined as the mean of H over all structural loci in the genome. However, the unbiased estimate of the expected heterozygosity at a locus is (if N < 50): HE = n 2N 2N 1 1 i=1 pi 2
  47. 47. Polymorphism Information Content (PIC) The polymorphism information content is another important measure of DNA polymorphism. Expected value of PIC for each locus is calculated as per (Botstein et al., 1980): n n-1 n PIC = 1 - Σ pi2 - Σ Σ 2 pi2 pj2 i=1 i=1 j=i+1
  48. 48. Genetic Distancing • Genetic distance expresses the genetic differences between two populations as a single number. • It is the basis for constructing phylogenetic trees • Different sets of data require different kinds of distance measures. • The different models are based on different assumptions each differing in certain assumptions of population divergence, and the basis of the estimation of breed relationship (co ‑ ancestry coefficient, proportion of shared number of alleles, probability of gene identity between two populations).
  49. 49. Methods of genetic distancing • Nei's (1972) standard genetic distance • Average • Delta square distance (Goldstein et al., 1995) mu squared (δμ)2 distance (Goldstein et al., 1995) • Reynold's • Slatkin's genetic distance (Reynold et al., 1983) (1995) genetic distance (Rst) • Cavalli-Sforza (Dkf) • Proportion and Bodmer's (1971) kinship coefficient distance of shared alleles distance (Dps) (Bowcock et al., 1994) • Cavalli-Sforza and Edwards (1967) chord distance (Dc)
  50. 50. Molecular data processing 1.GenAlex 2.POPGENE 3.GDA (Genetic Analysis) 4.GENEPOP 5.Phylip 6.Microsat 7.TreeView 8.FSTAT 9.BOTTLENECK 10.STRUCTURE Data
  51. 51. SNPs vs STR • Each SNP is less informative - Because only has two alleles • Need to genotype more SNPs to equal distinctive DNA profile Computationally: 25 to 45 SNPs equal 13 core STR loci Actual lab work: 50 or more SNPs equal 12 STRs

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