Genetic and linguistic correlation of the Kra-Dai-speaking groups in Thailand.

The Kra-Dai linguistic family includes Thai and Lao as well as a great number of languages spoken by ethnic minorities in Southeast Asia. In Thailand, a dozen of other Kra-Dai languages are spoken in addition to Thai, the national language. The genetic structure of the Kra-Dai-speaking populations in Thailand has been studied extensively using uniparentally inherited markers. To extend this line of genetic investigation, this study used 15 autosomal microsatellites of 500 individuals from 11 populations, belonging to nine Kra-Dai ethnicities, namely, the Kaleung, Phu Thai, Saek, Nyo, Lao Isan, Yuan, Black Tai, Phuan and Lue. These ethnolinguistic groups are dispersed in three different geographic regions of Thailand, that is, Northern, Northeastern and Central. The results show a very low average of pairwised F(st) (0.0099), as well as no population substructure based on STRUCTURE analysis, indicating genetic homogeneity within the Kra-Dai-speaking group, possibly owing to shared linguistic ancestry. The Mantel test, an analysis of molecular variance, and the approximate Bayesian computation procedure employed to evaluate potential factors for driving genetic diversity revealed that language is the predominant factor affecting genetic variations, whereas geography is not. The result of distance-based clustering analyses and spatial analysis of molecular variance revealed genetic distinctions of some populations, reflecting the effects of genetic drift and gene flow on allele frequency within populations, in concordance with the result of R-matrix regression. The genetic and linguistic affiliations of the contemporary Kra-Dai-speaking groups are consistent with each other despite certain deviation due to various evolutionary factors that may have occurred during their migrations and resettlements.

Allele frequencies for 15 autosomal STR loci and haplotype data for 17 Y-STR loci in a population from Belize.

Allele frequencies for 15 autosomal STR loci (N = 290) and haplotype data for 17 Y-STR loci (N = 157) were determined for an admixed population from Belize. There were no detectable departures from Hardy-Weinberg equilibrium expectations at any autosomal STR loci except for the D8S1179 locus (p = 0.002). The combined power of discrimination (PD) and combined power of exclusion (PE) were greater than 0.99999999 and 0.99999951, respectively. In addition, a total of 144 distinct Y-STR haplotypes were observed with 133 Y-STR haplotypes observed only once. The most common Y-STR haplotype was observed three times for two separate haplotypes. The various analyses of these forensically relevant STR loci showed that these markers are informative in the Belize population for forensic and parentage testing applications.

Validation of the PLEX-ID™ mass spectrometry mitochondrial DNA assay.

For very challenged biological samples, mitochondrial DNA (mtDNA) analysis can often provide results when the more traditional nuclear DNA markers fail. While reliable, the current method of mtDNA analysis by Sanger sequencing is expensive, labor-intensive, and time-consuming and is limited by its inability to quantify mixed samples. The Abbott PLEX-ID™ instrument, which enables analysis of mtDNA amplicons via electrospray ionization mass spectrometry (ESI-MS), produces comparable accuracy and sensitivity while offering a faster and less expensive alternative to Sanger sequencing. Unlike Sanger sequencing, this system is capable of quantifying DNA species and thus may be exploited for evaluating heteroplasmy and, possibly, mixture deconvolution. Validation studies of the PLEX-ID™ mtDNA assay confirmed that the instrument is highly sensitive and capable of yielding reproducible results. Samples commonly encountered in a forensic setting, as well as population samples, were typed correctly. The PLEX-ID™ mtDNA assay yields reliable results for single-source samples, which are the same sample types currently examined in forensic laboratories via Sanger sequencing, at a level that meets or exceeds that of the current method. While the instrument has the demonstrated capability to quantify mixed samples, the specific assay design for mtDNA analysis can be used only in a limited fashion to analyze mixtures due to the formation of chimeric mtDNA products.

First all-in-one diagnostic tool for DNA intelligence: genome-wide inference of biogeographic ancestry, appearance, relatedness, and sex with the Identitas v1 Forensic Chip.

When a forensic DNA sample cannot be associated directly with a previously genotyped reference sample by standard short tandem repeat profiling, the investigation required for identifying perpetrators, victims, or missing persons can be both costly and time consuming. Here, we describe the outcome of a collaborative study using the Identitas Version 1 (v1) Forensic Chip, the first commercially available all-in-one tool dedicated to the concept of developing intelligence leads based on DNA. The chip allows parallel interrogation of 201,173 genome-wide autosomal, X-chromosomal, Y-chromosomal, and mitochondrial single nucleotide polymorphisms for inference of biogeographic ancestry, appearance, relatedness, and sex. The first assessment of the chip's performance was carried out on 3,196 blinded DNA samples of varying quantities and qualities, covering a wide range of biogeographic origin and eye/hair coloration as well as variation in relatedness and sex. Overall, 95 % of the samples (N = 3,034) passed quality checks with an overall genotype call rate >90 % on variable numbers of available recorded trait information. Predictions of sex, direct match, and first to third degree relatedness were highly accurate. Chip-based predictions of biparental continental ancestry were on average ~94 % correct (further support provided by separately inferred patrilineal and matrilineal ancestry). Predictions of eye color were 85 % correct for brown and 70 % correct for blue eyes, and predictions of hair color were 72 % for brown, 63 % for blond, 58 % for black, and 48 % for red hair. From the 5 % of samples (N = 162) with <90 % call rate, 56 % yielded correct continental ancestry predictions while 7 % yielded sufficient genotypes to allow hair and eye color prediction. Our results demonstrate that the Identitas v1 Forensic Chip holds great promise for a wide range of applications including criminal investigations, missing person investigations, and for national security purposes.

Developing criteria and data to determine best options for expanding the core CODIS loci.

BACKGROUND: Recently, the Combined DNA Index System (CODIS) Core Loci Working Group established by the US Federal Bureau of Investigation (FBI) reviewed and recommended changes to the CODIS core loci. The Working Group identified 20 short tandem repeat (STR) loci (composed of the original CODIS core set loci (minus TPOX), four European recommended loci, PentaE, and DYS391) plus the Amelogenin marker as the new core set. Before selecting and finalizing the core loci, some evaluations are needed to provide guidance for the best options of core selection. METHOD: The performance of current and newly proposed CODIS core loci sets were evaluated with simplified analyses for adventitious hit rates in reasonably large datasets under single-source profile comparisons, mixture comparisons and kinship searches, and for international data sharing. Informativeness (for example, match probability, average kinship index (AKI)) and mutation rates of each locus were some of the criteria to consider for loci selection. However, the primary factor was performance with challenged forensic samples. RESULTS: The current battery of loci provided in already validated commercial kits meet the needs for single-source profile comparisons and international data sharing, even with relatively large databases. However, the 13 CODIS core loci are not sufficiently powerful for kinship analyses and searching potential contributors of mixtures in larger databases; 19 or more autosomal STR loci perform better. Y-chromosome STR (Y-STR) loci are very useful to trace paternal lineage, deconvolve female and male mixtures, and resolve inconsistencies with Amelogenin typing. The DYS391 locus is of little theoretical or practical use. Combining five or six Y-chromosome STR loci with existing autosomal STR loci can produce better performance than the same number of autosomal loci for kinship analysis and still yield a sufficiently low match probability for single-source profile comparisons. CONCLUSION: A more comprehensive study should be performed to provide the necessary information to decision makers and stakeholders about the construction of a new set of core loci for CODIS. Finally, selection of loci should be driven by the concept that the needs of casework should be supported by the processes of CODIS (or for that matter any forensic DNA database).

Variants observed for STR locus SE33: a concordance study.

Discordance of STR typing results can be expected between kits that employ different primers for amplification. The complex motif of the SE33 locus and its flanking regions can contribute to the degree of discordant results. Sequence-dependent conformational changes can manifest as length differences under certain electrophoretic conditions and/or use of different primers. The AmpFlSTR® NGM SElect™ PCR Amplification Kit (Life Technologies, Carlsbad, CA), PowerPlex® ESX 17 system (Promega Corporation, Madison, WI), and PowerPlex® ESI 17 system (Promega Corporation) were compared for concordance of allele calls for the SE33 marker in selected samples. A total of 16 samples were identified that were discordant at one of the SE33 alleles by an apparent one nucleotide in size. While the ESX 17 and NGM SElect™ kits yielded concordant results for these 16 samples, the ESI 17 kit generated alleles that differed. The discordant alleles were observed in individuals of African and European descent. Sequence analysis revealed that the one-base difference in size is not due to an indel but is instead the result of a single nucleotide polymorphism (SNP) in the flanking region of the SE33 repeat region. Three different SNPs were observed, one of which is novel. Although these migration anomalies were observed only with the ESI 17 kit, one cannot preclude that a similar phenomenon may occur with the other kits as data sets increase. The type and degree of discordance of STR allele calls among STR kits is an important issue when comparing STR profiles among laboratories and when determining search parameters for identifying candidate associations in national databases.

Extraction platform evaluations: a comparison of AutoMate Express™, EZ1® Advanced XL, and Maxwell® 16 Bench-top DNA extraction systems.

The DNA extraction performance of three low-throughput extraction systems was evaluated. The instruments and respective chemistries all use a similar extraction methodology that involves binding DNA to a coated magnetic resin in the presence of chaotropic salt, washing of the resin to remove undesirable compounds, and elution of DNA from the particles in a low-salt solution. The AutoMate Express™ (Life Technologies Corporation, Carlsbad, CA), EZ1® Advanced XL (Qiagen Inc., Valencia, CA), and Maxwell® 16 (Promega Corporation, Madison, WI) were compared using a variety of samples including: blood on swabs, blood on denim, blood on cotton, blood mixed with inhibitors (a mixture of indigo, hematin, humic acid, and urban dust) on cotton, blood on FTA® paper, saliva residue on cigarette butt paper, epithelial cells on cotton swabs, neat semen on cotton, hair roots, bones, and teeth. Each instrument had a recommended pre-processing protocol for each sample type, and these protocols were followed strictly to reduce user bias. All extractions were performed in triplicate for each sample type. The three instruments were compared on the basis of quantity of DNA recovered (as determined by real-time PCR), relative level of inhibitors present in the extract (shown as shifts in the C(T) value for the internal PCR control in the real-time PCR assay), STR peak heights, use of consumables not included in the extraction kits, ease of use, and application flexibility. All three systems performed well; however extraction efficiency varied by sample type and with the preprocessing protocol applied to the various samples.

Assessing a novel room temperature DNA storage medium for forensic biological samples.

The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies. A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625-200 ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze-thaw cycles (3 cycles). In addition, spiking of 1-4 × SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA. Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA.

Comparisons of familial DNA database searching strategies.

The current familial searching strategies are generally based on either Identity-By-State (IBS) (i.e., number of shared alleles) or likelihood ratio (i.e., kinship index [KI]) assessments. In this study, the expected IBS match probabilities given relationships and the logic of the likelihood ratio method were addressed. Further, the false-positive and false-negative rates of the strategies were compared analytically or by simulations using Caucasian population data of the 13 CODIS Short Tandem Repeat (STR). IBS ≥ 15, IBS ≥ 16, KI ≥ 1000, or KI ≥ 10,000 were found to be good thresholds for balancing false-positive and false-negative rates. IBS ≥ 17 and/or KI ≥ 1,000,000 can exclude the majority of candidate profiles in the database, either related or not, and may be an initial screening option if a small candidate list is desired. Polices combining both IBS and KI can provide higher accuracy. Typing additional STRs can provide better searching performance, and lineage markers can be extremely useful for reducing false rates.

Genetic composition of six miniSTR in a Brazilian Mulatto sample population.

The present study characterizes the genetic variability of Mulatto population based on the polymorphism of six miniSTR autosomal loci, known as Non Codis 01 and 02 (NC01 and NC02) and evaluate their applicability in forensic genetics. A sample of 102 unrelated Brazilian mulattoes were genotyped for miniSTR loci D1S1677, D2S441, D4S2364 (miniplex NC02) and 45 individuals for D10S1248, D14S1434, D22S1045 (miniplex NC01). No significant deviations from Hardy-Weinberg equilibrium expectations were detected. The combined power of discrimination (PD) and mean power of exclusion (PE) were 0.999996 and 0.98991, respectively. The results also support the effectiveness of the NC01and NC02 miniplexes for human identification.

Y-STR loci diversity in native Alaskan populations.

Y chromosome short tandem repeat (Y-STR) loci are important genetic markers for forensic biological evidence analyses. However, paternal inheritance, reduced effective population size, and lack of independence between loci can reduce Y-STR diversity and may yield greater population substructure effects on a locus-by-locus basis compared with the autosomal STR loci. Population studies are necessary to assess the genetic variation of forensically relevant markers so that proper inferences can be made about the rarity of DNA profiles. This study examined 16 Y-STRs in three sampled populations of Native Americans from Alaska: Inupiat, Yupik, and Athabaskan. Population genetic and statistical issues addressed were: (1) the degree of diversity at locus and haplotype levels, (2) determination of the loci that contribute more so to haplotype diversity, and (3) the effects of population substructure on forensic statistical calculations of the rarity of a Y-STR profile. All three population samples were highly polymorphic at the haplotype level for the 16 Y-STR markers; however, the Native Americans demonstrated reduced genetic diversity compared with major US populations. The degree of substructure indicated that the three populations were related and admixed in terms of paternal lineage. The examination of more polymorphic loci may be needed to increase the power of discrimination of Y-STR systems in these populations.

Population genetic analyses of the NGM STR loci.

The AmpFlSTR® NGM™ PCR Amplification Kit enables amplification of 15 autosomal short tandem repeat (STR) loci. The loci are the ten STRs in the SGM Plus® Kit plus the EDNAP and ENSFI recommended STRs D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Allele frequency and other forensically relevant statistics data were generated for the NGM loci in three US population groups (African Americans, Caucasians, and Hispanics). The analyses support that the NGM multiplex is one of the most informative STR multiplex kits available to the forensic science community. At the population level, there are no more detectable departures from expectations of the independence of alleles within as well as between loci than would be expected due to chance, even for the two syntenic loci vWA and D12S391; however, linkage analysis in three large pedigree families shows close linkage between these two loci with a recombination fraction of 0.108. Therefore, in contrast to the practices in calculating the rarity of a DNA profile, for kinship analyses independence between the loci, vWA and D12S391 cannot be assumed.

Pedigree likelihood ratio for lineage markers.

Lineage-based haplotype markers (e.g., Y chromosome STRs and mitochondrial DNA sequences) are important adjunct tools to the autosomal markers for kinship analysis and for specialized kinship applications such as database searching. Traditionally, the prosecution or kinship hypothesis considers the haplotypes in the same lineage and the probability of genotype data given the lineage hypothesis is simply set at 1 if the number of mismatched loci or nucleotides between the questioned person and the references is less than a predefined threshold. In this study, a kinship hypothesis based on a fixed relationship of the questioned person in the reference family is introduced. A graphical model is proposed to calculate the probability of the genotype data given the kinship hypothesis, which is the product of haplotype frequency of the founder in the pedigree and the transmission probability from the founder to all descendants. Proper mutation models are suggested for Y chromosome STRs and mitochondrial DNA sequence variants (i.e., SNPs) to calculate the transmission probability. The methods to infer the genotypes of the untyped individuals in the pedigree and the computational complexity of handling these untyped individuals are also addressed. Lastly, numerical examples of the applications are given to demonstrate the kinship hypothesis and the algorithms.

US forensic Y-chromosome short tandem repeats database.

A forensic Y-STR database generated in the US was compiled with profiles containing a portion or complete typing of 16 STR markers DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4. There were 17,447 samples in the version of database in which 77% and 20% were collected in North America and Asia, respectively. The database was separated into six general populations, African American, Asian, Caucasian, Hispanic, Indian, and Native American. Each population was further classified into subgroups according to geographic regions. Some subgroups were tested, found to be homogenous and merged together. Allele and haplotype frequencies, as well as sample sizes were summarized. Of the full haplotypes (i.e., 16 STRs without missing data), 93.7% in total population were distinct, 92.9% were population specific, and 89.3% were only observed once. The majority of shared haplotypes were found among North American populations as a result of admixture lasting the past few hundred years. The power of discrimination (PD), coancestry coefficient (F(st)), and coefficient of gene differentiation (G(st)) at locus and haplotype levels were also calculated. The most polymorphic marker was DYS385; this marker contains a tandem duplication and actually is composed of two loci. Both G(st) and F(st) estimates were very small with haplotypes composed of a high number of STRs haplotypes (e.g., 10-16 markers), although G(st) is slightly more conservative for these extended haplotypes. With Native American removed from the total population data set, the G(st) and F(st) estimates reduce further. PD was 0.9998 for the total population dataset for all 16 Y-STR markers. Three measures of Y-STR profile frequency were calculated: (1) unconditional haplotype frequency, (2) population substructure adjusted frequency, and (3) binomial upper bound of the haplotype frequency. The binomial upper bound is the most conservative estimate for most forensic applications. Estimates of the weight of a Y-STR haplotype can be estimated using population specific or total population databases.

Texas population substructure and its impact on estimating the rarity of Y STR haplotypes from DNA evidence*.

Three sampled populations of unrelated males--African American, Caucasian, and Hispanic, all from Texas-were typed for 16 Y short tandem repeat (STR) markers using the AmpFlSTR Yfiler kit. These samples also were typed previously for the 13 core CODIS autosomal STR loci. Most of the 16 marker haplotypes (2478 out of 2551 distinct haplotypes) were observed only once in the data sets. Haplotype diversities were 99.88%, 99.89%, and 99.87% for the African American, Caucasian, and Hispanic sample populations, respectively. F(ST) values were very small when a haplotype comprised 10-16 markers. This suggests that inclusion of substructure correction is not required. However, haplotypes consisting of fewer loci may require the inclusion of F(ST) corrections. The testing of independence of autosomal and Y STRs supports the proposition that the frequencies of autosomal and Y STR profiles can be combined using the product rule.

Validity of low copy number typing and applications to forensic science.

Low copy number (LCN) typing, particularly for current short tandem repeat (STR) typing, refers to the analysis of any sample that contains less than 200 pg of template DNA. Generally, LCN typing simply can be defined as the analysis of any DNA sample where the results are below the stochastic threshold for reliable interpretation. There are a number of methodologies to increase sensitivity of detection to enable LCN typing. These approaches encompass modifications during the polymerase chain reaction (PCR) and/or post-PCR manipulations. Regardless of the manipulations, when processing a small number of starting templates during the PCR exaggerated stochastic sampling effects will occur. The result is that several phenomena can occur: a substantial imbalance of 2 alleles at a given heterozygous locus, allelic dropout, or increased stutter. With increased sensitivity of detection there is a concomitant increased risk of contamination. Recently, a commission reviewed LCN typing and found it to be "robust" and "fit for purpose." Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing. The findings of the commission seem inconsistent with the nature of LCN typing. While LCN typing is appropriate for identification of missing persons and human remains and for developing investigative leads, caution should be taken with its use in other endeavors until developments are made that overcome the vagaries of LCN typing. A more in-depth evaluation by the greater scientific community is warranted. The issues to consider include: training and education, evidence handling and collection procedures, the application or purpose for which the LCN result will be used, the reliability of current LCN methods, replicate analyses, interpretation and uncertainty, report writing, validation requirements, and alternate methodologies for better performance.

Mutation rates at Y chromosome short tandem repeats in Texas populations.

Father-son pairs from three populations (African American, Caucasian, and Hispanic) of Texas were typed for the 17 Y STR markers DYS19, DYS385, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS456, DYS458, DYS635, DYS448, and Y GATA H4 using the AmpFlSTR YfilerTM kit. With 49,578 allele transfers, 102 mutations were detected. One three-step and four two-step mutations were found, and all others (95.1%) were one-step mutations. The number of gains (48) and losses (54) of repeats were nearly similar. The average mutation rate in the total population is 2.1 x 10(-3) per locus (95% CI (1.7-2.5)x10(-3)). African Americans showed a higher mutation rate (3.0 x 10(-3); 95% CI (2.4-4.0)x10(-3)) than the Caucasians (1.7 x 10(-3); 95% CI (1.1-2.5)x10(-3)) and Hispanics (1.5 x 10(-3); 95% CI (1.0-2.2)x10(-3)), but grouped by repeat-lengths, such differences were not significant. Mutation is correlated with relative length of alleles, i.e., longer alleles are more likely to mutate compared with the shorter ones at the same locus. Mutation rates are also correlated with the absolute number of repeats, namely, alleles with higher number of repeats are more likely to mutate than the shorter ones (p-value=0.030). Finally, occurrences of none, one, and two mutations over the father-son transmission of alleles were consistent with the assumption of independence of mutation rates across loci.

Null allele sequence structure at the DYS448 locus and implications for profile interpretation.

Null alleles can occur with any PCR-based STR typing system. They generally are due to deletions within the target region or primer binding sites or by primer binding site mutations that destabilize hybridization of at least one of the primers flanking the target region. Although not common, null types were detected at the DYS448 locus in seven out of 1,005 unrelated males in the Hispanic population. Of these DYS448 null types, four individuals displayed an apparent duplication at the DYS437 locus. The additional allele observed at the DYS437 locus is in actuality a smaller-sized DYS448 amplicon, which is the result of a deletion of the invariant N42 base pair domain and downstream repeats within the DYS448 locus. Thus, some DYS448 null types are not truly null. A true DYS448 null allele carried numerous primer binding site variants and a large deletion including the N42 base pair domain and surrounding or downstream repeat regions. The presence of null alleles is not a real concern for interpretation of Y STR loci evidence; current methods for interpreting Y STR profiles easily accommodate such phenomena.

A novel SNaPshot assay to detect the mdx mutation.

The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD). In order to evaluate possible treatments and to carry out genetic studies, it is essential to distinguish between mice that carry the dystrophic (mutant) or wild-type (wt) allele(s). The current amplification-resistant mutation system (ARMS) assay is labor intensive and yields false negatives, which reduces its efficiency as a screening tool. An alternate assay based on single-nucleotide polymorphism (SNP) primer extension technology (i.e., SNaPshot) is described. The SNaPshot assay has been optimized to identify both wild-type and mutant alleles, providing a robust, potentially automatable assay for high-throughput analysis.