The implementation and popularity of next generation sequencing (NGS) has led to the development of various rapid whole mitochondrial genome sequencing techniques. We summarise an efficient and cost-effective NGS approach for mitochondrial genomic DNA in humans using the Ion Torrent platform, and further discuss our bioinformatics pipeline for streamlined variant calling. Ion 316 chips were utilised with the Ion Torrent semi-conductor platform Personal Genome Machine (PGM) to perform tandem sequencing of mitochondrial genomes from the core pedigree (n = 315) of the Norfolk Island Health Study. Key improvements from commercial methods focus on the initial PCR step, which currently requires extensive optimisation to ensure the accurate and reproducible elongation of each section of the complete mitochondrial genome. Dual-platform barcodes were incorporated into our protocol thereby extending its potential application onto Illumina-based systems. Our bioinformatics pipeline consists of a modified version of GATK best practices tailored for mitochondrial genomic data. When compared with current commercial methods, our method, termed high throughput mitochondrial genome sequencing (HTMGS), allows high multiplexing of samples and the use of alternate library preparation reagents at a lower cost per sample (~1.7 times) when compared to current commercial methodologies. Our HTMGS methodology also provides robust mitochondrial sequencing data (>450X average coverage) that can be applied and modified to suit various study designs. On average, we were able to identify ~30 variants per sample with 572 variants observed across 315 samples. We have developed a high throughput sequencing and analysis method targeting complete mitochondrial genomes; with the potential to be platform agnostic with analysis options that adhere to current best practices.
Estimates of mutation rates for various regions of the human mitochondrial genome (mtGenome) vary widely, depending on whether they are inferred using a phylogenetic approach or obtained directly from pedigrees. Traditionally, only the control region, or small portions of the coding region have been targeted for analysis due to the cost and effort required to produce whole mtGenome Sanger profiles. Here, we report one of the first pedigree derived mutation rates for the entire human mtGenome. The entire mtGenome from 225 individuals originating from Norfolk Island was analysed to estimate the pedigree derived mutation rate and compared against published mutation rates. These individuals were from 45 maternal lineages spanning 345 generational events. Mutation rates for various portions of the mtGenome were calculated. Nine mutations (including two transitions and seven cases of heteroplasmy) were observed, resulting in a rate of 0.058 mutations/site/million years (95% CI 0.031–0.108). These mutation rates are approximately 16 times higher than estimates derived from phylogenetic analysis with heteroplasmy detected in 13 samples (n = 225, 5.8% individuals). Providing one of the first pedigree derived estimates for the entire mtGenome, this study provides a better understanding of human mtGenome evolution and has relevance to many research fields, including medicine, anthropology and forensics.
Many deployable forensic capabilities, including those used by the Australian Defense Force (ADF), employ mobile battery-operated fridge/freezers for DNA sample preservation that are not suitable for rapid response application due to their size and weight. These fridge/freezers are expensive, require regular specialised maintenance, and have a set payload. A variety of transport media are successful preservatives for DNA samples, however, there is no research specifically targeted to their suitability for operational environments where temperatures exceed 50 °C. This research examined whether sodium chloride (NaCl), ethanol, and dimethyl sulfoxide (DMSO) could preserve muscle and bone samples (fresh and early decomposition) as effectively as refrigeration, when stored at 21 °C, 45 °C, 55 °C, and 65 °C for at least one week. A total of 78 muscle and 78 bone samples were collected from an unknown deceased individual. Half of each tissue type was stored at 30 °C for 48 h to induce early decomposition. Following this, samples were stored in the transport media for one week at the above temperatures, and a control set of samples were refrigerated (-4 °C) without any transport media. Preserved samples would need to provide DNA profiles comparable to the refrigerated samples for the transport media to be considered a successful replacement method. NaCl and 70% ethanol preserved muscle samples (fresh and decomposed) up to 65 °C, as well as 70% ethanol and 20% DMSO for fresh bone samples. These results were comparable with refrigeration and therefore, these preservatives could be used in rapid response operations by the military and for disaster victim identification. Conversely, under the conditions of this study, 20% DMSO and 70% ethanol failed to consistently produce full DNA profiles from decomposed bone, and NaCl performed poorly at preserving DNA from fresh and decomposed bone samples.
Sanger sequencing of the mitochondrial DNA (mtDNA) control region was previously the only method available for forensic casework involving degraded samples from skeletal remains. The introduction of Next Generation Sequencing (NGS) has transformed genetic data generation and human identification using mtDNA. Whole mitochondrial genome (mtGenome) analysis is now being introduced into forensic laboratories around the world to analyze historical remains. Research into large pedigrees using the mtGenome is critical to evaluate currently available interpretation guidelines for mtDNA analysis, which were developed for comparisons using the control region. This study included mtGenomes from 225 individuals from the last four generations of the Norfolk Island (NI) genetic isolate pedigree consisting of 49 distinct maternal lineages. The data from these individuals were arranged into 2339 maternally related pairs separated by up to 18 meioses. Our results show that 97.3% of maternally related pairs were concordant at all nucleotide positions, resulting in the correct interpretation of "Cannot Exclude"; 2.7% of pairs produced an "Inconclusive" result, and there were no instances of false exclusion. While these results indicate that existing guidelines are suitable for multigenerational whole mtGenome analysis, we recommend caution be taken when classifying heteroplasmic changes as differences for human identification. Our data showed the classification of heteroplasmic changes as differences increases the prevalence of inconclusive identification by 6%, with false exclusions observed in 0.34% of pairs examined. Further studies of multigenerational pedigrees, however, are needed to validate mtGenome interpretation guidelines for historical case work to more fully utilize emerging advancements.
The Australian Army. 2017. "What We Do." 2017. www.army.gov.au/ourwork/unrecovered-war-casualties/what-we-do.
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