Abstract
BackgroundA population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets.ResultsWe developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences.ConclusionsThe described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing.
Highlights
A population reference database of complete human mitochondrial genome sequences is needed to enable the use of mitochondrial DNA coding region data in forensic casework applications
MtDNA sequencing has historically been limited to the non-coding control region (CR) or portions thereof, where the high concentration of fast-mutating sites presents the greatest opportunity for differentiation of samples representing distinct maternal lineages while minimizing data generation costs and effort
A recently published manual sequencing strategy generates high-quality Sanger sequence data with redundant coverage across the mitochondrial DNA (mtDNA) coding region (codR), and is perfectly suitable for the development of mtGenome reference data when combined with CR sequencing [12]
Summary
A population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. In forensics, mtDNA typing is most commonly employed when the nuclear DNA in an evidentiary sample is too limited or too damaged to develop sufficient nuclear data for forensic comparisons In this application, mtDNA sequencing has historically been limited to the non-coding control region (CR) or portions thereof, where the high concentration of fast-mutating sites presents the greatest opportunity for differentiation of samples representing distinct maternal lineages while minimizing data generation costs and effort. To ease the way for more rapid, high-volume generation of the complete mtGenome population reference data needed for forensics, accommodate different sample substrates and variable DNA quality/quantity, and further decrease the opportunities for human error inherent in manual sample handling, an entire mtGenome sequencing protocol and workflow designed for automated, high-throughput processing is necessary
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
