Open Access Open Badges Methodology article

A high-throughput Sanger strategy for human mitochondrial genome sequencing

Elizabeth A Lyons123, Melissa K Scheible12, Kimberly Sturk-Andreaggi12, Jodi A Irwin124 and Rebecca S Just125*

Author Affiliations

1 American Registry of Pathology, 120A Old Camden Rd., Camden DE 19934, USA

2 Armed Forces DNA Identification Laboratory, 115 Purple Heart Dr., Dover AFB, DE 19902, USA

3 Present affiliation: Michigan State Police, 7320 N. Canal Rd., Lansing, MI 48913, USA

4 Present affiliation: Federal Bureau of Investigation, 2501 Investigation Parkway, Quantico, VA 22135, USA

5 University of Maryland, College Park, 8082 Baltimore Ave., College Park, MD 20740, USA

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BMC Genomics 2013, 14:881  doi:10.1186/1471-2164-14-881

Published: 16 December 2013



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. 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.


We 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.


The 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.