Interpreting low template DNA profiles

We discuss the interpretation of DNA profiles obtained from low template DNA samples. The most important challenge to interpretation in this setting arises when either or both of “drop-out” and “drop-in” create discordances between the crime scene DNA profile and the DNA profile expected under the prosecution allegation. Stutter and unbalanced peak heights are also problematic, in addition to the effects of masking from the profile of a known contributor. We outline a framework for assessing such evidence, based on likelihood ratios that involve drop-out and drop-in probabilities, and apply it to two casework examples. Our framework extends previous work, including new approaches to modelling homozygote drop-out and uncertainty in allele calls for stutter, masking and near-threshold peaks. We show that some current approaches to interpretation, such as ignoring a discrepant locus or reporting a “Random Man Not Excluded” (RMNE) probability, can be systematically unfair to defendants, sometimes extremely so. We also show that the LR can depend strongly on the assumed value for the drop-out probability, and there is typically no approximation that is useful for all values. We illustrate that ignoring the possibility of drop-in is usually unfair to defendants, and argue that under circumstances in which the prosecution relies on drop-out, it may be unsatisfactory to ignore any possibility of drop-in.     

Validation issues around DNA typing of low level DNA

A recent ruling in the Crown Court of Northern Ireland, R v. Hoey, [R v Sean Hoey. 2007, Crown Court of Northern Ireland] has raised questions about the validity of one variant of DNA analysis, often termed LCN. The ruling and subsequent discussion also raises questions about what constitutes validation of a technique.This paper examines what can be achieved in a laboratory based validation study against the Daubert standard and against guidance given in the UK. There is a significant discrepancy between what can be achieved and the Daubert standard but much less of a discrepancy against the UK guidance. Much of the difference relates to differences in word usage, definitional difficulties, and a lack of mutual understanding and communication between the judiciary and forensic scientists. This highlights a gap that needs attention.     

Practical determination of the low template DNA threshold

The low template stochastic DNA threshold is used to infer the genotype of a single STR allelic peak. For example, within the context of the UK National DNA Database, the stochastic threshold is used to decide whether a DNA profile, consisting of a peak in position of allele a, is uploaded as aF or as an aa homozygote. The F designation acts as a ‘fail-safe’ wild card that is designed to capture the possibility of allele drop-out and to do this it will match any allele. If a profile is wrongly designated as an aa homozygote, the database search will be unnecessarily restricted and may fail to match a perpetrator reference sample on the database. If the stochastic threshold is too high, then this increases the number of adventitious matches, which in turn compromises the utility of the national DNA database. There are many different methods used to process DNA profiles. Often, the same stochastic threshold is used for each process (typically 150 rfu). But this means that more sensitive methods will have a threshold that is too low (and vice versa) and the risks of a wrongful designation are correspondingly greater. Recently, it was suggested that logistic regression could be used to relate the stochastic threshold to a defined probability of drop-out in order to properly evaluate the risks associated with a given stochastic threshold. In this article we introduce a new methodology to calculate the stochastic threshold that a practitioner could easily implement. The threshold depends on the sensitivity of the method employed, and is adjusted to be equivalent across all methods used to analyse DNA profiles. This ensures that risks associated with misdesignation are equivalent across all methods. In effect a uniformity of methods, underpinned by an analysis of risks associated with misdesignation can be achieved.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR(?) Identifiler(?) amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR(?) Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

Verifying likelihoods for low template DNA profiles using multiple replicates

To date there is no generally accepted method to test the validity of algorithms used to compute likelihood ratios (LR) evaluating forensic DNA profiles from low-template and/or degraded samples. An upper bound on the LR is provided by the inverse of the match probability, which is the usual measure of weight of evidence for standard DNA profiles not subject to the stochastic effects that are the hallmark of low-template profiles. However, even for low-template profiles the LR in favour of a true prosecution hypothesis should approach this bound as the number of profiling replicates increases, provided that the queried contributor is the major contributor. Moreover, for sufficiently many replicates the standard LR for mixtures is often surpassed by the low-template LR. It follows that multiple LTDNA replicates can provide stronger evidence for a contributor to a mixture than a standard analysis of a good-quality profile. Here, we examine the performance of the likeLTD software for up to eight replicate profiling runs. We consider simulated and laboratory-generated replicates as well as resampling replicates from a real crime case. We show that LRs generated by likeLTD usually do exceed the mixture LR given sufficient replicates, are bounded above by the inverse match probability and do approach this bound closely when this is expected. We also show good performance of likeLTD even when a large majority of alleles are designated as uncertain, and suggest that there can be advantages to using different profiling sensitivities for different replicates. Overall, our results support both the validity of the underlying mathematical model and its correct implementation in the likeLTD software.     

Comparison of different interpretation strategies for low template DNA mixtures

DNA typing protocols have been improved over the last few years and even mixtures from minute and low grade DNA stains do not necessarily preclude typing. Nevertheless, in those cases stochastic phenomena tend to hamper interpretation. In order to supplement the current discussion about the interpretation of such challenging data, we focused on different combinations of analyses as an attempt to overcome stochastic problems.We analyzed mixtures of two types of degraded DNA in low template amounts (50 and 100 pg DNA per contributor) using four types of multiplex STR typing kits: PowerPlex^® ESX 17, PowerPlex^® ESI 17, Investigator^® ESSplex SE Kit and P11, a non-commercial kit. We employed the results of double or triple analyses for a comparison of different types of interpretation rules based on reporting either only reproducible alleles (consensus interpretation) or all alleles, even if they are only observed once (composite interpretation). The interpreted and composed profiles were compared to the known alleles of the contributors and a “degree of validity” was calculated.When only two single amplifications were taken into account, we observed a higher degree of validity for composite profiles. The difference for consensus interpretation could be compensated when a minimum of three amplifications were carried out.Using the same kit for repeat analyses increases the chances to yield reproducible results required for consensus interpretation. Combining different kits in a complementing approach, on the other hand, offers the opportunity to reduce the number of drop-out alleles: differences in amplicon lengths for specific markers between kits can increase the resulting information. In the case of a few amplifications available this effect might only be visible with the composite method. Several markers like SE33 are particularly affected by this.Based on our observations the consensus interpretation method may not reflect the original profile in an optimal way in some special cases like low template, degraded mixture stains. In those cases the composite interpretation could yield more complete results. However, such a composite profile should be used with caution and only for limited purposes. Generally recommending the consensus interpretation thus seems not to be justified: a more differentiated approach appears to be worthwhile, e.g. the amount of drop-outs, the number of replicates, choice and combination of kits and even a marker specific procedure might be taken into account.     

Exploratory data analysis for the interpretation of low template DNA mixtures

The interpretation of DNA mixtures has proven to be a complex problem in forensic genetics. In particular, low template DNA samples, where alleles can be missing (allele drop-out), or where alleles unrelated to the crime-sample are amplified (allele drop-in), cannot be analysed with classical approaches such as random man not excluded or random match probability. Drop-out, drop-in, stutters and other PCR-related stochastic effects, create uncertainty about the composition of the crime-sample, making it difficult to attach a weight of evidence when (a) reference sample(s) is (are) compared to the crime-sample. In this paper, we use a probabilistic model to calculate likelihood ratios when there is uncertainty about the composition of the crime-sample. This model is essentially exploratory in the sense that it allows the exploration of LRs when two key-parameters, drop-out and drop-in are varied within their plausible ranges of variation. We build on the work of Curran et al. [8], and improve their probabilistic model to allow more flexibility in the way the model parameters are applied. Two new main modifications are brought to their model: (i) different drop-out probabilities can be applied to different contributors, and (ii) different parameters can be used under the prosecution and the defence hypotheses. We illustrate how the LRs can be explored when the drop-out and drop-in parameters are varied, and suggest the use of Monte Carlo simulations to derive plausible ranges for the probability of drop-out. Although the model is suited for both high and low template samples, we illustrate the advantages of the exploratory approach through two DNA mixtures (involving two and at least three individuals) with low template components.     

Assessment of mock cases involving complex low template DNA mixtures: A descriptive study

Complex DNA mixtures with low template (LT) components provide the most challenging cases to interpret and report. In this study, we designed such mixtures and we describe how reporting officers (ROs) at the Netherlands Forensic Institute (NFI) assess these when embedded in a mock case setting. DNA mixtures containing LT DNA from two to four contributors, sporadic contamination (mimicked by adding 6 pg of DNA, which represents once cell equivalent) and/or DNA of relatives (brothers), were amplified four-fold using the AmpFlSTR^® NGM? PCR Amplification Kit. Consensus profiles were then generated which included the alleles detected in at least half of the replicates. Four mock cases were created by including reference profiles of a hypothetical victim and suspect. The mock cases were assessed by eight ROs following the stepwise interpretation approach currently in use at the NFI. With this approach, the results of the comparisons between the DNA profiles of the evidentiary trace and the reference profiles are classified into four categories of evidential value [1]. The interpretations by the ROs were compared to the likelihood ratios (LRs) obtained from a probabilistic model that allows a calculation of LRs to assist the interpretation of LT DNA evidence and both were compared to the true composition of the designed mixtures.     

Consensus and pool profiles to assist in the analysis and interpretation of complex low template DNA mixtures

Forensic analysis of low template (LT) DNA mixtures is particularly complicated when (1) LT components concur with high template components, (2) more than three contributors are present, or (3) contributors are related. In this study, we generated a set of such complex LT mixtures and examined two methods to assist in DNA profile analysis and interpretation: the “n/2” consensus method (Benschop et al. 2011) and the pool profile approach. N/2 consensus profiles include alleles that are reproducibly amplified in at least half of the replications. Pool profiles are generated by injecting a blend of independently amplified PCR products on a capillary electrophoresis instrument. Both approaches resulted in a similar increase in the percentage of detected alleles compared to individual profiles, and both rarely included drop-in alleles in case mixtures of pristine DNAs were used. Interestingly, the consensus and the pool profiles often showed differences for the actual alleles detected for the LT component(s). We estimated the number of contributors using different methods. Better approximations were obtained with data in the consensus and pool profiles compared to the data of the individual profiles. Consensus profiles contain allele calls only, while pool profiles consist of both allele calls and peak height information, which can be of use in (statistical) profile analysis. All advantages and limitations of the various types of profiles were assessed, and based on the results we infer that both consensus and pool profiles (or a combination thereof) are helpful in the interpretation of complex LT DNA mixtures.     

DNA typing in cases of blood chimerism

A chimera is an organism whose cells derive from two or more distinct zygote lineages. and therefore two different blood cell populations circulate in one individual. To point out the potential pitfalls in forensic analysis, a set of triplets (a girl and two boys) who revealed blood chimerism was investigated with four STR systems using PCR. The results indicated that a DNA profile based on DNA extracted from blood can lead to a false determination because the band pattern of each triplet contained a mixture of the original genotype and the genotype of the siblings. Additional investigations on biological materials other than blood must be made in order to find out the real genetic characteristics of each child. Received: 17 July 1998 / Received in revised form: 27 November 1998     

Human tandem repeat sequences in forensic DNA typing

It has been 20 years since the first development of DNA fingerprinting and the start of forensic DNA typing. Ever since, human tandem repeat DNA sequences have been the main targets for forensic DNA analysis. These repeat sequences are classified into minisatellites (or VNTRs) and microsatellites (or STRs). In this brief review, we discuss the historical and current forensic applications of such tandem repeats.     

Improved specificity of Y-STR typing in DNA mixture samples

Y-STR loci are beneficial for the analysis of forensic samples especially in sexual assault cases or other situations where mixtures of male and female cells are present. However, the amplification of Y-chromosomal STRs is also known to result in the formation of artefactual amplification products, mainly due to insufficient PCR specificity. This is a major drawback of the method, as the sensitivity as well as the correct Y-STR interpretation are affected. In our study, the addition of a PCR enhancer to the reaction master-mix resulted in a significant increase of specificity of Y-STR typing. This was clearly demonstrated by a loss of artefactual signal with increasing enhancer concentration, while the peak heights of the Y-STR alleles were not significantly affected by the enhancer. Mixtures of up to 1:500 (200 pg male and 100 ng female DNA) gave correct Y-STR profiles when the PCR enhancer was added to the reaction, while artefactual amplification succeeded over Y-specific amplification when no PCR enhancer was present.     

Sequential multiplex amplification (SMA) of genetic loci: A method for recovering template DNA for subsequent analyses of additional loci

A method called Sequential Multiplex Amplification (SMA) has been developed whereby a limited amount of DNA extracted from a sample can be reutilized for several single polymerase chain reaction (PCR) amplifications. The method involves recovery of genomic template DNA by microfiltration of PCR-amplified samples. Up to 5 different loci have been typed, each in a single system PCR-based assay, beginning with a test quantity of 5 ng template DNA. Genotypes of the DNA donors were compared with those obtained from individual amplifications and shown to be identical. This could be a useful technique for typing a number of loci from a limited amount of DNa and to recover template DNA from samples previously subjected to PCR. Obviously, when small quantities of template DNA are available, this technique can prove quite useful.     

DNA typing in wildlife crime: recent developments in species identification

Species identification has become a tool in the investigation of acts of alleged wildlife crimes. This review details the steps required in DNA testing in wildlife crime investigations and highlights recent developments where not only can individual species be identified within a mixture of species but multiple species can be identified simultaneously. ‘What species is this?’ is a question asked frequently in wildlife crime investigations. Depending on the material being examined, DNA analysis may offer the best opportunity to answer this question. Species testing requires the comparison of the DNA type from the unknown sample to DNA types on a database. The areas of DNA tested are on the mitochondria and include predominantly the cytochrome b gene and the cytochrome oxidase I gene. Standard analysis requires the sequencing of part of one of these genes and comparing the sequence to that held on a repository of DNA sequences such as the GenBank database. Much of the DNA sequence of either of these two genes is conserved with only parts being variable. A recent development is to target areas of those sequences that are specific to a species; this can increase the sensitivity of the test with no loss of specificity. The benefit of targeting species specific sequences is that within a mixture of two of more species, the individual species within the mixture can be identified. This identification would not be possible using standard sequencing. These new developments can lead to a greater number of samples being tested in alleged wildlife crimes.     

Origin of DNA in human serum and usefulness of serum as a material for DNA typing

The aims of this study were to clarify the origin of DNA in human serum and to investigate whether serum is a material available for DNA typing in routine forensic practice. Blood was donated from 10 healthy adult volunteers and stored for up to 8 days, at 4 degrees C and at room temperature. The serum DNA concentration at zero time was in the range of 5.6 to 21.8 ng/ml with a mean of 12.2+/-1.6 ng/ml. The concentrations increased with storage time. On agarose gel electrophoresis, all serum samples showed ladder patterns and the size of each band was an integer multiple of approximately 180 bp considered to be characteristic of apoptosis. DNA typing from DNA released by apoptosis was possible. Exact DNA typing of D1S80, HLA DQA1, PM, CSF1PO, TPOX, TH01 and vWA was possible for each sample. These results indicate that serum contains fragmented DNA derived from apoptosis of leukocytes, especially neutrophils, and that fragmented DNA is an appropriate material for DNA typing.     

Identification in blood stains through DNA typing with C4 and HLA-DR probes

Summary A restriction fragment length polymorphism analysis using double digestion of DNA preparations with XbaI and BglII restriction enzymes and hybridization with C4 and HLA-DR probes is described. The typing conditions selected reveal extensive individual variation in both C4 and DR gene regions. In our panel of 46 unrelated individuals, 37 different phenotypic patterns were recognized when both probes were used, and preliminary discriminative power values of 0.865 and 0.914 were calculated for C4 and DR, respectively. The probability of a chance match using both systems is probably about 1.5 · 10^–2.The potential of this method for individual identification of blood stains was demonstrated on DNA prepared from 6-month-old dried blood stains from seven panel individuals. The seven individuals were all identified when comparing stain DNA patterns with panel control patterns. No RFLP pattern changes were observed following storage of blood stains. Based on these experiments with C4 and DR DNA typing under laboratory conditions, it is concluded that DNA typing with such probes may become a powerful tool in future stain identification analyses.     

Mitochondrial DNA extraction and typing from isolated dentin-experimental evaluation in a Korean population

This study reports mtDNA polymorphisms in both hypervariable segments HV1 and HV2 of the non coding D-loop region from 60 unrelated Koreans. In contrast to two previous Korean data base studies, mtDNA was extracted separately from pulp tissue and root dentin of teeth obtained from dentists. Dentin turned out to be a reliable source of mitochondrial DNA. This can be of practical importance in forensic identification case work after a long post-mortem interval since pulp decomposes rapidly. The extraction method is explained in detail. The mtDNA polymorphisms obtained from 60 teeth of unrelated Koreans were compared with the already existing Korean data base. Received: 27 October 1997 / Received in revised form: 2 Feburary 1998     

Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination

Validation studies were carried out with the commercially available HLA typing kit using a PCR-SPP (sequence-specific primer and probe) technique. This technique has made it possible to type class I (HLA-A and -B) and class II (HLA-DRB1 and -DQB1) alleles at low-resolution level with total 10 ng of template DNA, in addition to amplify directly from various forms of blood samples without DNA isolation procedure. Experimental examinations with bloodstains smeared on cotton cloth that were a week to 3 months old, bloodstains on gauze stored for 18 years, and buccal cells revealed that this HLA-SPP typing kit is a sensitive and reliable method for forensic investigations.