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Mixtures & Paternity - Coggle Diagram
Mixtures & Paternity
Mixed DNA samples
Currently
Sensitive testing: Most case samples are mixtures
Many are low level
Complicates interpretation process
Steps in mixture interpretation
Step 1: Identify the presence of a mixture
Step 2: Designate allele peaks
Step 3: Identify the number of possible contributors
Step 4: Estimate the relative ratio of indiv contributing to the mixture
Step 5: Consider all possible genotype combinations
Step 6: Compare reference samples
Steps
Step 1:
Is it a mixed profile
More than two peaks at a locus
Heterozygous imbalance
N-4 stutter peaks >15%
N+4 stutter peaks
All loci across the DNA profile need to be considered to make this decision
Step 2:
Designate allele peaks
Whether stutter peaks should be labelled or not
Step 3:
Identify the number of contributors
Examine whole profile
No more than four alleles at a locus
Two person mixture
Tri-allelic pattern at one locus could be trisomy
Up to 6 alleles per locus
Three-person mixture
Over 6 alleles per locus
Four or more contributors
Step 4:
Estimate the ratio of contributors
Using amelogenin
Male: Female mixture
If male = XY and female = XX then
X = three copies
Y = one copy
Assume 100% amplification efficiency
Male XY = 469 (Y) + 469 (X)
Female XX = 2272 - 469
Male total = 938
1803/938 = 1.9
RATIO = 2:1 (two times more female than male)
Using loci with 4 alleles
Step 5:
Consider all genotype combinations
1 peak - 2 homozygotes with the same allele
2 peaks
Both heterozygotes sharing both alleles
One heterozygote and one homozygote sharing one allele
Both homozygotes for separate alleles
3 peaks at a locus
Both heterozygotes and share one allele
One is homozygous and the other heterozygous for different alleles
4 peaks at a locus
They must be both heterozygous with no shared alleles
Unless more than 2 people
Step 6:
Compare reference samples
If there is a suspect, a lab must decide to include or exclude them
Victim samples can be helpful to eliminate their allele contributions from the mixture
Can lose valuable info from shared alleles
Determining genotypes - using peak heights
Peak heights can be used to work out whether genotype combinations are possible
Used in conjunction with calculated ratios
List of most likely genotypes
Doesn't rule out some combinations bust makes other combinations more likely
Can be used to determine probability of the profile
Complex mixtures
If more than two people at a similar ratio it may not be possible to assign genotypes
Can still report if a person is absent, or if they can not be excluded as being a contributor to the mixture
Automated Mixture Interpretations
Two main methods
Binary
Continuous
Probabilistic Genotyping
Using continuous methods
Use of biological modeling, statistical theory, computer algorithms, & probability ratios to calculate likelihood ratios (LRs)
Calculation of Weights
Determined using the Markov Chain Monte Carlo (MCMC)
In STRmix the MCMC is 'solving' the equation for genotype weights
Relies on random sampling
Paternity Testing
Outcome
More than 3 mismatches --> exclusion
No exclusions --> calculate likelihood
Same STR loci as in standard DNA testing
Tested sample is the offspring of the assumed mother & father
Tested offspring is not from the assumed mother & father
Any offspring must inherit one allele from the mother & one from the father, this is barring a mutation
What is the question?
In paternity testing the hypothesis are:
Either the tested male is the biological father of the offspring
Some other male is the biological father of the offspring
Paternity Index (PI)
Outcome of the likelihood ratio when used as a paternity calculation
Cumulative PI
Indiv PI values for each locus can be multiplied provided that the loci are all on different chromosomes or behave as if inherited independently
Cumulative PI can be converted into a probability if required