The peopling of South America and the trans-Andean gene flow of the first settlers.

Genetic and archaeological data indicate that the initial Paleoindian settlers of South America followed two entry routes separated by the Andes and the Amazon rainforest. The interactions between these paths and their impact on the peopling of South America remain unclear. Analysis of genetic variation in the Peruvian Andes and regions located south of the Amazon River might provide clues on this issue. We analyzed mitochondrial DNA variation at different Andean locations and >360,000 autosomal SNPs from 28 Native American ethnic groups to evaluate different trans-Andean demographic scenarios. Our data reveal that the Peruvian Altiplano was an important enclave for early Paleoindian expansions and point to a genetic continuity in the Andes until recent times, which was only marginally affected by gene flow from the Amazonian lowlands. Genomic variation shows a good fit with the archaeological evidence, indicating that the genetic interactions between the descendants of the settlers that followed the Pacific and Atlantic routes were extremely limited.

Genetic mapping of 15 human X chromosomal forensic short tandem repeat (STR) loci by means of multi-core parallelization.

Typing of X chromosomal short tandem repeat (X STR) markers has become a standard element of human forensic genetic analysis. Joint consideration of many X STR markers at a time increases their discriminatory power but, owing to physical linkage, requires inter-marker recombination rates to be accurately known. We estimated the recombination rates between 15 well established X STR markers using genotype data from 158 families (1041 individuals) and following a previously proposed likelihood-based approach that allows for single-step mutations. To meet the computational requirements of this family-based type of analysis, we modified a previous implementation so as to allow multi-core parallelization on a high-performance computing system. While we obtained recombination rate estimates larger than zero for all but one pair of adjacent markers within the four previously proposed linkage groups, none of the three X STR pairs defining the junctions of these groups yielded a recombination rate estimate of 0.50. Corroborating previous studies, our results therefore argue against a simple model of independent X chromosomal linkage groups. Moreover, the refined recombination fraction estimates obtained in our study will facilitate the appropriate joint consideration of all 15 investigated markers in forensic analysis.

Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq.

Sanger-type sequencing (STS) of mitochondrial DNA (mtDNA), specifically the control region (CR), is routinely employed in forensics in human identification and missing persons scenarios. Yet next-generation sequencing (NGS) has the potential to overcome some of the major limitations of STS processing, permitting reasonable paths forward for full mitochondrial genome (mtGenome) sequencing, while also offering higher-throughput and higher sensitivity capabilities. To establish the accuracy and reproducibility of NGS for the development of mtDNA data, 90 DNA extracts that were previously used to generate forensic quality full mtGenomes using STS were sequenced using Nextera XT library preparation and the Illumina MiSeq. Using the same amplicon product, replicate library sets were generated and sequenced at different laboratories, and analysis was performed in replicate using the CLC Genomics Workbench. Both sequencing sets resulted in 99.998% of positions with greater than 10X coverage when 96 samples (including controls) were multiplexed. Overall, 99.9996% concordance was observed between the NGS data and the STS data for the full mtGenome. The only "discordant" calls involved low level point heteroplasmies, with the differences resulting from stochastic variation and/or the increased sensitivity of NGS. Higher sensitivity also allowed for the detection of a mixed sample previously not detected with STS. Additionally, variant calls were reproducible between sequencing sets and between software analysis versions with the variant frequency only differing by 0.23% and 0.01%, respectively. Further validation studies and specialized software functionality tailored to forensic practice should facilitate the incorporation of NGS processing into standard casework applications. The data herein comprise the largest, and likely most thoroughly examined, complete mtGenome STS-NGS concordance dataset available.

Forensic typing of short tandem repeat markers on the X and Y chromosomes.

Short tandem repeat (STR) markers are the cornerstone of forensic identity and kinship testing. Markers located on the X and the Y chromosome can complement those found on the autosomes, which are commonly used in laboratories today. The distinctive inheritance pattern of the sex chromosomes affords advantages to the investigation of family pedigrees required by mass disaster victim identification or missing persons cases, as well as mixed samples typically recovered in sexual assault crimes. This review aims to provide an overview of how X and Y STR markers are currently being used in forensic laboratories, including the applications which target their use, the markers and multiplexes facilitating recovery of this genetic information, and the tools available to interpret the resulting data. Emerging research topics are considered as well as issues requiring further study for both marker systems.

Full mtGenome reference data: development and characterization of 588 forensic-quality haplotypes representing three U.S. populations.

Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S. population groups (African American, Caucasian and Hispanic) from anonymized, randomly-sampled specimens. Data production utilized an 8-amplicon, 135 sequencing reaction Sanger-based protocol, performed in semi-automated fashion on robotic instrumentation. Data review followed an intensive multi-step strategy that included a minimum of three independent reviews of the raw data at two laboratories; repeat screenings of all insertions, deletions, heteroplasmies, transversions and any additional private mutations; and a check for phylogenetic feasibility. For all three populations, nearly complete resolution of the haplotypes was achieved with full mtGenome sequences: 90.3-98.8% of haplotypes were unique per population, an improvement of 7.7-29.2% over control region sequencing alone, and zero haplotypes overlapped between populations. Inferred maternal biogeographic ancestry frequencies for each population and heteroplasmy rates in the control region were generally consistent with published datasets. In the coding region, nearly 90% of individuals exhibited length heteroplasmy in the 12418-12425 adenine homopolymer; and despite a relatively high rate of point heteroplasmy (23.8% of individuals across the entire molecule), coding region point heteroplasmies shared by more than one individual were notably absent, and transversion-type heteroplasmies were extremely rare. The ratio of nonsynonymous to synonymous changes among point heteroplasmies in the protein-coding genes (1:1.3) and average pathogenicity scores in comparison to data reported for complete substitutions in previous studies seem to provide some additional support for the role of purifying selection in the evolution of the human mtGenome. Overall, these thoroughly vetted full mtGenome population reference data can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via massively parallel sequencing) may be evaluated, and will provide a solid foundation for the generation of complete mtGenome haplotype frequency estimates for forensic applications.

Mutation rates of 15 X chromosomal short tandem repeat markers.

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.

Development of forensic-quality full mtGenome haplotypes: success rates with low template specimens.

Forensic mitochondrial DNA (mtDNA) testing requires appropriate, high quality reference population data for estimating the rarity of questioned haplotypes and, in turn, the strength of the mtDNA evidence. Available reference databases (SWGDAM, EMPOP) currently include information from the mtDNA control region; however, novel methods that quickly and easily recover mtDNA coding region data are becoming increasingly available. Though these assays promise to both facilitate the acquisition of mitochondrial genome (mtGenome) data and maximize the general utility of mtDNA testing in forensics, the appropriate reference data and database tools required for their routine application in forensic casework are lacking. To address this deficiency, we have undertaken an effort to: (1) increase the large-scale availability of high-quality entire mtGenome reference population data, and (2) improve the information technology infrastructure required to access/search mtGenome data and employ them in forensic casework. Here, we describe the application of a data generation and analysis workflow to the development of more than 400 complete, forensic-quality mtGenomes from low DNA quantity blood serum specimens as part of a U.S. National Institute of Justice funded reference population databasing initiative. We discuss the minor modifications made to a published mtGenome Sanger sequencing protocol to maintain a high rate of throughput while minimizing manual reprocessing with these low template samples. The successful use of this semi-automated strategy on forensic-like samples provides practical insight into the feasibility of producing complete mtGenome data in a routine casework environment, and demonstrates that large (>2kb) mtDNA fragments can regularly be recovered from high quality but very low DNA quantity specimens. Further, the detailed empirical data we provide on the amplification success rates across a range of DNA input quantities will be useful moving forward as PCR-based strategies for mtDNA enrichment are considered for targeted next-generation sequencing workflows.

Population genetic data for 15 X chromosomal short tandem repeat markers in three U.S. populations.

314 African American, 434 U.S. Caucasian, and 398 U.S. Hispanic individuals were typed at X STR markers DXS6795, DXS9902, DXS8378, DXS7132, DXS6803, DXS6789, DXS7424, DXS101, GATA172D05, DXS7130, GATA165B12, HPRTB, GATA31E08, DXS10147, DXS7423. High forensic efficiency parameter values confirm the potential usefulness of these markers in certain specific kinship situations involving female offspring as well as identity testing. Alleles previously unobserved in U.S. populations were noted in this study at 8 different markers. Additionally, null alleles and a triallelic pattern were observed and described. Pairwise comparisons indicated consistency with similar published populations at overlapping markers. These data represent a substantial increase in the quantity of U.S. X chromosomal short tandem repeat data available to the community.

An optimized protocol for forensic application of the PreCR™ Repair Mix to multiplex STR amplification of UV-damaged DNA.

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.

Development and characterization of two mini-X chromosomal short tandem repeat multiplexes.

This study presents the development and characterization of two X chromosomal short tandem repeat (STR) multiplexes utilizing reduced-size amplicons (less than 200 base pairs) for identity and kinship testing with degraded DNA. Approximately 1360 samples across 4 U.S. population groups were typed for 15 X chromosomal STR markers: DXS6789, DXS7130, DXS9902, GATA31E08, DXS7424, GATA165B12, DXS101, DXS6795, GATA172D05, DXS10147, DXS8378, DXS7132, DXS6803, HPRTB, and DXS7423. A high degree of polymorphism was observed for each marker and both multiplexes were sensitive down to 200pg of pristine DNA. The two proposed multiplexes are suitable for forensic use, and show potential for improved analysis of compromised bone samples.

Evaluating self-declared ancestry of U.S. Americans with autosomal, Y-chromosomal and mitochondrial DNA.

The current U.S. population represents an amalgam of individuals originating mainly from four continental regions (Africa, Europe, Asia and America). To study the genetic ancestry and compare with self-declared ancestry we have analyzed paternally, maternally and bi-parentally inherited DNA markers sensitive for indicating continental genetic ancestry in all four major U.S. American groups. We found that self-declared U.S. Hispanics and U.S. African Americans tend to show variable degrees of continental genetic admixture among the three genetic systems, with evidence for a marked sex-biased admixture history. Moreover, for these two groups we observed significant regional variation across the country in genetic admixture. In contrast, self-declared U.S. European and U.S. Asian Americans were genetically more homogeneous at the continental ancestry level. Two autosomal ancestry-sensitive markers located in skin pigmentation candidate genes showed significant differences in self-declared U.S. African Americans or U.S. European Americans, relative to their assumed parental populations from Africa or Europe. This provides genetic support for the importance of skin color in the complex process of ancestry identification.

WITHDRAWN: Development and Characterization of Two Mini-X Chromosomal Short Tandem Repeat Multiplexes.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Mitochondrial control region sequences from an African American population sample.

Entire mitochondrial control region data were generated for 248 African American individuals, which had been previously typed for 15 autosomal STRs [J.M. Butler, R. Schoske, P.M. Vallone, J.W. Redman, M.C. Kline, Allele frequencies for 15 autosomal STR loci on U.S. Caucasian, African American, and Hispanic populations, J. Forensic Sci. 48 (2003) 908-911].

Investigation of heteroplasmy in the human mitochondrial DNA control region: a synthesis of observations from more than 5000 global population samples.

Instances of point and length heteroplasmy in the mitochondrial DNA control region were compiled and analyzed from over 5,000 global human population samples. These data represent observations from a large and broad population sample, representing nearly 20 global populations. As expected, length heteroplasmy was frequently observed in the HVI, HVII and HVIII C-stretches. Length heteroplasmy was also observed in the AC dinucleotide repeat region, as well as other locations. Point heteroplasmy was detected in approximately 6% of all samples, and while the vast majority of heteroplasmic samples comprised two molecules differing at a single position, samples exhibiting two and three mixed positions were also observed in this data set. In general, the sites at which heteroplasmy was most commonly observed correlated with reported control region mutational hotspots. However, for some sites, observations of heteroplasmy did not mirror established mutation rate data, suggesting the action of other mechanisms, both selective and neutral. Interestingly, these data indicate that the frequency of heteroplasmy differs between particular populations, perhaps reflecting variable mutation rates among different mtDNA lineages and/or artifacts of particular population groups. The results presented here contribute to our general understanding of mitochondrial DNA control region heteroplasmy and provide additional empirical information on the mechanisms contributing to mtDNA control region mutation and evolution.

Complete mitochondrial genome sequences for 265 African American and U.S. "Hispanic" individuals.

Entire mitochondrial genome (mtGenome) sequences of 265 unrelated African American and U.S. "Hispanic" individuals were generated.

Mitochondrial control region sequences from a Vietnamese population sample.

Entire mitochondrial control region data were generated for 187 individuals from Vietnam. These samples have been previously typed for 16 autosomal short-tandem repeats (STRs) [1].

Mitochondrial control region sequences from northern Greece and Greek Cypriots.

Entire mitochondrial control region data were generated for population samples of 319 unrelated individuals from northern Greece and 91 unrelated individuals from Cyprus. The samples from northern Greece have been previously typed for 15 nuclear short tandem repeat (STR; Kovatsi et al., Forensic Sci. Int. 159:61-63, 2006).

Development and expansion of high-quality control region databases to improve forensic mtDNA evidence interpretation.

In an effort to increase the quantity, breadth and availability of mtDNA databases suitable for forensic comparisons, we have developed a high-throughput process to generate approximately 5000 control region sequences per year from regional US populations, global populations from which the current US population is derived and global populations currently under-represented in available forensic databases. The system utilizes robotic instrumentation for all laboratory steps from pre-extraction through sequence detection, and a rigorous eight-step, multi-laboratory data review process with entirely electronic data transfer. Over the past 3 years, nearly 10,000 control region sequences have been generated using this approach. These data are being made publicly available and should further address the need for consistent, high-quality mtDNA databases for forensic testing.

High efficiency DNA extraction from bone by total demineralization.

In historical cases, missing persons' identification, mass disasters, and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA typing. This is because of the physical and chemical barrier that the protein:mineral matrix of bone poses to environmental deterioration and biological attack. Most bone extraction protocols utilized in the forensic community involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the disposal of large quantities of undissolved bone powder. Here we present an extremely efficient protocol for recovery of DNA by complete demineralization, resulting in full physical dissolution of the bone sample. This is performed in a manner that retains and concentrates all the reagent volume, for complete DNA recovery. For our study, we selected 14 challenging bone samples. The bones were extracted side-by-side with our new demineralization protocol and the standard extraction protocol in use at AFDIL. A real-time quantification assay based on the amplification of a 143 bp mtDNA fragment showed that this new demineralization protocol significantly enhances the quantity of DNA that can be extracted and amplified from degraded skeletal remains. We have used this technique to successfully recover authentic DNA sequences from extremely challenging samples that failed repeatedly using the standard protocol.