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Massively Parallel Sequencing of the Mitogenome from Human Hair Shafts in Forensic Investigations

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The objective of this article is to provide a set of protocols for routine analysis of the mitochondrial genome sequence to assist the practitioner in the effective extraction of DNA from hair, accurate quantification of the mitochondrial DNA copies recovered, amplification of the mitogenome in small pieces using a multiplex approach, massively parallel sequencing, and data analysis using software specifically designed to report phylogenetically accurate mito-haplotypes.


This article highlights methods used to perform DNA extraction, mitochondrial DNA quantification, multiplex PCR amplification, amplicon-based massively parallel sequencing, and data analysis of the mitochondrial genome (mitogenome) from human hair shafts. The focus is on applications to forensic casework, but this set of protocols can be used for any purpose involving small cuttings (as small as one to five mm) of human hair shafts up to 40 years from the time of collection. The five basic protocols are: extraction of mitochondrial DNA from human hair shafts; quantification of mitochondrial DNA (copies/μl); multiplex amplification of the mitogenome; library preparation and sequencing of mitogenome amplicons; and data analysis of mitogenome haplotypes. Publisher Abstract Provided

Date Published: January 1, 2023