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DNA mixtures

In this video, Dr Gavin Turbett and Dr Vivek Sahajpal discuss DNA mixtures and the complications which arise in their interpretation.
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Another challenge that is commonly seen is mixed DNA profiles, and by definition, these are DNA profiles from two or more contributors in the one sample. In real casework samples, the contributors will almost certainly not be present in equal concentrations. And furthermore, individual contributors may share alleles by chance.
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Another complication is that the DNA from some contributors might be more degraded than the DNA from other contributors. It furthermore cannot be assumed that all of the DNA that is present in that mixed DNA profile was deposited onto the exhibit at the same time.
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In some instances, mixed DNA profiles may be so complicated that there is no confidence about the number of contributors that are actually present within that sample. But this is a mixed DNA profile, and you can see there are three, four, five or even six alleles per locus. So at the D21 locus, there are actually six alleles detectable. And the peak heights of each, highly variable. You can see at D5, for example, there are two alleles that are quite large and a third allele by itself, that’s quite tiny. Now, in reality, this was a mixture of DNA that was created in the laboratory for training purposes. And we actually know that this was a mixture of DNA from four people.
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So we took DNA from four staff volunteers and we mix their DNA in equal proportions. So this is one to one to one to one mixture of DNA from four people.
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Despite that, we only ever see up to six alleles at any one locus. If we’ve got four people, there could be theoretically as many as eight alleles detectable. So if we now show just one portion, this is the THO1 locus from the previous slide and it demonstrates that alleles can be shared by chance. Now, as I said, this was a mixture of DNA created in the laboratory. We know the genotypes, the DNA profiles of all four contributors. And as it turns out, all four donors all have an allele seven. Two of the donors actually have the same genotype at that locus. They were both alleles 7,9. Donor three has alleles 7,10 and donor four had alleles 7,8.
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So the four alleles that are detected are actually representing from four different people. There are eight alleles there, but only four actually detectable. And you can see that four people have contributed to that single allele 7 peak, one person contributed the allele 8, two people contributed the allele 9 and one person contributed the allele 10. So again, in mixed DNA profiles, alleles will be shared by chance. In mixed DNA profiles, it is very unlikely that the contributions made by each donor will be equal. In fact, it’s almost certain that they won’t be equal.
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And this typically means that in a mixed DNA profile, it’s quite often possible to see what we refer to as major contributors and minor contributors, and the different peak heights can sometimes be used to assist in interpretation. So we’ve got a small section of a DNA profile here on the right, and it’s the D3S1358 locus. And you can see that we’ve detected four alleles- 14, 16, 17 and 18. Now, if we assume that this is a two person mixed DNA profile, then there are three different combinations that could actually result in that pattern of four alleles. So one of the people could be a 14, 16 and the other a 17, 18.
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One could be a 14, 17 and the other a 16, 18 or one of the people could be a 14, 18 and the other being a 16, 17. So there’s three possibilities there that could account for that pattern of alleles. But once we take the peak heights into account, we can actually see that only one combination makes sense. So here we can see once the peak heights are taken into account, it is only the combination of alleles 14,18 coming from one person and alleles 16, 17 coming from the other person. That is the only combination that makes sense if it is a two person mixed DNA profile.
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Now, that would not apply if this was actually a three or four person mixed DNA profile, and there might be other parts of the DNA profile, more alleles detected elsewhere that make us believe that it’s a three or four person mixture. But for this example, if we assume that it is a two person mixture, then it would be roughly one contributor provided two thirds to three quarters of the DNA, and the other contributor provided about a third or a quarter of the DNA. So that it’s the only combination that makes sense is if one of the people is a 14,18.
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Those are the two alleles that are around 4000 units in height, and the other contributor has provided alleles 16, 17, and the alleles are 1600 to 1900 units in height. In some instances, the alleles may be roughly the same height. So we do have an example here where we have what appears to be a two person mixture and the two contributors have provided about the same amount of DNA each. So this is the D6S1043 locus and we’ve detected alleles 11, 12, 15 and 21. So there are actually six combinations that are possible. So we’ve got person 1 and person 2 and three of them are mirrors of the other.
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But nevertheless, that gives us the six combinations that could actually occur in this particular example. And given that the peak heights are all approximately equal, which of those pair-wise combinations is the most probable. The reality is because the peak heights are approximately equal, they are all equally probable. We can’t be certain which of them, which pair of alleles actually belong together. However, we may have additional information available to us that can help us to interpret or deconvolute this mixture. Let’s assume that we actually know this was a mixed DNA profile and it was taken from the skin of a of a particular person.
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So we now know that that person had alleles 11,21 at the D6 locus because we’ve tested their mouth swab, their reference DNA profile. So we now know for certain that this person that one of the two contributors provided alleles 11,21. Therefore, the only other possibility is for the unknown contributor to be alleles 12, 15 this particular locus. So if we have a two person mixed DNA profile, even if it can’t be deconvoluted by itself based on peak heights, once we know the DNA profile of one contributor and that perhaps might be the victim of crime, once we know that one DNA profile, we can infer the DNA profile of the other unknown contributor.
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The second type of scenario is when you are getting complex mixtures and when you don’t know the contributors. So these type of samples, they may include the non intimate samples, such as the swabs from public places, sidewalk bus, taxi, corridors, eatery tables or tools which have been handled by multiple people. These mixtures are typically the most difficult category to interpret. These mixtures are sometimes very complex, as such cannot be interpreted based just on peak height. Applying statistical interpretation is easier when one contributor is known, when you have a major component. So statistical evaluation of complex mixtures involves the use of likelihood ratio or probability of inclusion and exclusion. Mixture interpretation softwares are useful for mixture interpretation.
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However, very, very complex mixtures are almost impossible to interpret.

In case of a DNA mixture, there are two or more contributors to the DNA profile. The deconvolution process or the process by which the DNA examiner interprets the possible allele pairs or genotype in the DNA mixture can be highly subjective and complicated.

In 2016, the President’s Council of Advisors on Science and Technology, a council of expert’s to the US President, published a report called Forensic Science in Criminal Courts: Ensuring Scientific Validity of Feature-Comparison Methods (PCAST). The report examined a range of forensic disciplines that were based on ‘feature comparison’ methods, such firearm analysis, hair microscopy analysis, latent fingerprint analysis, bitemark analysis, footwear analysis and DNA analysis. The report found that forensic DNA analysis of single source DNA and simple mixture of two individuals was an objective method and foundationally valid. On the other hand, when it came to DNA analysis of complex DNA mixtures, the report found that the traditionally used combined probability of inclusion (CPI) method was subjective, prone to error and not foundationally valid. CPI method requires the scientists to make subjective choices about what can be considered alleles, possible allele pairings as well as major and minor contributors in a mixture. Each of these choices affect the final conclusion and opinion of the scientist. Due to the subjective nature of this method, there has been a move towards using probabilistic genotyping softwares (PGS) by scientists to deconvolute complex DNA mixtures. The PCAST report found that the process may be objective but there was a requirement for additional studies to examine its foundational and as applied validity.

In 2021, a report published by the National Institute of Standards and Technology, DNA Mixture Interpretation: A NIST Scientific Foundation Review, found that PGS utilises more information than traditional binary approaches (like CPI). The report is the first of its kind, where experts in the discipline have conducted a scientific foundation review of DNA mixture analysis methods. The report also found that interpretation of DNA mixtures varied across analysts and laboratories adding a lot of variance to the process. One of the key takeaways from this report is that there is not enough publicly available data to analyse the foundational validity of the PGS method of analysis.

Based on the reports mentioned above, it is important to keep the following things in mind:

  • Due to increased sensitivity of forensic DNA techniques, minute amounts of DNA can now also be detected. In such cases, it may be difficult to ascertain major and minor contributors, further complicating DNA mixture interpretation.
  • Biological evidence found at crime scenes may often be degraded and generate partial DNA profiles, which further complicates deconvolution of DNA mixtures
  • Interpretation of the raw data and electropherograms in a mixed DNA profile case requires the experts to make certain assumptions. It is therefore important that experts specify the assumptions made as part of this step in their reports.
  • It is important to note that experts often do not comment on all the possible genotypes found in the crime scene sample, instead only on the genotypes with greater statistical significance.

Research surrounding deconvolution of complex DNA mixtures is still at a nascent stage. Scientists are yet to validate the PGS method, while traditional binary methods have multiple elements of subjectivity. Therefore, from a legal perspective, it is important to assess how the interpretation of a DNA mixture has been carried out in a particular case and carefully evaluate it against established guidelines.

In this video, Dr Turbett and Dr Sahajpal further aid your understanding of the challenges in interpretation of DNA mixtures.

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Decoding Forensics for Legal Professionals

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