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Interlaboratory studies are a type of collaborative exercise in which many laboratories are presented with the same set of data to interpret, and the results they produce are examined to get a "big picture" view of the effectiveness and accuracy of analytical protocols used across participating laboratories. In 2005 and again in 2013, the Applied Genetics Group of the National Institute of Standards and Technology (NIST) conducted interlaboratory studies involving DNA mixture interpretation. In the 2005 NIST MIX05 study, 69 laboratories interpreted data in the form of electropherograms of two-person DNA mixtures representing four different mock sexual assault cases with different contributor ratios. In the 2013 NIST MIX13 study,108 laboratories interpreted electropherogram data for five different case scenarios involving two, three, or four contributors, with some of the contributors potentially related. This paper describes the design of these studies, the variations observed among laboratory results, and lessons learned.

MIX13 was an interlaboratory exercise directed by NIST in 2013. The goal of the exercise was to evaluate the general state of interpretation methods in use at the time across the forensic community within the US and Canada and to measure the consistency in mixture interpretation. The findings were that there was a large variation in analysts’ interpretations between and within laboratories. Within this work, we sought to evaluate the same mock mixture cases analyzed in MIX13 but with a more current view of the state-of-the-science. Each of the five cases were analyzed using the Identifiler™ multiplex and interpreted with the combined probability of inclusion, CPI, and four different modern probabilistic genotyping systems. Cases 1–4 can be interpreted without difficulty by any of the four PG systems examined. Cases 1 and 4 could also be interpreted successfully with the CPI by assuming two donors. Cases 2 and 3 cannot be interpreted successfully with the CPI because of potential of allele dropout. Case 3 demonstrated the need to consider relevant background information before interpretation of the profile. This case does not show that there is some barrier to interpretation caused by relatedness beyond the increased allelic overlap that can occur. Had this profile been of better template it might have been interpreted using the CPI despite the (potential) relatedness of contributors. Case 5 suffers from over-engineering. It is unclear whether reference 5C, a non-donor, can be excluded by manual methods. Inclusion of reference 5C should be termed an adventitious match not a false inclusion. Beyond this statement this case does not contribute to the interlaboratory study of analyst/laboratory interpretation method performance, instead, it explores the limits of DNA analysis. Taken collectively the analysis of these five cases demonstrates the benefits of changing from CPI to a PG system.

Current approaches to mixture deconvolution of complex biological samples at times do not have the capability to resolve component contributors in DNA evidence. Additional short tandem repeat (STR) loci were sought that may improve the forensic genetic analysis of mixtures. This study presents exploratory data of a multiplex comprised of 73 highly polymorphic STRs (referred herein as the 73Plex) that were selected because of their high diversity due to sequence variation. These STRs (or a subset of them) may be considered as candidates that may augment current core markers capabilities for DNA mixture deconvolution. Population genetics analyses were performed for each locus using DNA samples from 451 individuals comprising three U.S. populations. Sequence-based heterozygosities ranged from 72% to 98%, where only two loci (D10A97 and D6A7) fell below 80%. Mixture deconvolution capabilities for two-person mixtures were assessed based on complete allele resolution per locus (i.e., four alleles observed) of pairwise mixtures using in silico methods. A subset of 20 highly informative loci (referred herein as the 20Plex) from the 73Plex was compared to the 20 CODIS core loci on all population samples with full DNA profiles for both panels (i.e., no locus dropout; n = 443). Based on proportion of loci displaying four alleles, the 20Plex outperformed the CODIS core loci with increases of 82.6% and 89.3% using length-based and sequence-based alleles, respectively. A combination of 17 STR from the 20Plex and 3 CODIS loci gave the highest capacity for resolving allelic components per locus. These data illustrate the increased value of utilizing sequenced-based alleles of additional STR loci. Furthermore, there are a number of candidate STR loci that could notably augment the current core STR loci and enhance mixture interpretation capabilities.

Damage to the DNA molecule can occur through exposure to environmental conditions such as ultraviolet light, heat and humidity. Forensic samples are particularly prone to such damage due to their prolonged exposure after deposition at crime scenes or mass disasters. Current methods for typing such samples rely heavily on the intact DNA template, and can be adversely affected by damage that is present. Proposed solutions center around increased access to the smaller remaining fragments and/or increased sensitivity. However, all rely on the polymerase chain reaction to copy the starting material; the required polymerase can be impeded by certain types of damage such as dimer-formation after ultraviolet light exposure, resulting in stochastic effects that can complicate interpretation. In vitro repair of such damage offers the ability to generate high quality profiles using traditional methods without changes to the current amplification reagents or conditions. Typically, repair reactions required large quantities of starting material and a separate repair reaction. Forensic samples, however, usually consist of small quantities, and quality control measures necessitate laboratory procedures that minimize sample manipulation. Here, an optimized protocol for forensic application of the PreCR™ Repair Mix to current typing methods is demonstrated for samples damaged by ultraviolet light exposure.

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 amplicon sizes. This study constructed a partial miniSGM panel and investigated the performance of four miniSTR loci, D2S1338, D16S539, D18S51 and FGA, in three ethnic populations residing in Singapore. The suitability of the miniSTR primers for Singapore populations was assessed for loci D16S539, D18S51 and FGA.

The International Commission on Missing Persons (ICMP) conducts high throughput STR profiling on degraded skeletal remains, primarily recovered from mass graves relating to conflicts from 1992 to 1999 in the former Yugoslavia. To date, over 11,000 individuals have been identified through comparison of bone profiles to a large database of profiles from family members of the missing. To increase success rates in STR recovery, three short amplicon STR multiplexes (a 7-plex, a 6-plex, and a 5-plex) have been devised and implemented. These target loci from large commercial multiplexes, with an average decrease in amplicon size of 144 bp. The ICMP “miniplexes” have proven to provide substantially greater recovery of DNA data from a certain subset of difficult samples. However, the circumstances under which miniplexes provide additional data are restricted, and their advantages do not outweigh those of large commercial multiplexes for a majority of cases. The miniplexes, however, also have a very powerful use in DNA testing to support large scale reassociation of commingled, partial skeletons recovered from secondary mass graves.

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.

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.

Recently, it has been recognized that accessing information in the mtDNA coding region can provide additional forensic discrimination with respect to the standard typing of the D-loop region, augmenting the sometimes rather limited forensic power of mtDNA testing. Here, we discuss considerations relating to maximally effective approaches for recovering additional discrimination in the coding region, bearing in mind that (1) DNA quality and quantity in typical mtDNA casework usually restrict the amount of additional sequence that can be obtained, and (2) the need for additional discrimination primarily arises when common HV1/HV2 types are encountered. Most investigators have sought additional discrimination by sequencing short segments of coding region that are thought to be particularly variable. Unfortunately, efforts in this regard have generally failed to appreciate that most variation in the coding region is redundant with information already present in HV1/HV2 and have therefore overvalued the potential of this approach for providing additional discrimination. An alternative single nucleotide polymorphism-based approach [Int J Legal Med 118:137-146, 2004] has been to identify specific bases that provide resolution in specific common HV1/HV2 types (and related sequences). We investigate several highly relevant data sets wherein the latter approach performs appreciably better than sequencing selected short portions of the coding region. This is true even when only synonymous variation is targeted to minimize the potential for problems arising from discovery of mutations that have reportedly been related to disease.

A number of studies have demonstrated that successful analysis of degraded DNA specimens from mass disasters or forensic evidence improves with smaller sized polymerase chain reaction (PCR) products. We have scanned the literature for new STR loci, unlinked from the CODIS markers, which can generate amplicons less than 125 bp in size and would therefore be helpful in testing degraded DNA samples. New PCR primers were designed and tested for the STR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434, and D22S1045, arranged into two miniSTR triplexes. All loci show a moderate degree of polymorphism among 474 U.S. population samples tested and were reliable and sensitive to at least 100 pg of DNA template under controlled laboratory conditions and pristine DNA samples. The utility of these new loci were confirmed in comparing the success of the miniSTR assays for typing degraded bone samples while partial profiles were observed with the majority of the samples using a commercial STR kit.

The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) can help resolve individuals with an identi- cal HV1/HV2 mitotype. A set of 11 SNPs selected for dis- tinguishing individuals of the most common Caucasian HV1/HV2 mitotype were incorporated in an allele spe- cific primer extension assay. The assay was optimized for multiplex detection of SNPs at positions 3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477 and 14470 in the mtGenome. Primers were designed to allow for simultaneous PCR amplification of 11 unique regions in the mtGenome and subsequent primer extension. By enzymatically incorporating fluorescently labeled dideoxy- nucleotides (ddNTPs) onto the 3' end of the extension primer, detection can be accomplished with a capillary- based electrophoresis (CE) platform common in most forensic laboratories. The electrophoretic mobility for the extension primers was compared in denaturing POP4 and POP6 CE running buffers. Empirical adjustment of exten- sion primer concentrations resulted in even signal inten- sity for the 11 loci probed. We demonstrate that the assay performs well for heteroplasmy and mixture detection, and for typical mtDNA casework samples with highly de- graded DNA.

Though allele frequency data for a variety of X chromosomal short tandem repeat (STR) markers in a range of populations have been reported, fewer studies of mutation rates in these same markers or populations are available. In order to address possible mismatches during kinship analysis due to mutation, a robust estimate of the rate of mutation must be established. Here, mutation rates in three US populations have been determined for a total of 15 markers (DXS6789, DXS9902, DXS7132, DXS7130, DXS6795, DXS10147, DXS8378, DXS7423, HPRTB, DXS101, DXS7424, GATA31E08, GATA172D05, GATA165B12, and DXS6803). Eighteen mutations over 20,625 meioses were observed, and the overall X STR mutation rate in this study was found to be 8.73 × 10(-4) (95 % CI, 5.2-13.8 × 10(-4)). A review of published mutation rate studies revealed similar findings in other global populations, and allowed the compilation of a combined dataset of 81,310 meioses which can be employed by the forensic community.

The forensic analysis of DNA is most often undertaken by the amplification of short tandem repeats (STR) using the polymerase chain reaction (PCR). DNA amplification can result in production of the target allele amplicon and a by-product called stutter. Stutter is the result of the miscopy of the target allele and is typically one repeat smaller. Stutter is traditionally described as a ratio of stutter and allele height; stutter ratio (SR). The challenge to DNA profile interpretation is most serious whenever stutter products are of a similar height to the minor allelic peaks in a mixed DNA profile. An accurate assignment of peaks and the prediction of their height is important when objectively interpreting forensic DNA profiles. The longest uninterrupted stretch (LUS) of tandem repeats within the allele has previously been shown to be a good predictor of stutter ratio. LUS is determined by sequencing a range of observed alleles at a locus. The locus D6S1043 is a relatively new locus to appear in commercial forensic DNA testing kits. To date however, there has been no comprehensive report of sequencing of this locus. In this work, we sequence a sample of D6S1043 alleles to determine LUS values and investigate allele repeat number and LUS as explanatory variables for SR.

The PowerPlex Y23 kit contains 23 Y-chromosomal loci including all 17 of the markers in the Yfiler Y-STR kit plus six additional markers: DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643. We have typed 1032 unrelated population samples from four self-declared US groups: African Americans, Asians, Hispanics, and Western European Caucasians. An analysis of the population genetic parameters and the improvement of adding additional Y-STR markers to the dataset are described.

The amplification of short tandem repeat (STR) markers throughout the human nuclear DNA genome are used to associate crime scene evidence to the perpetrator's profile in criminal investigations. For highly challenged or compromised materials such as stains exposed to the elements, skeletal remains from missing persons cases, or fragmented and degraded samples from mass disasters, obtaining a full STR profile may be difficult if not impossible. With the introduction of short amplicon STR or "miniSTR" typing, it is possible to obtain STR genetic information from highly challenged samples without the need to sequence the hypervariable regions of the mitochondrial DNA (mtDNA) genome. Non-Combined DNA Index System (CODIS) STR markers have been developed to obtain information beyond the core CODIS loci. This chapter will focus on the steps necessary to prepare and use one of the non-CODIS (NC) multiplexes, NC01 (Coble and Butler 2005), for analysis on capillary electrophoresis instrumentation.

Seventeen autosomal STRs were analyzed (D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, Penta D, and Penta E) in the Lebanese population. A total of 192 unrelated individuals were genotyped for the 15 autosomal STRs in the Promega PowerPlex 16 STR kit. An additional 275 unrelated individuals were genotyped for the Applied Biosystems AmpFlSTR Identifiler and SGM+STR kits. Allele frequencies for the shared CODIS 13 loci among the three STR kits tested were not significantly different among individuals within the Lebanese population. Forensic and population genetic parameters for the 17 loci were calculated. We also compared the allele frequencies from this population with other populations in the same geographic vicinity.

X chromosomal short tandem repeat (STR) loci can be used in combination with autosomal STRs to solve cases of complex kinship. In order to properly apply STR results to forensic cases, population databases must be established to which comparisons can be made and the rarity of profiles determined. Here, a population from Bosnia and Herzegovina was studied to determine the appropriate forensic efficiency parameters with 14 widely used X chromosomal STR loci.

To characterize the data produced using a modified amplification protocol for the AmpFlSTR Yfiler PCR Amplification Kit (Applied Biosystems) and explore the potential of Y-chromosomal short tandem repeat (Y-STR) recovery from severely degraded skeletal remains encountered at the Armed Forces DNA Identification Laboratory.Experiments were performed using two sets of Yfiler amplification parameters. One set of parameters reflected the manufacturer's recommendations. The second set of parameters included twice the recommended Taq concentration and 6 additional cycles. Recovery of authentic alleles and the incidence of drop-in alleles were assessed for 3 data sets: 8 different quantities of pristine DNA, 8 artificially-degraded samples, and 31 non-probative case samples.Samples tested with both protocols from all 3 data sets yielded twice as many authentic alleles under the modified parameters than under the standard parameters (62% vs 31%), with only a nominal associated increase in the occurrence of non-authentic alleles (1.36% of all alleles detected). When applied to a range of representative casework samples, the modified protocol leveraged 9 or more reproducible alleles from over half of the specimens tested.Reproducible and informative Y-STR profiles can be recovered from a broad range of degraded and inhibited skeletal remains extracts when a commercially available kit is employed under modified amplification parameters.

In order to promote mitochondrial DNA (mtDNA) testing in Sweden we have typed 296 Swedish males, which will serve as a Swedish mtDNA frequency database. The tested males were taken from seven geographically different regions representing the contemporary Swedish population. The complete mtDNA control region was typed and the Swedish population was shown to have high haplotype diversity with a random match probability of 0.5%. Almost 47% of the tested samples belonged to haplogroup H and further haplogroup comparison with worldwide populations clustered the Swedish mtDNA data together with other European populations. AMOVA analysis of the seven Swedish subregions displayed no significant maternal substructure in Sweden (F (ST) = 0.002). Our conclusion from this study is that the typed Swedish individuals serve as good representatives for a Swedish forensic mtDNA database. Some caution should, however, be taken for individuals from the northernmost part of Sweden (provinces of Norrbotten and Lapland) due to specific demographic conditions. Furthermore, our analysis of a small sample set of a Swedish Saami population confirmed earlier findings that the Swedish Saami population is an outlier among European populations.

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.

A number of studies have demonstrated that successful analysis of degraded DNA specimens from mass disasters or forensic evidence improves with smaller sized polymerase chain reaction (PCR) products. We have scanned the literature for new STR loci, unlinked from the CODIS markers, which can generate amplicons less than 125 bp in size and would therefore be helpful in testing degraded DNA samples. New PCR primers were designed and tested for the STR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434, and D22S1045, arranged into two miniSTR triplexes. All loci show a moderate degree of polymorphism among 474 U.S. population samples tested and were reliable and sensitive to at least 100 pg of DNA template under controlled laboratory conditions and pristine DNA samples. The utility of these new loci were confirmed in comparing the success of the miniSTR assays for typing degraded bone samples while partial profiles were observed with the majority of the samples using a commercial STR kit.

We performed a study on the forensic utility of allele-discriminatory quantitative real-time PCR (rtPCR) using Minor Groove Binder TaqMan probes, targeting the highly variable mitochondrial single nucleotide polymorphism 16519T/C. The apparent single-cycle PCR efficiency was virtually 100% for both 16519 alleles. The allele designations made by rtPCR were concordant with the results obtained in a previous study by sequencing analysis. In heteroplasmic samples, minor allele proportions down to 9% were unambiguously detected and quantified. The variation in allele proportion estimates was essentially the same within and between different rtPCR runs, and the differences between total copy number estimates found for rerun samples were comparable to those found with non-allele-discriminatory quantitative rtPCR assays.

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was designed for use in the standardization of forensic and paternity quality assurance procedures for fragment-based typing short tandem repeat (STR) alleles generated by the polymerase chain reaction (PCR). Certified genotypes of the 6 components A–F were assigned for 24 autosomal and 17 Y-STR markers plus Amelogenin using concordance testing between commercial kits. Selected Sanger sequencing characterization was performed for the alleles of 11 STR markers when only one PCR primer set was available for fragment-based typing. The goal is to characterize the remaining 30 STR loci in components A–C by Sanger sequencing methods for the STR repeat regions and adjacent flanking regions. Additional characterization of the SRM is intended to support the emerging interest in next-generation sequencing technologies for forensic typing applications. Sanger methods have detected underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. The sequenced regions include the commercial or known PCR binding sites commonly implemented in fragment-based typing.