Original Research Paper| Volume 15, P8-15, March 2015

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

Published:November 18, 2014DOI:


      • We present a midi amplicon protocol involving two PCR multiplexes and amplicon sizes of 300–500 bp for massively parallel sequencing (MPS) of entire mitochondrial (mt)genomes from hair shaft samples.
      • We evaluate alternative protocols using larger amplification products (2–3 kbp) as well as MPS from hair samples without prior amplification.
      • Our study demonstrates that massively mtGenome sequencing is feasible from hair samples, which would significantly increase the discrimination power in forensic mtDNA testing.


      Though shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, DNA testing has historically focused on the recovery of just about 600 base pairs of the mitochondrial DNA control region. Here, we describe our success in recovering complete mitochondrial genome (mtGenome) data (∼16,569 bp) from single shed hairs. By employing massively parallel sequencing (MPS), we demonstrate that particular hair samples yield DNA sufficient in quantity and quality to produce 2–3 kb mtGenome amplicons and that entire mtGenome data can be recovered from hair extracts even without PCR enrichment. Most importantly, we describe a small amplicon multiplex assay comprised of sixty-two primer sets that can be routinely applied to the compromised hair samples typically encountered in forensic casework. In all samples tested here, the MPS data recovered using any one of the three methods were consistent with the control Sanger sequence data developed from high quality known specimens. Given the recently demonstrated value of complete mtGenome data in terms of discrimination power among randomly sampled individuals, the possibility of recovering mtGenome data from the most compromised and limited evidentiary material is likely to vastly increase the utility of mtDNA testing for hair evidence.


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        • Bender K.
        • Schneider P.
        Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots.
        Forensic Sci. Int. Genet. 2006; 161: 52-59
        • Holland M.M.
        • Parsons T.J.
        Mitochondrial DNA sequence analysis–Validation and use for forensic casework.
        Forensic Sci. Rev. 1999; 11: 21-50
        • Parson W.
        • Dür A.
        EMPOP - A forensic mtDNA database.
        Forensic Sci. Int. Genet. 2007; 1: 88-92
        • Coble M.D.
        • Just R.S.
        • O’Callaghan J.E.
        • Letmanyi I.H.
        • Peterson C.T.
        • Irwin J.A.
        • et al.
        Single nucleotide polymorphisms over the entire mtDNA genome that increase the power of forensic testing in Caucasians.
        Int. J. Legal Med. 2004; 118: 137-146
        • Just R.S.
        • Leney M.D.
        • Barritt S.M.
        • Los C.W.
        • Smith B.C.
        • Holland T.D.
        • et al.
        The use of mitochondrial DNA single nucleotide polymorphisms to assist in the resolution of three challenging forensic cases.
        J. Forensic Sci. 2009; 54: 887-891
        • Sturk K.A.
        • Coble M.D.
        • Barritt S.M.
        • Parsons T.J.
        • Just R.S.
        The application of mtDNA SNPs to a forensic case.
        Forensic Sci. Int. Genet. Suppl. Ser. 2008; 1: 295-297
        • Irwin J.A.
        • Edson S.M.
        • Loreille O.
        • Just R.S.
        • Barritt S.M.
        • Lee D.A.
        • Holland T.D.
        • Parsons T.J.
        • Leney M.D.
        DNA identification of Earthquake McGoon 50 years postmortem.
        J. Forensic Sci. 2007; 52: 1115-1118
        • King J.L.
        • LaRue B.L.
        • Novroski N.
        • Stoljarova M.
        • Seo S.B.
        • Zeng X.
        • et al.
        High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq.
        Forensic Sci. Int. Genet. 2014; 12: 128-135
      1. R. Just, S. Fast, M. Scheible, K. Sturk-Andreaggi, A. Röck, J. Bush, et al., Full mtGenome reference data: development and characterization of 588 forensic-quality haplotypes representing three U.S. populations, Forensic Sci. Int. Genet. (under review).

        • Irwin J.A.
        • Parson W.
        • Coble M.D.
        • Just R.S.
        mtGenome reference population databases and the future of forensic mtDNA analysis.
        Forensic Sci. Int. Genet. 2011; 5: 222-225
        • Van Neste C.
        • Van Nieuwerburgh F.
        • Van Hoofstat D.
        • Deforce D.
        Forensic STR analysis using massive parallel sequencing.
        Forensic Sci. Int. Genet. 2012; 6: 810-818
        • Bornman D.M.
        • Hester M.E.
        • Schuetter J.M.
        • Kasoji M.D.
        • Minard-Smith A.
        • Barden C.A.
        • et al.
        Short-read, high-throughput sequencing technology for STR genotyping.
        BioTechniques. 2012; : 1-6
        • Parson W.
        • Strobl C.
        • Huber G.
        • Zimmermann B.
        • Gomes S.M.
        • Souto L.
        • et al.
        Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM).
        Forensic Sci. Int. Genet. 2013; 7: 543-549
        • Mikkelsen M.
        • Hansen R.F.
        • Hansen A.J.
        • Morling N.
        Massively parallel pyrosequencing 454 methodology of the mitochondrial genome in forensic genetics.
        Forensic Sci. Int. Genet. 2014; 12: 30-37
        • McElhoe J.A.
        • Holland M.M.
        • Makova K.D.
        • Su M.S.
        • Paul I.M.
        • Baker C.H.
        • Faith S.A.
        • Young B.
        Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq.
        Forensic Sci. Int. Genet. 2014; 13: 20-29
        • Scheible M.K.
        • Loreille O.
        • Just R.S.
        • Irwin J.A.
        Short tandem repeat typing on the 454 platform: Strategies and considerations for targeted sequencing of common forensic markers.
        Forensic Sci. Int. Genet. 2014; 12: 107-119
        • Dalsgaard S.
        • Rockenbauer E.
        • Buchard A.
        • Mogensen H.S.
        • Frank-Hansen R.
        • Borsting C.
        • et al.
        Non-uniform phenotyping of D12S391 resolved by second generation sequencing.
        Forensic Sci. Int. Genet. 2014; 8: 195-199
        • Rockenbauer E.
        • Hansen S.
        • Mikkelsen M.
        • Borsting C.
        • Morling N.
        Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing.
        Forensic Sci. Int. Genet. 2014; 8: 68-72
        • Weber-Lehmann J.
        • Schilling E.
        • Gradl G.
        • Richter D.C.
        • Wiehler J.
        • Rolf B.
        Finding the needle in the haystack: differentiating identical twins in paternity testing and forensics by ultra-deep next generation sequencing.
        Forensic Sci. Int. Genet. 2014; 9: 42-46
        • Bintz B.J.
        • Dixon G.B.
        • Wilson M.R.
        Simultaneous detection of human mitochondrial DNA and nuclear-inserted mitochondrial-origin sequences (NumtS) using forensic mtDNA amplification strategies and pyrosequencing technology.
        J. Forensic Sci. 2014; 59: 1064-1073
        • Briggs A.W.
        • Good J.M.
        • Green R.E.
        • et al.
        Targeted retrieval and analysis of five Neandertal mtDNA genomes.
        Science. 2009; 325: 318-321
        • Krause J.
        • Briggs A.W.
        • Kircher M.
        • et al.
        A complete mtDNA genome of an early modern human from Kostenki, Russia.
        Curr. Biol. 2010; 20: 231-236
        • Loreille O.
        • Koshinsky H.
        • Fofanov V.Y.
        • Irwin J.A.
        Application of next generation sequencing technologies to the identification of highly degraded unknown soldiers’ remains.
        Forensic Sci. Int. Genet. Suppl. Ser. 2011; 3: e540-e541
        • Templeton J.E.
        • Brotherton P.M.
        • Llamas B.
        • Soubrier J.
        • Haak W.
        • Cooper A.
        • et al.
        DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification.
        Investig. Genet. 2013; 4 (26-2223-4-26)
      2. QIAGEN® User-Developed Protocol. Purification of total DNA from nails, hair, or feathers using the DNeasy® Blood & Tissue Kit.

        • Burnside E.S.
        • Bintz B.J.
        • Wilson M.R.
        Improved extraction efficiency of human mitochondrial DNA from hair shafts and its implications for sequencing of the entire mtGenome from a single hair fragment,.
        in: Proceedings of the American Academy of Forensic Sciences 65th Annual Meeting, Washington, DCFebruary 18–23 2012
        • Kavlick M.F.
        • Lawrence H.S.
        • Merritt R.T.
        • Fisher C.
        • Isenberg A.
        • Robertson J.M.
        • Budowle B.
        Quantification of human mitochondrial DNA using synthesized DNA standards.
        J. Forensic Sci. 2011; 56: 1457-1463
        • Lyons E.A.
        • Scheible M.K.
        • Sturk-Andreaggi K.
        • Irwin J.A.
        • Just R.S.
        A high-throughput Sanger strategy for human mitochondrial genome sequencing.
        BMC Genomics. 2013; 14 (881-2164-14-881)
        • Andrews R.M.
        • Kubacka I.
        • Chinnery P.F.
        • Lightowlers R.N.
        • Turnbull D.M.
        • Howell N.
        Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.
        Nat. Genet. 1999; 23: 147
        • Hellmann A.
        • Rohleder U.
        • Schmitter H.
        STR typing of human telogen hairs - a new approach.
        Int. J. Legal Med. 2001; 114: 269-273
        • Bauer C.M.
        • Niederstätter H.
        • McGlynn G.
        • Stadler H.
        • Parson W.
        Comparison of morphological and molecular genetic sex-typing on mediaeval human skeletal remains.
        Forensic Sci. Int. Genet. 2013; 7: 581-586
        • Fendt L.
        • Zimmermann B.
        • Daniaux M.
        • Parson W.
        Sequencing strategy for the whole mitochondrial genome resulting in high quality sequences.
        BMC Genomics. 2009; 10: 139
        • Bandelt H.-J.
        • Parson W.
        Consistent treatment of length variants in the human mtDNA control region: a reappraisal.
        Int. J. Legal Med. 2008; 122: 11-21
        • Parson W.
        • Gusmao L.
        • Hares D.R.
        • Irwin J.A.
        • Mayr W.R.
        • Morling N.
        • Pokorak E.
        • Prinz M.
        • Salas A.
        • Schneider P.M.
        • Parsons T.J.
        DNA Commission of the International Society for Forensic Genetics: revised and extended guidelines for mitochondrial DNA typing.
        Forensic Sci. Int. Genet. 2014; 13: 134-142
        • Berger C.
        • Parson W.
        Mini-midi-mito: Adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples.
        Forensic Sci. Int. Genet. 2009; 3: 149-153
      3. Primer 3:

      4. Oligo Analyzer 3.1:

      5. UCSC In-silico PCR:

        • Vallone P.M.
        • Butler J.M.
        AutoDimer: a screening tool for primer-dimer and hairpin structures.
        Biotechniques. 2004; 37: 226-231
        • Li H.
        • Durbin R.
        Fast and accurate short read alignment with Burrows–Wheeler transform.
        Bioinformatics. 2009; 25: 1754-1760
      6. picardtools:

        • DePristo M.A.
        • Banks E.
        • Poplin R.
        • Garimella K.V.
        • Maguire J.R.
        • Hartl C.
        • et al.
        A framework for variation discovery and genotyping using next-generation DNA sequencing data.
        Nat. Genet. 2011; 43: 491-498
        • Thorvaldsdóttir H.
        • Robinson J.T.
        • Mesirov J.P.
        Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration.
        Briefings Bioinform. 2013; 14: 178-192
        • Just R.S.
        • Scheible M.K.
        • Fast S.A.
        • Sturk-Andreaggi K.
        • Higginbotham J.L.
        • Lyons E.A.
        • et al.
        Development of forensic-quality full mtGenome haplotypes: Success rates with low template specimens.
        Forensic Sci. Int. Genet. 2014; 10: 73-79
        • Anderson S.
        • Bankier A.T.
        • Barrell B.G.
        • de Bruijn M.H.
        • Coulson A.R.
        • Drouin J.
        • et al.
        Sequence and organization of the human mitochondrial genome.
        Nature. 1981; 290: 457-465
        • Rasmussen M.
        • Li Y.
        • Lindgreen S.
        • Pederson J.
        • Albrechtsen A.
        • Moltke I.
        • Mestpalu M.
        • et al.
        Ancient human genome sequence of an extinct Palaeo-Eskimo.
        Nature. 2010; 463: 757-762
        • Gilbert M.T.P.
        • Tomsho L.P.
        • Rendulic S.
        • Packard M.
        • Drautz D.I.
        • Sher A.
        • et al.
        Whole-genome shotgun sequencing of mitochondria from ancient hair shafts.
        Science. 2007; 317: 1927-1930
        • Miller W.
        • Drautz D.I.
        • Ratan A.
        • Pusey B.
        • Qi J.
        • Lesk A.M.
        • et al.
        Sequencing the nuclear genome of the extinct woolly mammoth.
        Nature. 2008; 456: 387-390