Validation of a 52-mtSNP minisequencing panel for haplogroup classification of forensic DNA samples.

Mitochondrial DNA (mtDNA) is a useful tool in forensic investigation as it provides information about the matrilineal ancestry of individuals. In addition, mtDNA can be analyzed when the analysis of other nuclear markers is underperforming. Recently, we developed a minisequencing panel for the simultaneous analysis of 52 mtDNA SNPs to classify maternal lineages into the main haplogroups and their phylogeographic origin. In order to make this panel suitable for forensic genetics laboratories, a validation study has been performed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, including species specificity, reproducibility, sensitivity, and stability tests. The results demonstrate that the panel of 52 mtDNA SNPs is highly sensitive, since it enables to obtain complete genetic profiles of samples containing minimal amounts of DNA (1 pg). Furthermore, it provides sufficient genetic information to detect the matrilineal biogeographical origin of highly degraded samples, i.e., ancient dating skeletal remains, and samples with the presence of inhibitors, such as hematin and humic acid. In addition, this panel can detect mixtures in samples whose mtDNA haplogroups of contributors are different. Overall, the results of this study demonstrate the suitability of this minisequencing panel of 52 mtDNA SNPs to be used in forensic cases, with samples of low amount or degraded DNA.

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays.

The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol-chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A260/A280 and A260/A230), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol-chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.

Development of a mitochondrial DNA real-time polymerase chain reaction assay for quality control of pathogen reduction with riboflavin and ultraviolet light.

BACKGROUND AND OBJECTIVES: Transfusion is associated with a risk of infection and alloimmunization. Pathogen reduction using riboflavin and UV light (Mirasol treatment) inactivates pathogens and leucocytes. With increasing adoption of the technology in clinical use, regulatory agencies have recommended the introduction of quality control measures to monitor pathogen reduction efficacy. We sought to develop a real-time PCR-based assay to document the impact of pathogen reduction on the mitochondrial genome in blood components. MATERIALS AND METHODS: DNA was extracted from platelet and plasma components before and after treatment with riboflavin and UV light. Inhibition of PCR amplification of mitochondrial DNA (mtDNA) in short- and long-amplicon target regions, ranging from under 200 base pairs (bp) to over 1800 bp, was measured in treated relative to untreated components. RESULTS: Pathogen reduction of platelets using riboflavin and UV light resulted in inhibition of PCR amplification of long-amplicon mtDNA targets, demonstrating approximately 1 log reduction of amplification relative to untreated products. Amplification of short-amplicon mtDNA targets was not affected by treatment. Evaluation of 110 blinded platelet samples from the PREPAReS clinical trial resulted in prediction of treatment status with 100% accuracy. Pathogen reduction of plasma components resulted in similar levels of PCR inhibition, while testing of 30 blinded plasma samples resulted in prediction of treatment status with 93% accuracy. CONCLUSION: A differential sized amplicon real-time PCR assay of mitochondrial DNA effectively documents nucleic acid damage induced by Mirasol treatment of platelets. The use of the assay for plasma product pathogen reduction requires further investigation.

A new method for DNA extraction from feces and hair shafts of the South China tiger (Panthera tigris amoyensis).

It is commonly known that tigers (Panthera tigris) groom themselves by licking their coats, which leads to an abundance of hairs in their feces. These hairs are designated specially as "fecal hairs". In our study, in order to explore fecal hairs potential as a DNA source for genetic analysis, 55 fecal hair samples were collected from 23 captive South China tigers (P. t. amoyensis). According to the amplification of mitochondrial primers loop F and loop R, DNA quality of noninvasive samples were grouped into three grades: grade I-the highest-quality DNA, grade II--high-quality DNA, and grade III--poor-quality DNA. No failed amplifications on microsatellite primers and only 0.27% genotyping errors occurred with grade I fecal hair DNA, as compared with 9.4% failed amplifications on microsatellite primers and 9.5% genotyping errors with grade II fecal hair DNA. It was found that 25.45% of fecal hair DNA was grade I and 65.45 and 10.00% of fecal hair DNA were grades II and III, respectively, as compared with 4.35% grade I fecal DNA and 34.78 and 60.87% grades II and III fecal DNA, respectively. Thus, higher-quality DNA can be extracted from fecal hairs than feces. In addition, DNA could be extracted from hair shafts of tigers and a minimum of 2000 hair shafts were required for visible DNA bands on a 1% agarose gel. These findings demonstrate that fecal hairs may serve as a convenient and reliable genomic DNA source for genotype analysis.

Characterization of NIST human mitochondrial DNA SRM-2392 and SRM-2392-I standard reference materials by next generation sequencing.

Standard Reference Materials SRM 2392 and 2392-I are intended to provide quality control when amplifying and sequencing human mitochondrial genome sequences. The National Institute of Standards and Technology (NIST) offers these SRMs to laboratories performing DNA-based forensic human identification, molecular diagnosis of mitochondrial diseases, mutation detection, evolutionary anthropology, and genetic genealogy. The entire mtGenome (∼16569bp) of SRM 2392 and 2392-I have previously been characterized at NIST by Sanger sequencing. Herein, we used the sensitivity, specificity, and accuracy offered by next generation sequencing (NGS) to: (1) re-sequence the certified values of the SRM 2392 and 2392-I; (2) confirm Sanger data with a high coverage new sequencing technology; (3) detect lower level heteroplasmies (<20%); and thus (4) support mitochondrial sequencing communities in the adoption of NGS methods. To obtain a consensus sequence for the SRMs as well as identify and control any bias, sequencing was performed using two NGS platforms and data was analyzed using different bioinformatics pipelines. Our results confirm five low level heteroplasmy sites that were not previously observed with Sanger sequencing: three sites in the GM09947A template in SRM 2392 and two sites in the HL-60 template in SRM 2392-I.

Mitochondrial haplogroups and expression studies of commonly used human cell lines.

We developed a multiplex fragment length analysis (MFLA) for clearly assigning mitochondrial haplogroups mostly endemic in Europe for future cardiac diagnostics. As a technical proof, 23 commonly used human cell lines were haplotyped as reference standards. The functional analysis on mtDNA copies per cell revealed no correlation to haplogroups but a relatively high rate of mitochondria per cell and at the same time a very low expression of all mitochondrial and some nuclear encoded mitochondrial related genes. Established MFLA is an easy to handle method for analysing European mitochondrial haplogroups to perform epidemic studies and elucidate correlations to distinct diseases.

Mitogenome metadata: current trends and proposed standards.

Mitogenome metadata are descriptive terms about the sequence, and its specimen description that allow both to be digitally discoverable and interoperable. Here, we review a sampling of mitogenome metadata published in the journal Mitochondrial DNA between 2005 and 2014. Specifically, we have focused on a subset of metadata fields that are available for GenBank records, and specified by the Genomics Standards Consortium (GSC) and other biodiversity metadata standards; and we assessed their presence across three main categories: collection, biological and taxonomic information. To do this we reviewed 146 mitogenome manuscripts, and their associated GenBank records, and scored them for 13 metadata fields. We also explored the potential for mitogenome misidentification using their sequence diversity, and taxonomic metadata on the Barcode of Life Datasystems (BOLD). For this, we focused on all Lepidoptera and Perciformes mitogenomes included in the review, along with additional mitogenome sequence data mined from Genbank. Overall, we found that none of 146 mitogenome projects provided all the metadata we looked for; and only 17 projects provided at least one category of metadata across the three main categories. Comparisons using mtDNA sequences from BOLD, suggest that some mitogenomes may be misidentified. Lastly, we appreciate the research potential of mitogenomes announced through this journal; and we conclude with a suggestion of 13 metadata fields, available on GenBank, that if provided in a mitogenomes's GenBank record, would increase their research value.

A review of the DNA standard reference materials developed by the National Institute of Standards and Technology.

The Standard Reference Materials Program at the US National Institute of Standards and Technology (NIST) has three human DNA standard reference materials (SRM 2390, SRM 2391a, and SRM 2392) currently available [1, 2]. Both the DNA profiling SRM 2390 and the polymerase chain reaction (PCR)-based DNA profiling SRM 2391a are intended for use in forensic and paternity identifications, for instructional law enforcement, or for non-clinical research purposes and are not intended for clinical diagnostics. The mitochondrial DNA (mtDNA) SRM 2392 is to provide standardization and quality control when performing PCR and sequencing any segment or the entire 16,569 base pairs that comprise human mitochondrial DNA. SRM 2392 is designed for use by the forensic, medical, and toxicological communities for human identification, disease diagnosis or mutation detection.