Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin.

Mayra Eduardoff,Christina Strobl,Walther Parson,Andrea Casas-Vargas,Catarina Xavier

doi:10.3390/genes8100237

Abstract

The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories.

Highlights

MitochondrialDNA is present in a higher copy number in the cell than nuclear DNA, which is why its analysis can be useful in forensic cases, where the evidentiary material does not contain enoughGenes 2017, 8, 237; doi:10.3390/genes8100237 www.mdpi.com/journal/genesGenes 2017, 8, 237 nuclear DNA for conventional autosomal Short Tandem Repeat (STR) typing [1]. mitochondrial DNA (mtDNA) haplotypes are less informative than combined autosomal genotypes, as they are shared by maternally related individuals and do not undergo recombination
Note that Sanger-type Sequencing (STS) was performed previous to the primer extension capture (PEC). Analysis in another laboratory by different staff, and only the tube containing the DNA extract was handed to the PEC Massively Parallel Sequencing (MPS) laboratory. Both hair shaft samples resulted in mtDNA haplotypes typical for the West Eurasian phylogeny (T2b6 and U5b2b, respectively; Table S3) with plausible haplotypes after quality control according to quality criteria established through the EDNAP
Since the PEC assay targeted a region around position 8342 and extended both ends of the control region (CR), as well as taking into account random non-targeted mtDNA reads from non-removed background DNA, the coverage levels of the mitogenome reached up to 70%

Summary

Introduction

Mitochondrial (mt)DNA is present in a higher copy number in the cell than nuclear DNA, which is why its analysis can be useful in forensic cases, where the evidentiary material does not contain enoughGenes 2017, 8, 237; doi:10.3390/genes8100237 www.mdpi.com/journal/genesGenes 2017, 8, 237 nuclear DNA for conventional autosomal Short Tandem Repeat (STR) typing [1]. mtDNA haplotypes are less informative than combined autosomal genotypes, as they are shared by maternally related individuals and do not undergo recombination. Mitochondrial (mt)DNA is present in a higher copy number in the cell than nuclear DNA, which is why its analysis can be useful in forensic cases, where the evidentiary material does not contain enough. MtDNA haplotypes are less informative than combined autosomal genotypes, as they are shared by maternally related individuals and do not undergo recombination. Its meaningful application in the forensic context is restricted to cases that involve (a reduced number of) maternally unrelated individuals. The separate amplification and sequencing of these segments turned out to be prone to sample mix-up (aka artificial recombination) when performed manually [7], which is why the amplification of the entire CR in one fragment [8] and sequencing of the amplicon with internal primers is preferred [7,9]. For degraded mtDNA, smaller amplicon strategies were developed to target ‘medium-sized’ fragments (approximately 300 bp [10])

Methods

Results

Discussion

Conclusion