forensic biochemistry 2 exam 4

assigning DNA profiles, probability, frequency, mixture interpretation, next generation sequencing, testimony

defined as the probability of discriminating between 2 unrelated individuals

power of discrimination

formula for probability of match (PM)?

sum of (frequency of genotype at a locus)²

power of discrimination formula?

1- PM

steps for determining the probability of a single source DNA profile?

determine alleles at each locus, find allele frequency from relevant populations, calculate expected genotype frequency, report multilocus results

when calculating expected genotype frequency using Hardy Weinburg, what are the potential subpopulation corrections?

correction for profile probability or correction for match probability

aggregates and harmonizes data across mnay large scale sequencing projects to create summary allele frequency statistics

gnomAD database

_______________ is distinct from either race or ethnicity

genetic ancestry

reflects an individual’s demographic history and refers to the specific lines of decent through a family tree by which an individual inherited DNA from specific ancestors

genetic ancestry

sociopolitical constructs used to group individuals based on perceived shared ancestry, biological characteristics , or on perceived shared cultural heritage

race and ethnicity

genetic ancestry is a ____________________

continuous measure

homozygote loci formula without population substructure correction

p²

heterozygote loci formula without population substructure correction

p²j²

homozygote loci formula with inbreeding population

p²+pF(1-p)

heterozygote loci formula with inbreeding population

2pq(1-F)

subpopulation theory is similar to inbreeding but F (which is really ______) becomes __________

Fis, Fst

F_ST refers to

probability that 2 alleles randomly drawn from the population are identical by decent

5 main points of the 196 NRC Report on Forensic DNA Evidence

validated DNA evidence, new formulas to calculate the likelihood of a match for better understanding for jurors, protecting suspects from false incrimination, recommending the use of a DNA profile database specific to the racial background of the sample, assuring DNA profiling is reliable

conservative value to be used for θ (in the US) in this formula p² +p(1-p)θ, when the exact genotype can be determined

0.01

recommendation 4.1 of the NRC report stated

profile frequency of heterozygotes need to use H-W without theta correction

heterozygote formula to be used per the 4.1 recommendation?

2pipj

why did recommendation 4.1 change the formula for heterozygotes?

formula with theta was overestimating the frequency of a genotype

homozygote formula with a subpopulation?

pi² + pi(1-pi)thetaii

heterozygote formula with a subpopulation?

2pipj(1-thetaij)

what did recommendation 4.2 of the NRC report say?

use allele frequencies from the subgroup the sample came from. if unknown subgroup, use the formula

signs that sample is a mixture

loci with more than 2 alleles, severe peak imbalance, abnormally high stutter

expected severe peak balance of _______% in a mixture sample

60-70

expected high stutter of _______% in mixture samples

15-20

minimum height requirement at and above which detected peaks can be reliably distinguished from background noise

analytical threshold

the analytical threshold or AT is typically around _______ RFUs

25-50

peak height value below which is reasonable to assume that, at a given locus, allelic dropout of a sister allele in a heterozygous pair may have occurred

stochastic threshold

stochastic threshold or ST is typically around _______ RFUs

200

steps for interpreting a mixture

identify presence of a mixture, designate allele peaks, identify number of contributors, estimate relative ratio of individuals contributing to the mixture, consider all possible genotypes, compare reference samples, statistical interpretation

formula for determining minimum number of alleles

Nalleles/2 then rounded up

relative ratio considers

the peak heights of the whole profile

all possible genotypes for 4 peaks (A, B, C, D)

A, B + C, D

A, C + B, D

A, D + B, C

all possible genotypes for 3 peaks (A, B, C)

A, A + B, C

B, B + A, C

C, C + A, B

A, B + A, C

B, C + A, C

A, B + B, C

all possible genotypes for 2 peaks (A, B)

A, A + A, B

A, B + A, B

A, A + B, B

A, B + B, B

accounts for if a single peak below the stochastic threshold results from the homozygous genotype or the heterozygous genotype

2p rule

one or more of the mixture components could comprise low template DNA, as such we need to take into account

allele drop out and drop in

2p rule is used to calculate if

an actual allele dropped out or if the sample is a homozygote

2p rule formula

2pa-pa² < 2pa

probability that the DNA of a randomly chosen person has the same DNA profile as the DNA of the casework sample

RMP or random match probability

sum of the probabilities for all of the genotypes that represent the possible contributors to a DNA mixture under the assumption of a defined number of contributors

RMP calculation or modified RMP

how is modified RMP different from the combined probability of Inclusion (CPI)?

doesn’t use assumptions to determine number of contributors

estimate of the probability that a randomly selected, unrelated individual would be included as a possible contributor to a mixture

combined probability of inclusion or CPI

probability that a randomly selected, unrelated individual would be excluded as a contributor to the mixture

combined probability of exclusion or CPE

if it is determined that there is allele dropout at a given locus, the locus ______________

will be excluded from the match probability

steps for calculating the likelihood ratio for a 2-person mixture

condition the number of contributors, state the alternative hypothesis, evaluate the probability of the evidence under the defense proposition, evaluate the probability of the casework sample under the prosecution proposition, calculate the likelihood ratio, report the likelihood ratio

refers to the use of biological modeling, statistical theory computer algorithms, and probability distributions to calculate likelihood ratios and/or infer genotypes for the DNA typing results of forensic samples

probabilistic genotyping

why do we use probabilistic genotyping?

statistically interprets mixture samples

PG continuous models consider _______ as a continuous variable

peak heights

probabilistic genotyping genotyping considers _________ in order to deconvolute a DNA profile into a list of genotype sets

observable data, models, calibration data, and unknowable

specific for a set of laboratory hardware and DNA typing kit

calibration data

refers to the specifics of the actual DNA profile being analyzed

unknowables

the unknowables of PG continuous models include

number of contributors, DNA amounts of each contributor, degradation of each contributor, amplification efficiency of each locus, replicate amplification strength, level of peak height variability within the sample

“mass parameters” or the total allelic product within PG continuous models includes

DNA amounts of each contributor, degradation of each contributor, amplification efficiency of each locus, replicate amplification strength

assumes degradation is exponential but that each contributor to have different curves

total allelic product modeling

total allelic product modeling tests different mass parameters to form a ____________

probability density

iterative re-sampling process-in each iteration, genotype combinations and biological parameters (mass parameters) are proposed to describe the profile

Markov Chain Monte Carlo

how does the Markov Chain Monte Carlo deconvolution work?

genotype and set of values is proposed for every iteration and compared to observed results to see how well they explain the data

preliminary MCMC run to ensure the post burn-in MCMC begins in an area of high probability space

burn-in

parameters for MCMC burn-in?

8 independent chains must reach 100,000 accepted iterations

occurs after burn-in and uses the same number of chain to acheive ~50,000 accepted iterations

post burn-in

occurs at completion of MCMC and normalizes the number of genotype sets accepted during post-burn in

weight

an MCMC weight of 0 means

observed data cannot be explained by the proposed genotype set

an MCMC weight of 1 means

only genotype set that explains the DNA profile

the progression fo the MCMC is influenced by a “seed” set by a __________

random number generator

process of using calculating the probability density of each peak in the profile, comparing it with the proposed model, measuring it’s “fit” , and accepting or rejecting the proposed values

Metropolis-Hastings

the Metropolis-Hastings Algorithm operates

within the Markov Chain Monte Carlo framework

when working with the Metropolic-Hastings algorithm, the ________ the probability density the better fit of the parameter values to the observed profile

higher

within the Metropolis-Hastings algorithm, the proposed values for the genotypes and mass parameters are either accepted or rejected depending on ________

probability density

after deconvolution, a likelihood ratio can be assigned to any POI based on ____________

propositions considered

parameters requiring optimization for probabilistic genotyping

analytical threshold, stutter ratios, saturation limit, drop-in parameters, allele/stutter peak height variance, LSAE variance, relevant population parameters

year QIAGEN developed the first DNA purification method in forensics

1998

year QIAGEN launched its first STR kit

2010

QIAGEN workflow steps

collection, pre-treatment, sample preparation, array setup, quantification, STR/NGS analysis

traditional DNA analysis workflow

sample collection, extraction and quantification, PCR, CE & data analysis

why use next generation sequencing over CE?

add more loci targets, not limited by ampicon bp size, can use STRs and SNPs, visible trait estimation

ForenSeq Human Identification workflow?

sample collection, extraction & quantification, library preparation, sequencing & data analysis

why sequence STRs?

smaller amplicons, looks at the whole sequence not length, can target STRs and SNPs

the ForenSeq Signature Plus is the only QIAGEN machine that has

STR analysis, kinship, and externally visible characteristics

SNPs are used over STRs bc

need way more for a match

the MainstAY and MainstAY SE kits can identify relatives of the ________ degree

first

the SIgnature Plus kit can identify relatives to the ________ degree

first or second

Kintelligence can identify relatives to the _________ degree

fourth or fifth

how are libraries prepared?

amplify and tag targets, attach indexes and adapters, purify, dilute sample to make loci all the same concentration

what is the purpose of indexes in QIAGEN NGS?

provide a unique marker specific to that allele and sample

how does the sequencing part of the QIAGEN NGS work?

samples get pulled onto the flowcell, make a U shape on the cell to be read, one nucleotide is added and read during each cycle

a really special feature about sequencing is that it able to

easily determine number of contributors

steps of PCR

extraction, quantification, amplification, analysis

STRmix is used to

help declutter mixture samples

forensic scientists/biologists can only speak to the _______ level of testimony

source or sub-source

occurs when the conclusion is restated in a manner that bolsters the hypothesis of the prosecutor, typically by transposing the conditional and making the evidence seem more exclusive

prosecutor’s fallacy

error in logic on the part of the defense counsel that bolster’s the defense’s hypothesis and favors the defendant, typically by relating the probability to a specific population to make the profile seem more inclusive

defendant’s fallacy

fallacy in which the statistic is bolstered by relating it directly to the profile being compared in relation to the general population

uniqueness fallacy

occurs when the probability statement is taken from one level within the hierarchy of propositions to a higher level

association fallacy