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 1 to 5 mm) of human hair shafts up to 40 years from the time of collection. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of mitochondrial DNA from human hair shafts Basic Protocol 2: Quantification of mitochondrial DNA (copies/μl) Basic Protocol 3: Multiplex amplification of the mitogenome Basic Protocol 4: Library preparation and sequencing of mitogenome amplicons Basic Protocol 5: Data analysis of mitogenome haplotypes.
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