Working towards implementation of whole genome mitochondrial DNA sequencing into routine casework

Churchill, Jennifer D.; Peters, Daniel R.; Capt, Christina; Strobl, Christina; Parson, Walther; Budowle, Bruce

doi:10.1016/j.fsigss.2017.09.167

Cited by 15 publications

(12 citation statements)

References 10 publications

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“…The CE-based Sanger sequencing system, although limited in its ability to sequence large expanses of the mtGenome on a practical level, has been demonstrated to be highly reliable. However, with the development and maturation of massively parallel sequencing (MPS) technologies [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ], there is interest in enhancing mtDNA analysis, with some laboratories already tackling the validation and implementation process [ 20 , 21 , 22 , 23 , 24 ]. Analysis of the mtGenome offers a good first step to transition MPS workflows as current CE-based workflows are already sequence-based, making data analysis of MPS results a more familiar and similar process.…”

Section: Introductionmentioning

confidence: 99%

“…Additionally, the increase in information, provided by evaluating the entire mtGenome, offers the potential for an increase in discrimination power and phylogenetic resolution [ 10 , 26 , 27 , 28 ]. As databases with whole mtGenome data continue to grow [ 10 , 14 , 24 , 26 , 27 , 29 , 30 , 31 , 32 ], some laboratories are opting to employ multiplexes that target the entire mtGenome [ 21 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

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Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel

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Amory

Lagacé

et al. 2020

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For the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology—Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge—has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel

Cihlar

Amory

Lagacé

et al. 2020

Genes

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“…It has now become widely feasible to sequence all 16,568 mitogenome sites as part of a forensic investigation [ 6 – 8 ]. For autosomal short tandem repeat (STR) profiles, there are two alleles per locus and because of the effects of recombination, the alleles at distinct loci are treated as independent, after any adjustments for sample size, coancestry and direct relatedness [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

How many individuals share a mitochondrial genome?

Andersen

Balding

2018

PLoS Genet

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Mitochondrial DNA (mtDNA) is useful to assist with identification of the source of a biological sample, or to confirm matrilineal relatedness. Although the autosomal genome is much larger, mtDNA has an advantage for forensic applications of multiple copy number per cell, allowing better recovery of sequence information from degraded samples. In addition, biological samples such as fingernails, old bones, teeth and hair have mtDNA but little or no autosomal DNA. The relatively low mutation rate of the mitochondrial genome (mitogenome) means that there can be large sets of matrilineal-related individuals sharing a common mitogenome. Here we present the simulation software that we use to describe the distribution of the number of mitogenomes in a population that match a given mitogenome, and investigate its dependence on population size and growth rate, and on a database count of the mitogenome. Further, we report on the distribution of the number of meioses separating pairs of individuals with matching mitogenome. Our results have important implications for assessing the weight of mtDNA profile evidence in forensic science, but mtDNA analysis has many non-human applications, for example in tracking the source of ivory. Our methods and software can also be used for simulations to help validate models of population history in human or non-human populations.

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“…The introduction of MPS has made the use of mtDNA as a forensic tool more tractable by facilitating the sequencing of full mtGenomes and generating large amounts of quantitative genetic data in general [3,4,20,22,23]. Linkage information and related quantitative data allow for potential reconstruction of individual haplotypes and detection of novel and low-frequency variation [3,4,10,24].…”

Section: Introductionmentioning

confidence: 99%

A Continuous Statistical Phasing Framework for the Analysis of Forensic Mitochondrial DNA Mixtures

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Cihlar

Mandape

et al. 2021

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Despite the benefits of quantitative data generated by massively parallel sequencing, resolving mitotypes from mixtures occurring in certain ratios remains challenging. In this study, a bioinformatic mixture deconvolution method centered on population-based phasing was developed and validated. The method was first tested on 270 in silico two-person mixtures varying in mixture proportions. An assortment of external reference panels containing information on haplotypic variation (from similar and different haplogroups) was leveraged to assess the effect of panel composition on phasing accuracy. Building on these simulations, mitochondrial genomes from the Human Mitochondrial DataBase were sourced to populate the panels and key parameter values were identified by deconvolving an additional 7290 in silico two-person mixtures. Finally, employing an optimized reference panel and phasing parameters, the approach was validated with in vitro two-person mixtures with differing proportions. Deconvolution was most accurate when the haplotypes in the mixture were similar to haplotypes present in the reference panel and when the mixture ratios were neither highly imbalanced nor subequal (e.g., 4:1). Overall, errors in haplotype estimation were largely bounded by the accuracy of the mixture’s genotype results. The proposed framework is the first available approach that automates the reconstruction of complete individual mitotypes from mixtures, even in ratios that have traditionally been considered problematic.

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Working towards implementation of whole genome mitochondrial DNA sequencing into routine casework

Cited by 15 publications

References 10 publications

Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel

Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel

How many individuals share a mitochondrial genome?

A Continuous Statistical Phasing Framework for the Analysis of Forensic Mitochondrial DNA Mixtures

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