A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing.

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.

DNA Commission of the International Society for Forensic Genetics: Recommendations on the validation of software programs performing biostatistical calculations for forensic genetics applications.

The use of biostatistical software programs to assist in data interpretation and calculate likelihood ratios is essential to forensic geneticists and part of the daily case work flow for both kinship and DNA identification laboratories. Previous recommendations issued by the DNA Commission of the International Society for Forensic Genetics (ISFG) covered the application of bio-statistical evaluations for STR typing results in identification and kinship cases, and this is now being expanded to provide best practices regarding validation and verification of the software required for these calculations. With larger multiplexes, more complex mixtures, and increasing requests for extended family testing, laboratories are relying more than ever on specific software solutions and sufficient validation, training and extensive documentation are of upmost importance. Here, we present recommendations for the minimum requirements to validate bio-statistical software to be used in forensic genetics. We distinguish between developmental validation and the responsibilities of the software developer or provider, and the internal validation studies to be performed by the end user. Recommendations for the software provider address, for example, the documentation of the underlying models used by the software, validation data expectations, version control, implementation and training support, as well as continuity and user notifications. For the internal validations the recommendations include: creating a validation plan, requirements for the range of samples to be tested, Standard Operating Procedure development, and internal laboratory training and education. To ensure that all laboratories have access to a wide range of samples for validation and training purposes the ISFG DNA commission encourages collaborative studies and public repositories of STR typing results.

D5S2500 is an ambiguously characterized STR: Identification and description of forensic microsatellites in the genomics age.

In the process of establishing short tandem repeat (STR) sequence variant nomenclature guidelines in anticipation of expanded forensic multiplexes for massively parallel sequencing (MPS), it was discovered that the STR D5S2500 has multiple positions and genomic characteristics reported. This ambiguity is because the marker named D5S2500 consists of two different microsatellites forming separate components in the capillary electrophoresis multiplexes of Qiagen's HDplex (Hilden, Germany) and AGCU ScienTech's non-CODIS STR 21plex (Wuxi, Jiangsu, China). This study outlines the genomic details used to identify each microsatellite and reveals the D5S2500 marker in HDplex has the correctly assigned STR name, while the D5S2500 marker in the AGCU 21plex, closely positioned a further 1643 nucleotides in the human reference sequence, is an unnamed microsatellite. The fact that the D5S2500 marker has existed as two distinct STR loci undetected for almost ten years, even with reported discordant genotypes for the standard control DNA, underlines the need for careful scrutiny of the genomic properties of forensic STRs, as they become adapted for sequence analysis with MPS systems. We make the recommendation that precise chromosome location data must be reported for any forensic marker under development but not in common use, so that the genomic characteristics of the locus are validated to the same level of accuracy as its allelic variation and forensic performance. To clearly differentiate each microsatellite, we propose the name D5S2800 be used to identify the Chromosome-5 STR in the AGCU 21plex.

The mitochondrial landscape of African Americans: An examination of more than 2500 control region haplotypes from 22 U.S. locations.

The mitochondrial DNA (mtDNA) control region (16024-576) was Sanger-sequenced for a total of 2563 self-identified African Americans, using automated processing techniques and data review standards exceeding guidelines for forensic applications. Genetic diversity ranged from 0.9952 to 0.9998 in 22 population samples from 20 different states. Haplogroups of African ancestry, found in 82.48% of individuals overall, were most concentrated in the Southeast U.S. and decreased to the north and west. West African and West Central African haplotypes were well-represented in the population samples, especially in the southern U.S. states, while East African haplogroups were observed in low-frequency clusters in a handful of locations across the country. East Asian, Native American, and West Eurasian admixture was present in 3.16%, 2.93%, and 11.43% of samples, respectively. While some geographic substructure was detected across the population samples as clines in admixture frequencies, 20 of the 22 population samples were found to be statistically indistinguishable by pairwise comparisons and AMOVA calculations. Datasets from Hawaii and Idaho, however, were clear outliers. Overall, these more than 2500 control region sequences represent the most comprehensive regional sampling of African American mtDNA diversity to date, and are suitable for use in a forensic mtDNA database. The population data are made available via EMPOP (www.empop.org) and GenBank.

Autosomal SNP typing of forensic samples with the GenPlex™ HID System: results of a collaborative study.

The GenPlex™ HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250-500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power.

Allele frequencies of six miniSTR loci of three ethnic populations in Singapore.

MiniSTR loci has demonstrated to be an effective approach to recover genetic information from degraded sample, due to the improved PCR efficiency of their reduced PCR product sizes. This study investigated the allele frequency of six miniSTR loci, D1S1677, D2S441, D4S2364, D10S1248, D14S1434 and D22S1045, in three Singapore populations. All loci showed a moderate degree of polymorphism with observed heterozygosity >0.6 for all three populations. The allele frequencies, forensic parameters and heterozygosity comparison with other CODIS STR in similar populations are presented.

Forensic application of the Affymetrix human mitochondrial resequencing array.

In the field of forensic DNA testing, sequencing regions of the mitochondrial genome is performed when insufficient genomic DNA is present for traditional autosomal short tandem repeat (STR) testing. Sequencing coding region polymorphisms in the mitochondrial genome can be useful for resolving individuals who have the identical HV1 and HV2 control region sequence. Various methods and strategies have been established to interrogate coding region polymorphisms. These range from SNP assays probing sites most likely to differentiate individuals based on their HV1/HV2 sequence to the use of mass spectrometry to pyrosequencing. Here we evaluate the potential of the Affymetrix GeneChip Mitochondrial Resequencing Array (version 2.0) for forensic applications.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

Naming the Dead - Confronting the Realities of Rapid Identification of Degraded Skeletal Remains.

The Armed Forces DNA Identification Laboratory (AFDIL) is one of the leading laboratories in the world for the processing of degraded skeletal remains. Extended efforts have been made to develop protocols and standards that will hold up to the intense scrutiny of both the scientific world and the U.S. legal system. Presented in this paper are the specifics of the in-house systems and procedures that have allowed AFDIL to streamline the processing of degraded skeletal remains and family references for mitochondrial DNA (mtDNA) analysis. These include the development of our in-house bioinformatics systems by which every package and sample that passes through the laboratory is tracked; protocols designed specifically for both questioned and reference samples; and the difficulties inherent in this type of organization. Two case studies presented involve one of ancient remains and one on the recent event of September 11, 2001. Finally, future directions available to both AFDIL and the DNA analysis community as a whole are discussed.

Increasing the forensic discrimination of mitochondrial DNA testing through analysis of the entire mitochondrial DNA genome.

The principal limitation in forensic mitochondrial DNA (mtDNA) testing is the low power of discrimination that is obtained when common "mtDNA types" are involved in a case. Currently, an "mtDNA type" refers to the sequence within hypervariable regions I and II (HV1/HV2) of the control region, approximately 610 bp. In Caucasians, the most common HV1/HV2 type is found in approximately 7% of the population and there are 12 additional types found at greater than approximately 0.5% (ignoring HV2 C-stretch polymorphism). We are performing large scale sequencing of the entire mtDNA genome (mtGenome), approximately 16,569 bp, of individuals who have common HV1/HV2 types. Of 31 individuals with the most common HV1/HV2 type, only 3 still match after mtGenome sequencing. Similar high discrimination is seen in other common HV1/HV2 types. The sites that discriminate the various common HV1/HV2 types are generally not those that are known to vary widely in more diverse population samples. This indicates that complete mtGenome sequencing of selected HV1/HV2 types may stand as the best way for identifying maximally useful single nucleotide polymorphism sites outside of the control region. Our strategy for identifying SNP sites is useful in resolving U.S. Caucasian, Hispanic, and African American mtDNAs is discussed. We also discuss the development of homogeneous fluorogenic polymerase chain reaction assays that target phenotypically neutral sites for practical use in casework.

Comparative identity and homogeneity testing of the mtDNA HV1 region using denaturing gradient gel electrophoresis.

A denaturing gradient gel electrophoresis (DGGE) assay has been developed for comparative identity and homogeneity testing of the mtDNA HV1 region. A total of 49 pairs of sequences, each pair differing by a single unique polymorphism, were tested to verify the reliability of the assay. Discrimination between all pairings was achieved as judged by the resolution of the mismatch-containing heteroduplexes from the fully base-paired homoduplexes. In all but two pairings, resolution of the fully base-paired homoduplexes was also obtained. Sequence pairs differing by multiple polymorphisms were also tested and resulted in a greater separation between the homo- and heteroduplexes. Additional information derived from the technique includes the identification of co-amplifying contaminating or heteroplasmic samples in the independent samples lanes. Thirteen heteroplasmic samples, six at positions distinct from those analyzed in the pairwise comparison study, were analyzed and the heteroplasmic positions identified unambiguously by sequencing the excised bands. The technique constitutes a conceptually simple, accurate, and inexpensive test for determining whether two sequences match within the mtDNA HV1 region, while providing a more definitive control for the identification of co-amplifying contaminating or heteroplasmic sequences than is presently available.