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Effect of Drop-in on False Positive and Rank-Order Likelihood Ratios (LR)

Calculated for a Mixture of Touch DNA


Presenting Author: Clinton Hughes
Affiliation: Senior Staff Attorney, DNA Unit, The Legal Aid Society, New York, NY
Co-Authors and Affiliations: Dr. Eli Shapiro, DNA Consultant for the Legal Aid Society,
Former Assistant Director and Training Coordinator for the New York Office of the Chief
Medical Examiners Forensic Biology Department and Arthur Speiser, B.S. candidate,
Columbia University
Abstract
We have examined the LR statistical approach to analysis of low template DNA mixtures
exhibiting drop-out and drop-in used in casework by the NYC Office of Chief Medical Examiner
Department of Forensic Biology (OCME), called Forensic Statistical Tool (FST)1. We used
Microsoft Excel to create a program to reproduce the results of FST on a three-person mock
case mixture. We used the Excel add-on DigDB to create the roughly 200,000 possible
combinations necessary to recreate the FST results of a suspect comparison to this mixture.
Drop-out, when allele(s) that are part of a DNA sample are not called in the laboratory results,
can be caused by a number of factors, including low DNA template amount. Recent work2
showed that drop-out can generate positive LR for DNA profiles of known non-contributors, and
that these false positives can have higher LR than known contributor profiles. The number of
false positives increased with increasing number of alleles in the mixture and increased number
of drop-outs. The effect of drop-in was not considered in these studies.
Allelic drop-in is defined by John Butler as contamination from an unknown source.3 In the
FST program artifactual stutter alleles are lumped into the drop-in probability. There is general
agreement in the field that drop-in should be a rare event. FST values for the probability of
drop-in were derived from empirical studies using mixtures of pristine single-source buccal swab
extracts. For 31-cycle amplification, FST expects the low probabilities of 0.035 single allele
drop-ins per locus, and 0.005 drop-in of two or more alleles per locus.
We examined the effects of drop-in on the calculated LR using an OCME laboratory validation
sample created by three known touch DNA contributors. We also examined one of the false
positives for this sample found in a search of 1256 database profiles, including the NIST
population database.
The mixture we examined contained 60pg of DNA in each of three amplifications, and was
called a deducible 3-person mixture by the OCME analyst. This meant that FST used drop-out
rates determined for the minor contributors to a 5:1:1 ratio mixture. The drop-in rates were as
described above. The mixture generated false-positive associations with known noncontributors. The highest LR for database sample was 157, for sample JB from the NIST
Caucasian database. By contrast, the two minor known contributors had LR of 4 and 10e-5.
This mixture provided a good example of drop-in. Out of 78 total alleles in the mixture, 20 were
drop-in alleles not associated with any known contributor. 13 drop-in alleles were found in the

59 alleles in the consensus profile, being present in at least two of three amplifications. Out of
the 45 individual loci, 14 loci showed one drop-in allele. An additional 13 loci showed two or
more drop-in alleles. The FST drop-in rate predicts approximately 2 loci with one drop-in. FST
expects no loci with drop-in of two or more alleles. Thus, the JB mixture had 10 times more
foreign and stutter alleles than the FST program predicts.
Analysis of the JB mixture showed that drop-in affects LR in the same way that has been
demonstrated for drop-out. Drop-in caused a strong false positive LR, which was higher than
the LR for the known minor contributors.
Although the FST does not provide LRs for alternative suspects, our Excel spreadsheets showed
the LRs of all possible genotypes, showing the effect of drop-in in the JB mixture upon all
possible profiles. Drop-in created a highest-ranking false positive profile, with an LR of 10e15.
Locus by locus analysis of the mixture is not provided by FST. Our Excel calculations showed
that at many loci, drop-in caused the highest LR. Examining allele frequencies at loci with 5 or 6
alleles in the mixture, along with the calculation of the percentage of DNA profiles giving LR
greater than 1, showed that a locus with too many alleles can lose its probative value, yet still
provide high LR solely conditioned on the genotype of the comparison sample.
Over 20 percent of the alleles in the consensus profile were drop-in alleles, calling into question
the use of consensus profiles and multiple replication, since this approach does not eliminate all
spurious alleles, and creates opportunities for single drop-ins that weaken the statistical analysis.
The values we obtained using our Excel FST program were close to the values obtained by FST
but not exact. We would be happy to review our calculations with OCME to directly compare to
the FST.
Given the poor description of the actual touch DNA mixture, the unreliable FST results are not
surprising. Artificially lowering the drop-in rate exaggerates the strength of the evidence by
inflating confidence that any allele is not an artifact. In casework samples, it is not possible to
distinguish drop-in alleles from alleles coming from a contributor. Our analysis shows the need
for better ways to account for and control drop-in when evaluating low-template and touch DNA
mixtures.
1. Mitchell, et. al., (2012) FSI Genetics 6, pp 749-761.
2. Gill, et. al., (2014) FSI Genetics 13, pp 167-175.
3. Butler, J. (2010) Fundamentals of Forensic DNA Typing, page 440, Appendix 1.