Global survey on evaluative reporting on DNA evidence with regard to activity-level propositions.

For many criminal cases, the source of who deposited the DNA is not what the prosecutor and the defense are trying to dispute. In court, the question may be how the DNA was deposited at the crime scene rather than who the DNA came from. Although laboratories in many countries have begun to evaluate DNA evidence given formal activity-level propositions (ALPs), it is unknown how much other forensic practitioners know and what they think about activity-level evaluative reporting (ALR). To collect this information, a survey with 21 questions was submitted to international forensic science organizations across Europe, Australia, South America, Canada, Asia, and Africa. The survey combined open-ended and multiple-choice questions and received 162 responses. Responses revealed a wide range of knowledge on the topic. Overall, most respondents were somewhat knowledgeable about ALR, ALP, and current practices in court and expressed their support of the concept. A majority of participants identified gaps and obstacles regarding ALR they would like to see addressed. Examples include (1) need for more education/training at all stakeholder levels, (2) need for more DNA evidence-related data under realistic case scenarios, (3) need to internally implement and validate a formalized and objective approach for reporting, and (4) in some countries the need to achieve court admissibility. This global survey gathered the current concerns of forensic DNA practitioners and outlined several operational concerns. The information can be used to advance the implementation of ALR in laboratories and court testimony worldwide.

A global snapshot of current opinions of next-generation sequencing technologies usage in forensics.

The future of forensic DNA testing is being shaped by the research and usage of next-generation systems, which have increased the multiplexing capabilities of the field and the type and amount of genetic data that can be utilized for investigations. The NGS adoption for casework has been slow, albeit the plethora of data that has been published. This study evaluated the current opinions on sequencing in forensics. A 20-question online-survey focusing on NGS knowledge, training, and usage was distributed to 6001 forensic DNA researchers and practitioners worldwide. A total of 367 responses were obtained from all continents (North/South America (69.8%), Europe (21.2%), Asia (5.5%), Oceania (2.5%), and Africa (1%)). The respondents consisted of 50% practitioners, 31% researchers, and 19% both. Of these, 38% already own a next-gen sequencing instrument, and 13% are planning to purchase one. Overall, there exists an extensive knowledge on next-gen sequencing within the forensic community, including among laboratories that have not yet implemented this high-throughput technology in their workflows. Current usage focuses primarily on SNP analysis for investigative leads and mitochondrial DNA analysis while future applications included both STR and SNP testing applied to general casework. The major overall concerns respondents have for implementing a sequencing instrument include limited funding, staffing, lack of time, and the cost-effectiveness of providing this service. Specific technical concerns that the respondents had are the lack of training, statistical applications, bioinformatics support, and of rigorous guidelines and recommendations. Most of the respondents do believe there will be a technology shift from using CE only to the use of NGS on casework in 5-10 years. In addition, around 66% of respondents believe that it is moderately to very likely that the court will accept sequencing analysis. Sixteen percent fell in the middle, and the remaining 15% believe it is more unlikely, with 3% of respondents believing it is very unlikely. In conclusion, this work outlines current analytical challenges experienced by the global forensic DNA community and addresses different strategies for the implementation of next-gen sequencing technologies in casework.

American forensic DNA practitioners' opinion on activity level evaluative reporting.

The technical advancements made in DNA profiling now allow for very low DNA amounts to be analyzed. Accordingly, the argument often made in criminal courts is not who the DNA belongs to but rather how it was deposited. Despite the complexity of the relevant DNA transfer, persistence, prevalence, and recovery issues, forensic laboratories in some European countries have used evaluative reports with activity level propositions, while this is not current practice in the United States. The purpose of this study was to gain an overview of the opinions about activity level reporting (ALR) held by forensic biologists in the United States. A seventeen-question survey was distributed to members of the American Society of Crime Laboratory Directors and U.S. members of the International Society for Forensic Genetics. The survey included multiple-choice and open-response questions and received 54 responses. The majority of responses expressed moderate support of ALR. Participants mentioned six major concerns to be addressed prior to implementing ALR in the United States: (1) effect of number of variables involved; (2) need of education for practitioners/legal system; (3) inadequate number of activity studies with realistic scenarios; (4) difficulty of achieving admissibility in court; (5) need for standardized approaches/guidelines; and (6) requisite shift in perspective as to the validity of ALR. Overall, this small segment of U.S. forensic DNA practitioners appear to be willing to implement ALR once these concerns are fully addressed and resolved. As a follow-up, it would be worthwhile exploring these and other questions with a larger group and also other disciplines.

Retrofitting massively parallel sequencing (MPS) for HLA-DQA1 and polymarker (PM) in forensic casework.

Genotype profiling has played a major role in forensics for decades. The technology for detection and discrimination has advanced substantially, from serology to DNA sequence analysis. Currently, there may be situations where there is a need for re-analysis of forensic DNA data that was produced using methodology that is no longer available. An example of this is the allele-specific oligonucleotide hybridization assays used in the 1990s. In the study presented herein, we have developed a multiplex system combining PCR and massively parallel sequencing (MPS) technologies to identify DNA polymorphisms. Our results are consistent with those found in the widely utilized AmpliType PM + DQA1 Amplification and Typing Kit originally marketed by Perkin Elmer. During the course of our studies, it became apparent that paralogous genes for two of the loci, GYPA and HBG2 (formerly HBGG), could have confounded the interpretation of the original assays, and we describe the technical solutions we developed to overcome ambiguity in genotype assignment. This study results in a novel resource enabling the re-analysis of DNA profiling results produced decades past using current day technology.

Population genetic data of 74 microhaplotypes in four major U.S. population groups.

Microhaplotypes (microhaps or MHs) are novel forensically relevant genetic markers that demand large and appropriate allele frequency datasets for their implementation in casework. In this study we report on the allele frequency data of 74 microhap loci (230 SNPs) included in a newly developed 74-plex assay. The panel was tested on the Ion S5 system on a total of 347 samples from four main U.S. population groups of African, European, East Asian and Southwest Hispanic descent. Overall, frequencies of individual alleles at each locus varied considerably among the different population groups. An increase in the average value of gene diversity was also observed as the number of SNPs per locus increased. Most microhap markers showed no significant deviation from Hardy-Weinberg ratios within any of the individual population samples displaying an average power of discrimination between 0.74 and 0.81 and an average probability of exclusion between 0.32 and 0.39. Moreover, the four population groups had no clear genetic affinities with the exception of U.S. European and U.S. Southwest Hispanic populations, which showed the lowest FST value. STRUCTURE and principal component analyses (PCA) analysis resulted in effective clustering of the four populations with the U.S. European and Southwest Hispanic showing some overlap. These results support the potential use of this sequence-based 74plex-microhaplotype assay for ancestry inference in addition to previously reported human identification and mixture deconvolution capabilities.

A sequence-based 74plex microhaplotype assay for analysis of forensic DNA mixtures.

Microhaplotypes are emerging biomarkers for forensic applications. In this study, a sequence-based multiplex assay of 74 microhaplotypes (230 SNPs) was developed on the Ion Torrent S5™ (Thermo Fisher Scientific) system and the potential for its application to mixture deconvolution was explored. The 74 loci are distributed across the autosomal human genome and have Ae (i.e., effective number of alleles) values ranging from 1.307 to 6.010 (median = 2.706) and In (i.e., informativeness) values ranging from 0.096 to 0.660 (median = 0.251); the amplicon sizes range between 157 and 325 bp. The typing performance of the panel was evaluated on a series of in-silico two to five-person DNA mixtures and results were compared to fragment and sequence-based STRs. The 74plex-locus assay was found sensitive down to 0.05 ng of input DNA and effective for the analysis of mixtures at different contributor ratios and input DNA amounts. As expected, none or very partial minor CE-STR profile(s) were reported for highly imbalanced two-person and high-order DNA mixtures while sequencing of STRs enabled the detection of more individual minor alleles. For microhaplotypes, a full minor profile was detected down to a 20:1 ratio at 10 ng and minimal allele dropout at 1 ng of input DNA. A higher rate of allele dropout from the minor donor(s) was reported at 1 ng than 10 ng for three-person mixtures while for four- and five-person mixtures, the same number of dropouts was observed for almost all minor donors. Overall this microhaplotype panel is a powerful tool that can complement and enhance size- and sequence-based STR analysis of forensic DNA mixtures.

Forensic molecular biomarkers for mixture analysis.

The deconvolution of DNA mixtures has gathered the attention of forensic DNA scientists for over two decades. To enhance mixture deconvolution capabilities, a new generation of sensitive DNA-typing approaches has been recently proposed. In this review, we describe novel, forensically relevant multi-SNP loci (i.e., microhaplotypes or microhaps), compound markers (i.e., DIP-STRs, SNP-STRs and DIP-SNPs) and lineage markers (i.e., rapidly mutating Y chromosome STRs) that improve the deconvolution of two and more than two-person mixtures typed using conventional STR, binary and non-binary loci. We explore the features and applications of these emerging molecular biomarkers with respect to their ability to forensically detect same-or-opposite sex donors. Finally, we discuss the impact of initial massively parallel sequencing (MPS) investigations of STR, microhaplotype and SNP/indel assays for DNA mixture profiling.

Correction to: Mixture deconvolution by massively parallel sequencing of microhaplotypes.

The original version of this article contained an author name error. In this article, Katrina Madella has been corrected to Katrina Maddela.

Mixture deconvolution by massively parallel sequencing of microhaplotypes.

Short tandem repeat polymorphisms (STRs) are the standard markers for forensic human identification. STRs are highly polymorphic loci analyzed using a direct PCR-to-CE (capillary electrophoresis) approach. However, STRs have limitations particularly when dealing with complex mixtures. These include slippage of the polymerase during amplification causing stutter fragments that can be indistinguishable from minor contributor alleles, preferential amplification of shorter alleles, and limited number of loci that can be effectively co-amplified with CE. Massively parallel sequencing (MPS), by enabling a higher level of multiplexing and actual sequencing of the DNA, provides forensic practitioners an increased power of discrimination offered by the sequence of STR alleles and access to new sequence-based markers. Microhaplotypes (i.e., microhaps or MHs) are emerging multi-allelic loci of two or more SNPs within < 300 bp that are highly polymorphic, have alleles all of the same length, and do not generate stutter fragments. The growing number of loci described in the literature along with initial mixture investigations supports the potential for microhaps to aid in mixture interpretation and the purpose of this study was to demonstrate that practically. A panel of 36 microhaplotypes, selected from a set of over 130 loci, was tested with the Ion S5™ MPS platform (Thermo Fisher Scientific) on single-source samples, synthetic two-to-six person mixtures at different concentrations/contributor ratios, and on crime scene-like samples. The panel was tested both in multiplex with STRs and SNPs and individually. The analysis of single-source samples showed that the allele coverage ratio across all loci was 0.88 ± 0.08 which is in line with the peak height ratio of STR alleles in CE. In mixture studies, results showed that the input DNA can be much higher than with conventional CE, without the risk of oversaturating the detection system, enabling an increased sensitivity for the minor contributor in imbalanced mixtures with abundant amounts of DNA. Furthermore, the absence of stutter fragments simplifies the interpretation. On casework-like samples, MPS of MHs enabled the detection of a higher number of alleles from minor donors than MPS and CE of STRs. These results demonstrated that MPS of microhaplotypes can complement STRs and enhance human identification practices when dealing with complex imbalanced mixtures.

Microhaplotypes in forensic genetics.

Microhaplotype loci (microhaps, MHs) are a novel type of molecular marker of less than 300 nucleotides, defined by two or more closely linked SNPs associated in multiple allelic combinations. The value of these markers is enhanced by massively parallel sequencing (MPS), which allows the sequencing of both parental haplotypes at each of the many multiplexed loci. This review describes the features of these multi-SNP markers and documents their value in forensic genetics, focusing on individualization, biogeographic ancestry inference, and mixture deconvolution. Foreseeable applications also include missing person identification, relationship testing, and medical diagnostic applications. The technique is not restricted to humans.

Application of DIP-STRs to sexual/physical assault investigations: Eight case reports.

DIP-STRs are compound markers formed by a deletion/insertion polymorphism linked to a microsatellite. They enable the deconvolution of unbalanced DNA mixtures from two individuals, up to 1000 fold excess of one contributor. In practice, this novel tool allows to test for the presence of a DNA of interest in traces appearing not useful because of the masking effect of the major DNA contributor. Thus far two sets of DIP-STRs have been published: the first set was described as proof-of-principle, while the second set was specifically developed for forensic applications. Here, we report on the first use of these markers in casework to show advantages and limitations in real examples. Traces, suggestive of containing unbalanced DNA mixtures (beyond standard STR mixture resolution), were selected from eight cases submitted to the Forensic Genetics Unit of the University Center of Legal Medicine of Lausanne-Geneva. Using 18 validated DIP-STRs, two to ten markers were selected for each case. A minor DNA contributor - undetected using conventional STRs - was detected for the trace samples of six cases. DIP-STR results contributed to each case, either by complementing Y-STRs results or by producing novel investigative leads. This was especially true with same sex unbalanced DNA mixtures, female minor/male major unbalanced DNA mixtures or when the source of the DNA mixture was said to come either from the suspect and the female complainant or from his brother and the female complainant. Interestingly, these markers were found to be more sensitive and specific than previously known. Positive results were obtained at 16,000-fold excess of major DNA using few picograms of input DNA, as well as from traces collected several months after the alleged offence. Likelihood ratios assigned to measure the strength of DIP-STRs' DNA evidence were modest (10), when accounted by only two DIP-STRs, and high (106) when determined by six markers. In some cases the detection of extra alleles from additional minor DNA contributors or because of extremely unbalanced DNA ratios, limited the interpretation of the results. In conclusion, the DIP-STRs often provide additional value to the analysis of traces that cannot be exploited by the use of standard methods.

Sensitive DIP-STR markers for the analysis of unbalanced mixtures from "touch" DNA samples.

Casework samples collected for forensic DNA analysis can produce genomic mixtures in which the DNA of the alleged offender is masked by high quantities of DNA coming from the victim. DIP-STRs are novel genetic markers specifically developed to enable the target analysis of a DNA of interest in the presence of exceeding quantities of a second DNA (up to 1000-fold). The genotyping system, which is based on allele-specific amplifications of haplotypes formed by a deletion/insertion polymorphism (DIP) and a short tandem repeat (STR), combines the capacity of targeting the DNA of an individual with a strong identification power. Finally, DIP-STRs are autosomal markers therefore they can be applied to any combination of major and minor DNA. In this study we aimed to assess the ability of DIP-STRs to detect the minor contributor on challenging "touch" DNA samples simulated with representative crime-associated substrates and to compare their performance to commonly used male-specific markers (Y-STRs). As part of a comprehensive study on the relative DNA contribution of two persons handling the same object, we selected 71 unbalanced contact traces of which 14 comprised a male minor DNA contributor mixed to a female major DNA contributor. Using a set of six DIP-STRs, one to four markers were found to be informative for the minor DNA detection across traces. When compared to Y-STRs (14 traces), the DIP-STRs showed similar sensitivity in detecting the minor DNA across substrate materials with a similar occurrence of allele drop-out. Conversely, because of the sex combination of the two users of the object, 57 remaining traces could only be investigated by DIP-STRs. Of these, 30 minor DNA contributors could be detected by all informative markers while 12 traces showed events of allele drop-out. Finally, 15 traces showed no amplification of the minor DNA. These last 15 samples were mostly characterized by a combination of short handling time of the object, low DNA recovery and/or one single informative DIP-STR. In conclusion, the DIP-STRs represent alternative markers to help solving unbalanced two-source DNA mixtures, and also those produced from contact stains. These markers, in addition to a novel set of 10 DIP-STRs specifically developed according to forensic technical standards, will offer a valuable tool complementary to Y-STR markers.

A novel set of DIP-STR markers for improved analysis of challenging DNA mixtures.

Currently available molecular biology tools allow forensic scientists to characterize DNA evidence found at crime scenes for a large variety of samples, including those of limited quantity and quality, and achieve high levels of individualization. Yet, standard forensic markers provide limited or no results when applied to mixed DNA samples where the contributors are present in very different proportions (unbalanced DNA mixtures). This becomes an issue mostly for the analysis of trace samples collected on the victim or from touched objects. To this end, we recently proposed an innovative type of genetic marker, named DIP-STR that relies on pairing deletion/insertion polymorphisms (DIP) with standard short tandem repeats (STR). This novel compound marker allows detection of the minor DNA contributor in a DNA mixture of any gender and cellular origin with unprecedented resolution (beyond a DNA ratio of 1:1000). To provide a novel analytical tool useful in practice to common forensic laboratories, this article describes the first set of 10 DIP-STR markers selected according to forensic technical standards. The novel DIP-STR regions are short (between 146 and 271 bp), include only highly polymorphic tri-, tetra- and pentanucleotide tandem repeats and are located on different chromosomes or chromosomal arms to provide statistically independent results. This novel set of DIP-STR can target the amplification of 0.03-0.1 ng of DNA when mixed with a 1000-fold excess of major DNA. DIP-STR relative allele frequencies are estimated based on a survey of 103 Swiss individuals. Finally, this study provides an estimate of the occurrence of informative alleles and a calculation of the corresponding random match probability of the detected minor DIP-STR genotype assessed across 10,506 pairwise conceptual mixtures.

Completion of a worldwide reference panel of samples for an ancestry informative Indel assay.

The use of ancestry informative markers (AIMs) in forensic analysis is of considerable utility since ancestry inference can progress an investigation when no identification has been made of DNA from the crime-scene. Short-amplicon markers, including insertion deletion polymorphisms, are particularly useful in forensic analysis due to their mutational stability, capacity to amplify degraded samples and straightforward amplification technique. In this study we report the completion of H952 HGDP-CEPH panel genotyping with a set of 46 AIM-Indels. The study adds Central South Asian and Middle Eastern population data, allowing a comparison of patterns of variation in Eurasia for these markers, in order to enhance their use in forensic analyses, particularly when combined with sets of ancestry informative SNPs. Ancestry analysis using principal component analysis and Bayesian methods indicates that a proportion of classification error occurs with European-Middle East population comparisons, but the 46 AIM-Indels have the capability to differentiate six major population groups when European-Central South Asian comparisons are made. These findings have relevance for forensic ancestry analyses in countries where South Asians form much of the demographic profile, including the UK, USA and South Africa. A novel third allele detected in MID-548 was characterized - despite a low frequency in the HGDP-CEPH panel samples, it appears confined to Central South Asian populations, increasing the ability to differentiate this population group. The H952 data set was implemented in a new open access SPSmart frequency browser - forInDel: Forensic Indel browser.