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

Peck, Michelle A.; Brandhagen, Michael D.; Marshall, Charla; Diegoli, Toni M.; Irwin, Jodi A.; Sturk-Andreaggi, Kimberly

doi:10.1016/j.fsigen.2016.06.003

Cited by 43 publications

(44 citation statements)

References 31 publications

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“…Additional GeneMarker HTS analysis options employed included filter settings (both region filters and variant filters) and amplicon settings. Settings were based on vendor and published paper recommendations [5,7,28,29] as well as preliminary evaluations of the PowerSeq assay. The region filters, which define the range of mtDNA sequence to be analyzed, were set to include nucleotide positions 16013-592 of the mtDNA CR.…”

Section: Discussionmentioning

confidence: 99%

“…Mitochondrial DNA (mtDNA) testing in forensic casework is generally performed with amplicons targeting the non-coding control region (CR) and capillary electrophoresis-based Sanger sequencing. However, over the past five or so years, next generation sequencing (NGS) has been shown to be an equally robust technology for the development of forensic quality mitochondrial DNA sequence data [1][2][3][4][5][6][7][8][9][10][11]. NGS not only offers the capability to reduce workflows, but it also allows for the generation of larger, more informative genetic data sets at higher throughput and overall lower cost per nucleotide than capillary electrophoresis-based methods [12,13].…”

Section: Introductionmentioning

confidence: 99%

“…The recovery of entire mitochondrial genome (mtGenome) data and the additional discriminatory power those data offer is arguably the greatest benefit that NGS brings to forensic mtDNA analysis. MtGenome data development via NGS is technically feasible [1][2][3]5,9,10,[14][15][16], and may be more cost-effective than current Sanger-based control region sequencing. As a result, many laboratories, including the FBI Laboratory, are evaluating mtGenome assays for casework implementation.…”

Section: Introductionmentioning

confidence: 99%

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Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

Brandhagen

Just

Irwin

2020

Forensic Science International: Genetics

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As a first step towards integrating next generation sequencing (NGS) technology into the FBI Laboratory's operational casework, the PowerSeq™ CRM Nested System, an NGS-based mitochondrial DNA (mtDNA) control region assay, was developmentally and internally validated. The validation studies were conducted in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines for Forensic DNA Analysis Methods, and the FBI's Quality Assurance Standards (QAS) for Forensic DNA Testing Laboratories. The assay was shown to be highly reproducible, with variant frequencies across intra and inter-run replicates of the same sample differing, on average, by just 0.3% for substitutions and point heteroplasmies and 1.5% for insertions and deletions. The assay was also shown to be extremely sensitive, yielding complete control region sequence data from as few as 2000 copies of mtDNA. This is a more than 20-fold increase in sensitivity when compared to the FBI Laboratory's current Sanger sequencing-based protocols and, based on mtDNA quantitation values of samples routinely encountered in mtDNA casework, suggests that the percentage of questioned samples from which full control region data can be recovered will increase from our current 20% to approximately 90% success with NGS technology. In addition, the assay requires on average only 30% of the extract volume typically required to develop control region profiles from degraded samples via Sanger sequencing. Overall, these studies establish the reliability of the PowerSeq™ CRM Nested System for accurate mtDNA control region typing and can serve as a model for laboratories seeking to validate NGS protocols for forensic mtDNA analysis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

Brandhagen

Just

Irwin

2020

Forensic Science International: Genetics

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“…Many other studies on the use of NGS technologies for forensic genetics and mtDNA analysis have been published (Chaitanya et al, 2015;Churchill, Stoljarova, King, & Budowle, 2018;Hollard et al, 2017;Just, Irwin, & Parson, 2015;Just, Scheible, Fast, Sturk-Andreaggi, Higginbotham, et al, 2014;Just, Scheible, Fast, Sturk-Andreaggi, Röck, et al, 2014;Lopopolo, Børsting, Pereira, & Morling, 2016;Ma et al, 2018;Marshall et al, 2017;Ovchinnikov, Malek, Kjelland, & Drees, 2016;Park et al, 2017;Templeton et al, 2013;Young, King, Budowle, & Armogida, 2017). However, further validation studies and specialized software functionality tailored to forensic practice should be produced in order to facilitate the incorporation of NGS processing into standard casework applications (Amorim & Pinto, 2018;Peck et al, 2016). In the meantime, and according to current international guidelines (W. Parson et al, 2014;Prinz et al, 2007), Sanger sequencing still continues to be an adequate method for mtDNA analysis for forensic human identification, and is used in most casework laboratories worldwide (Ballard, 2016).…”

Section: Mitochondrial Dna Sequencing Methodologiesmentioning

confidence: 99%

Peer Review #1 of "Mitochondrial DNA in human identification: a review (v0.1)"

2019

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Mitochondrial DNA (mtDNA) presents several characteristics useful for forensic studies, especially related to the lack of recombination, to a high copy number, and to matrilineal inheritance. mtDNA typing based on sequences of the control region or full genomic sequences analysis is used to analyze a variety of forensic samples such as old bones, teeth and hair, as well as other biological samples where the DNA content is low. Evaluation and reporting of the results requires careful consideration of biological issues as well as other issues such as nomenclature and reference population databases. In this work we review mitochondrial DNA profiling methods used for human identification and present their use in the main cases of humanidentification focusing on the most relevant issues for forensics.

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“…The NGS workflows were selected for this study as they are commonly used in forensics for mitogenome sequencing (e.g. [42][43][44][45][46][47][48][49][50]). These well-established NGS methods offer a quantitative analysis of mitogenome sequence data, which allowed for an investigation of the effects of the sequencing platform as well as enrichment strategy on LHP.…”

Section: Introductionmentioning

confidence: 99%