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DNA Analysis & Interpretation > Mixtures > Flashcards

Mixtures Flashcards

1
Q
  1. Describes a suite of emerging DNA sequencing Technologies were sensitive test can be done simultaneously.
  2. Three or more peaks at a locus in indicative of?
  3. Involves searching a DNA database for profiles that partly match the crime scene sample
  4. DNA – wholy maternally inherited.
  5. DNA that is accidentally transferred?
  6. A ;likelihood ratio of between 1,000 and 10,000 provides this level of support?
A
  1. Next generation sequencing describes a suite of emerging DNA sequencing Technologies were sensitive test can be done simultaneously.
  2. Three or more peaks at a locus in indicative of a mixed profile.
  3. Familial searching involves searching a DNA database for profiles that partly match the crime scene sample
  4. DNA – wholy maternally inherited is mitochondrial.
  5. DNA that is accidentally transferred is contaminated.
  6. A likelihood ratio of between 1,000 and 10,000 provides strong level of support.
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2
Q

STR Markers

A
  • Contain a repeating unit (called the motif)
  • composed of four nucleotide bases, such as AGTC or ATTC.
  • These “tetranucleotide” markers make up the majority of STRs used in forensic testing.
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3
Q

Tandem

A

The term “tandem” within STR refers to the fact that the short sequences are repeated sequentially at a locus.

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4
Q

Chromosomes

A

Chromosomes are in pairs, one is inherited from each parent.

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5
Q

Alleles are said to be a match when…

A
  • Alleles are said to be a match:
  • When their peaks fall at the same (left-to-right) position on the EPG.
  • When comparing profiles from unrelated people, it would not be unusual to find that they have a few matching alleles, just as it wouldn’t be unusual to match one or two numbers in a lottery.
  • But it would be incredibly unlikely for all the alleles to match. The more base pairs we target with our multiplex the more unlikely this is.
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6
Q

DNA 17 multiplex

A
  • Extremely sensitive optimized for less than 100 cells (400 – 500 pg.).
  • Amount of DNA in a cell ~6 pg.
  • So, the ability to generate good profiles from only a few cells is possible.
  • The previous multiplex was optimized for 1000 pg. of DNA.
  • This has implications in the determination of how and when matching DNA was deposited - direct versus indirect transfer.
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7
Q

What happens if two different DNA multiplexes were used?

A

It can still be compared, but only at the loci in common between the two tests.

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8
Q
  1. Case mentioned by Evett and Pope in para 2 (page 1)
  2. When a crime sample consists of good quality DNA from a single person its comparison is said to be?
  3. The ratio of probabilities between the prosecution and defence propositions are referred to as?
  4. When dealing with mixtures the prosecution may put forward that to the defendant is one of three contributors to the mixture. The defence may say that?
  5. Presenting a number of matching components between the defendant and a mixture can foster is what type of view of the evidence?
A
  1. Case mentioned by Evett and Pope is Dlugosz.
  2. When a crime sample consists of good quality DNA from a single person its comparison is said to be straightforward.
  3. The ratio of probabilities between the prosecution and defence propositions are referred to as likelihood ratios.
  4. When dealing with mixtures the prosecution may put forward that to the defendant is one of three contributors to the mixture. The defence may say that is a mixture of three unknown people.
  5. Presenting a number of matching components between the defendant and a mixture can foster is a prejudicial view of the evidence.
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9
Q

What is a DNA mixture?

A

They are described as mixed as they contain DNA from two or more individuals.

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10
Q

Major issues associated with complex interpretation.

A

The major issues associated with this can include:
* Incomplete information which leads to the generation of only partial profiles with some genetic information missing.
* Scenarios where relatives are connected with the crime profile which makes it difficult to separate these from the crime profile.
* Mixtures.

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11
Q

Whats the problem with DNA mixtures?

A
  • Rarely provide enough information to determine nature of transfer (direct versus indirect transfer) or in what order any DNA was deposited.
  • If an individuals reference DNA profile has been compared to a mixed DNA result and no statistical evaluation of potential match has been possible then the result must be considered evidentially inconclusive.
  • Nowadays, more use of probabilistic genotyping is undertaken in order to apply the likelihood ratio to mixed and incomplete profiles.
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12
Q

Whats the issue with low level DNA transfer?

A
  • We sometimes lack information.
  • We can’t tell the order of deposition.
  • A mixed and incomplete DNA profile may prevent statistical evaluation.
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13
Q

Issues with high sensitivity?

A
  • We often shed small amounts of DNA when we talk, sneeze and touch things.
  • As a result, many surfaces are likely to contain mixtures of minute amounts of DNA from several people.
  • These mixtures have always been present at crime scenes, but when sensitivity was lower, they wouldn’t have been detected or, if they were, labs would not have attempted to interpret them.
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14
Q
  1. A mixed profile is more likely to be found in the?
  2. STR markers contain repeating units called?
  3. Forensic STR marker D8S1179 is located on?
  4. Multiple STRs combined in one forensic testing kit is called?
  5. Fundamental unit of double-stranded nucleic acid, consisting of two nuclear bases bound to each other by hydrogen bonds?
  6. DNA mixtures are one factor which increases the complexity of interpretation.. Another issue is a profile containing?
A
  1. A mixed profile is more likely to be found in the evidential sample.
  2. STR markers contain repeating units called motif.
  3. Forensic STR marker D8S1179 is located on chromosome eight.
  4. Multiple STRs combined in one forensic testing kit is called multiplex.
  5. Fundamental unit of double-stranded nucleic acid, consisting of two nuclear bases bound to each other by hydrogen bonds is called a base pair.
  6. DNA mixtures are one factor which increases the complexity of interpretation.. Another issue is a profile containing incomplete information.
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15
Q

Principles of evidence interpretation

A

This is, Pr(E | H,I), where I is not the DNA evidence. These are assumptions an analyst has made during the interpretation.

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16
Q

Probability for independant events

A
  • ## Events are independent if the probability of one occurring has no effect on the probability of the other occurring.
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17
Q

If events A and B are independent, then the probability of A and B occurring is:

A

If events A and B are independent, then the probability of A and B occurring is:
* Pr (A and B) = Pr (A) × Pr (B)
* Instead of the word “and” you may see the intersection symbol ∩ from set theory
* Pr (A ∩ B) You may also see this written as Pr (AB) or Pr (A,B).

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18
Q

Probability when independance can’t be assumed

A
  • Then Pr(A and B) = Pr(A) × Pr(B | A)
  • This is the general form of the third law of probability, and it can be read as the probability of event A occurring multiplied by the probability of event B occurring given event A has occurred.
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19
Q

Pr(B | A)

A
  • The term Pr(B | A) is a conditional probability.
  • The conditioning bar | means “given” or “if” and events behind this bar are held true.
  • If the order in which A and B occur does not matter, then Pr(A and B) = Pr(B) × Pr(A | B)
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20
Q

How do you convert a probability into odds?

A
  • To convert a probability into odds is a very simple equation: odds = probability/(1 − probability).
  • For example, a probability of 0.6 has odds of 0.6/0.4 = 6/4. This would be expressed as odds of 6 to 4 in favour of the proposition.
  • A probability of 0.5 has odds of 0.5/0.5 = 1. This is expressed as 1 to 1 or evens.
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21
Q

Principles of evidence interpretation

Propositions

A
  • To evaluate the uncertainty of any given proposition it is necessary to consider at least one alternate proposition. These propositions are also referred to as hypotheses, H. In the forensic context, these alternate propositions conveniently align with the two sides of an adversarial case with one aligning with the prosecution’s argument and one the defence’s. Generally, one is inclusionary with respect to the POI and one is exclusionary. These are variously called Hp (hypothesis of the prosecution) or H1 and Hd (hypothesis of the defence), H2, or Ha (alternate proposition).
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22
Q

Likelihood ration using the principles of evidene interpretation

A

Pr(E | Hp)/Pr(E | Hd)

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23
Q

If a scientist decides that this is a mixture of DNA from three people and has observed the components of the defendants DNA at all loci, what would the prosecution proposition be?

A

“…that the crime sample is a mixture of DNA of the (a) defendant and (b) two unknown people”.

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24
Q

Proxy defence proposition

A
  • At the time of her/his analysis, the scientist will probably have been unaware of what the defence position would be at Court
  • In this position it is standard practice for the scientist to adopt provisional defence propositions.
  • The defence proposition, in this instance, is that the crime sample is a mixture of the DNA of two/three unknown people.
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25
Q

What do questions should a scientist address relating to the probability of the observations?

A
  • The scientist first asks “…what is the probability of observing this profile if the prosecution proposition were true”
  • And “…what is the probability I would observe a profile like this if the defence proposition were true”.
  • It is the ratio of the answers to these two questions that determines the weight of the evidence.
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26
Q

What is the statisitcal technique of choice for a single source DNA profile?

A

Random match probability

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27
Q

What is the statistic of choice for a mixed profile?

A

Likelihood ratio

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28
Q

Likelihood ratio using hypothesis

A

LR = (Probability of evidence under prosecution hypothesis) / probability of the evidenc eunder defense hypothesis.

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29
Q

Likelihood ratio = 1

A

Evidence is neutral

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30
Q

Likelihood ratio >1

A

The evidence supports the prosecution hypothesis

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31
Q

Likelihood ratio <1

A

The evidence supports the defense hypothesis

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32
Q

A big LR value

A

The bigger the value, the grater the support for the prosecution proposition

33
Q

A small LR value

A

The smaller the value, the greater the support for the defence.

34
Q

What happens to the probability of a mach when there is a blood relation to the defendant.

A

The probability of a match is much greater if the unknown person were a blood relation of the defendant, say a brother.

35
Q

ENFSCI recommendations for mixed DNA profiles

A
  • If possible, mixed DNA profiles should be interpreted and designated into their contributing DNA profiles.
  • Mixed profiles from (known) to victims and (unknown) donors can be resolved because the alleles of the DNA profile of the victim can be subtracted from the mixed profile.
  • The remaining alleles must belong to the unknown donor.
36
Q

What happens to the value of evidence for a mixed DNA sample if you can’t calculater a statistic or separate out the DNA profile?

A

If you can’t separate a mixed DNA profile out or put a statistic then its no value in court.

37
Q

Ian Evett, Sue Pope et al

A

In summary:
1. Prosecution – the defendant is one of three contributors to a mixture – or
2. Defence – the mixture is from three unknown persons
* Presenting the number of matching components between the defendant and the DNA mixtures fosters a prejudicial view of the evidence
* Scientists are not trained to provide qualitative assessments of evidential weight of DNA mixtures, and no systems exist for assessing the robustness of such assessments

38
Q

What mixtures can be easy to intrepret

A
  • Two-person mixtures can be relatively easy to interpret.
  • Those mixtures would not be considered complex.
39
Q

What happens if the evidence contains a trace amount of DNA

A

When the evidence contains only a trace amount of DNA, it is sometimes impossible to know if that DNA came from only one individual or from multiple people.

40
Q

3 main factors affecting DNA evidence and its evidential weighting.

A
  1. How many people contributed DNA to the mixture? More contributors make a mixture more complex, and therefore, more difficult to interpret.
  2. How much DNA did each person contribute? Even if a mixture contains plenty of DNA overall, one or several people might have contributed only a tiny amount. The lower those amounts, the more complex the mixture.
  3. How degraded is the DNA? DNA degrades over time and with exposure to the elements. This can also increase complexity.
41
Q

Difficulty with trace DNA

A

Evidence that contains trace amounts of DNA or a DNA mixture can require a lot of interpretation.

42
Q
  1. If the probability of one event has no effect on the probability of the other events occurring it is said to be?
  2. The term Pr (B I A) is?
  3. Probability divided by one, minus the probability =
  4. A likelihood ratio of 1 provides this level of support.
  5. Three main factors affecting complex DNA profiles are associated with how many people contributed; how much DNA each person contributed and what else?
  6. Whereas for a single source DNA profile the random match probability is the statistic of choice. What would be statistic of choice were mixed profile is observed?
A
  1. If the probability of one event has no effect on the probability of the other events occurring it is said to be independant.
  2. The term Pr (B I A) is conditional probability
  3. Probability divided by one, minus the probability = odds
  4. A likelihood ratio of 1 provides neutral level of support.
  5. Three main factors affecting complex DNA profiles are associated with how many people contributed; how much DNA each person contributed and degradation.
  6. Whereas for a single source DNA profile the random match probability is the statistic of choice. What would be statistic of choice were mixed profile is observed? Likelihood ratio
42
Q

Dlugosz (2011)

Conviction

A
  • Convicted of burglary, robbery, and manslaughter & sentenced to imprisonment for public protection for seven years.
  • Subsequently retried and convicted of manslaughter & robbery and sentenced to 9 years in prison.
43
Q

Dlugosz (2011)

Case background information

A
  • On 27th November 2008 an elderly lady’s house in North London was burgled.
  • On 1 January 2009 the victim was found dead in her house.
  • She had been tied up and being unable to free herself had died from hypothermia.
  • Her ankles and wrists had been bound with silver tape and her mouth had been gagged with the same tape to stop her screaming.
  • The house had been ransacked.
44
Q

Kuba Dlugosz

European arrest warrant

A
  • Kuba Dlugosz, 33, of no fixed address, was wanted on a European Arrest Warrant for skipping prison in Poland when he attacked the 83-year-old at her £1million house in Leweston Place, where she had lived since childhood.
  • The convicted robber fled to the UK in 2007.
45
Q

Kuba Dlugosz (2011)

Forensic examination

A
  • No fingerprints were found.
  • It was assumed the offenders had worn gloves.
  • The only forensic evidence was a very small quantity of DNA on two of the three chisels left at the premises.
  • The amounts were less than 200 pg.
  • The results showed a mixed profile.
  • He was arrested in July 2010.
46
Q

Kuba Dlugosz (2011)

Co-offender

A
  • Whilst on remand his telephone calls were recorded.
  • In September 2010 his co-offender Wyrostek was arrested for the same offence.
  • He had admitted his part in the burglary and admitted they put masking tape on the victims face.
47
Q

Kuba Dlugosz (2011)

DNA evidence

A
  • He applied to exclude the DNA evidence based on the fact it was incomplete, low level and a mixture.
  • His previous convictions were put before the jurt allowing an element of biased to be introduced. (This process is called a bad character application)
  • He didn’t go into the witness box to explain himself. Of course, this is the right of any individual. We call this the burden of proof which, in UK law always rests on the prosecution to prove their case to the jury so they are certain.
  • The defendant has the absolute right to say nothing.
48
Q

Kuba Dlugosz (2011)

Defence proposition

A
  • The Defence accepted that Dlugosz could have made a contribution to these samples.
  • However, the number of contributors to the DNA profiles could not be determined.
  • It was impossible to resolve these mixed DNA profiles into individual profiles of the contributors.
  • It was therefore not possible to calculate a robust statistic indicating the strength of the link between Dlugosz and DNA in the two samples.
  • Although they provided an investigative lead, they did not provide cogent evidence.
49
Q

Kuba Dlugosz (2011)

Proseuction case

A
  • The prosecution scientist, in an effort to be helpful expressed the view:
  • “…To evaluate these results I have considered the following two propositions”
  • DNA from Dlugosz was present on the swabs.
  • Or DNA from Dlugosz was not present on the swabs.
  • In my opinion, the DNA profile results provide support for the presence of DNA from Dlugosz on the swab but I am unable to quantify the level of this support.
50
Q

Kuba Dlugosz (2011)

DNA exclusion

A

The defence sought to exclude the evidence of DNA:
* Because without statistical evaluation.
* Its significance could not be evaluated.
* It should not be placed before the jury.
However:
* The judge heard this defence and allowed the evidence to be heard in the absence of a statistic.

51
Q

Kuba Dlugosz (2011)

Prosecution expert witnesses

A
  • The jury heard the evidence of the two experts and were in a position to use that evidence in accordance with a clear and careful summing up. Bearing in mind the difference between the two experts was not that great.
  • One saying it was rare and the other saying it was somewhat unusual.
  • Evidence nowadays would not be allowed in this format.
52
Q

RFU

A

relative fluorescent units

53
Q

Uncertainty 1 in DNA interpretation

When is a peak a peak?

A
  • When the amount of DNA is very low, the peaks can be very small.
  • Some peaks can be so small that they disappear entirely (they drop out of the profile).
  • Also, small blips in the data can be mistaken for real peaks (they drop in, to the profile).
  • Many of these effects are random, and they can make it difficult to interpret the evidence.
54
Q

Uncertainty 2 in DNA interpretation

Whose peak is it?

A
  • When analysing a DNA mixture, the alleles from all the contributors show up on the same chart.
  • This can make it difficult to tease apart the DNA profiles of the individual contributors.
  • To understand why this makes things complicated, recall that after amplifying the DNA, the forensic scientist has a test tube with millions of copies of the alleles in solution.
55
Q

DNA mixtures

Appearance of alleles

A

Just because a person’s alleles appear in a mixture does not mean that person contributed to it. The alleles may have come from some combination of other people who, between them, have all the allele types in the suspect’s profile

56
Q

A scientist decides that this is a mixture of DNA from three people and has observed the components of the defendants DNA at all loci.

Prosecution proposition

A

“…that the crime sample is a mixture of DNA of the (a) defendant and (b) two unknown people”

57
Q

The random match probability

A
  • The sample contains a mixed profile: Eliminate the alleles that are contributed by innocent parties.
  • Determine which suspect the remaining alleles belong to.
  • Calculate the likelihood of the profile belonging to anyone else.
58
Q

Why are binary methods of forensic DNA interpretation restricted?

A
  • Binary methods of forensic DNA interpretation are restricted as they are unable to deal completely with complex low-level or mixed DNA profiles. These types of data have become more prevalent as DNA typing technologies and STR multiplex chemistries become more sensitive.
  • The shortcomings of binary methods led to the development of improved models that use PG to interpret profiles.
59
Q

Binary methods of DNA interpretation

A
  • Early methods of DNA profile interpretation were described as binary as the probability of the evidence given a proposed genotype was assigned as zero (genotype excluded) or one (genotype included).
  • In a binary method of interpretation all included genotype combinations are considered equally likely.
60
Q

Probabilisitc genotyping

A

Uses:
- biological modelling
- probability distributions
- Statistical theory
- To infer geno types for DNA profiles from forensic profiles and calculate LRs

61
Q

What is probabilistic genotyping software & how does it help?

A
  • Scientists have developed computer programs to help interpret complex mixtures
  • Probabilistic genotyping software (PGS) uses statistical and biological models to calculate probabilities. For instance, the software is designed to account for drop-in, drop-out and other effects by using mathematics to approximate what happens in a real mixture.
  • PGS also considers the fact that some alleles are more common in the population than others.
62
Q

What can affect the results in probabilistic genotyping

A
  • The type of software used, how the software is configured, and which models the software runs can all affect the results. Different labs might produce different results when interpreting the same evidence.
  • There is no consensus on how to identify those limits.
63
Q

Probabilistic methods

Continuous

A
  • Uses all of the data present, including allele and peak height information, and incorporates biological parameters such as peak height ratios, mixture ratios, and stutter percentages.
  • This method uses the quantitative information from peak heights to calculate the probability of the observed peak heights given all possible genotype combinations.
  • This type of software requires numerous calculations and may use simulations to model the observed data and produce statistics and may have a longer analysis time.
  • The time for such analysis varies by software program.
64
Q

Probabilistic methods

Semi continuous

A
  • Uses the peak height information and alleles present in a mixture without considering biological parameters such as peak height ratios, mixture ratios, and stutter percentages.
  • This model accounts for the probability of allele drop-out (non-appearance of an allele) and drop-in (appearance of an additional non-reproducible allele).
  • Software programs that use this model generally conduct fast calculations, but the model does not use all of the available data.
65
Q

The analytical threshold (AT)

A
  • Allows the analyst to reliably distinguish a peak as being allelic versus an artifact (noise) from the CE instrument.
  • The first threshold is the analytical threshold (AT). The AT allows the analyst to reliably distinguish a peak as being allelic versus an artifact (noise) from the CE instrument.
  • Laboratories determine their AT empirically from validation studies for their STR kit and CE combination. There are a number of ways the AT can be determined
66
Q

Stochastic threshold

A
  • Any single allele above the ST would be considered homozygous.
  • Any allele below the ST is treated as having a missing sister allele.
  • Like the AT, the ST is determined from empirical data.
  • The ST provides some assurance to the analyst that a sister peak has not fallen below the AT and a homozygote genotype can be called with some confidence.
  • Any single allele above the ST would be considered homozygous, and any allele below the ST is treated as having a missing sister allele.
67
Q

Clayton Rules

A
  1. Identify the presence of a mixture
  2. Identify artefacts vs alleles
  3. Identify the number of contributors to a mixture
  4. Determine the approximate ratio of the components.
  5. Determine the possible pairwise genotype combinations for the different contributors to the mixture.
  6. Compare the resultant genotype profiles for the contributors with those from the reference samples.
  7. Perform statistical analyses.
68
Q

Most common artefact

A
  • Stutter is the most common artifact within a profile and can confound mixture interpretation.
  • Especially when the height of the stutter peaks are similar to a minor contributor within the profile.
  • Other artifacts include pull-up, where the RFU signal from one dye colour (e.g., green) is so strong it “bleeds” into another dye channel (e.g., blue) creating a peak that may fall in the region of an allele.
69
Q

How to determine approximate ratios of contributors of a mixture?

A

Use AT and ST

70
Q

Heterzygote single source LR

A
  • Given the peak heights, it is reasonable to assume that there is no dropout of alleles and no drop-in within the profile.
  • There are a maximum of two alleles (and their corresponding stutter peaks) at each locus and no unreasonable imbalance between the two alleles at locus one.
  • Therefore, it is reasonable to assume based on the profile that it has originated from only one individual.
71
Q

How confident can one be that the DNA is related to the crime?

A
  • While PGS can tell you who might have contributed DNA to a mixture, it can’t tell you how or when their DNA got there.
  • If the evidence contains a lot of DNA, this might not be a problem.
72
Q

What is allele dropout?

A
  • Allele dropout occurs when a sample is produced and one or more alleles are not present.
  • Describes when allelic peaks fall below the analytical threshold of an instrument.
  • It is generally set to some amount above the lower detection limit of the instrument, where an analyst can reliably assign a peak as allelic with a low or nil risk of the peak being a baseline artifact.
  • Low-level DNA template and/or degraded DNA results in alleles not amplifying above this threshold, resulting in incomplete or partial profiles.
73
Q

What factors can cause allele dropout?

A

This can be due to a variety of factors – namely
* The initial input quantity of DNA is too low, resulting in a failure to amplify one or more alleles in the sample
* A mutation in the primer binding site is present, which causes of failure in the amplification of the allele
* An allele sizes outside of the normal calling range for a particular locus and goes undetected

74
Q
  1. Two thresholds used to quality assure peak heights. One of these is referred to as the analytical threshold. The higher threshold is referred to as?
  2. Any single allele above the stochastic threshold would be considered?
  3. Probabilistic genotyping methods making use of peak height information I’ll refer to as?
  4. Probabilistic genotyping methods using all data present including allele and peak height information, peak height ratios and stutter percentages are referred to as?
  5. Seven steps used to analyse mixtures are referred to as?
  6. MaSTR is one technology software used in?
A
  1. Two thresholds used to quality assure peak heights. One of these is referred to as the analytical threshold. The higher threshold is referred to as the stochastix threshold.
  2. Any single allele above the stochastic threshold would be considered homozygous.
  3. Probabilistic genotyping methods making use of peak height information is referred to as semi continuous.
  4. Probabilistic genotyping methods using all data present including allele and peak height information, peak height ratios and stutter percentages are referred to as continuous methods.
  5. Seven steps used to analyse mixtures are referred to as clayton rules.
  6. MaSTR is one technology software used in probabilistic genotype.
75
Q

Profile frequency calculation

A
  • Calculated by multiplying all of the loci frequencies together.
  • How common is this profile in the population
76
Q

Match probability

A

This is done by dividing 1 by the profile frequency.

77
Q

The responsibilities of SWGDAM are:

A

The responsibilities of SWGDAM are:
* To recommend revisions, as necessary, to the Quality Assurance Standards for Forensic DNA Testing Laboratories and the Quality Assurance Standards for DNA Databasing Laboratories;
* To serve as a forum to discuss, share, and evaluate forensic biology methods, protocols, training, and research to enhance forensic biology services; and
* To recommend and conduct research to develop and/or validate forensic biology methods.

DNA Analysis & Interpretation (19 decks)

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