Forensic Population Genetics - Original Research Full mtGenome reference data: Development and characterization of 588 forensic-quality haplotypes representing three U.S. populations

Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S. population groups (African American, Caucasian and Hispanic) from anonymized, randomly-sampled specimens. Data production utilized an 8-amplicon, 135 sequencing reaction Sanger-based protocol, performed in semi-automated fashion on robotic instrumentation. Data review followed an intensive multi-step strategy that included a minimum of three independent reviews of the raw data at two laboratories; repeat screenings of all insertions, deletions, heteroplasmies, transversions and any additional private mutations; and a check for phylogenetic feasibility. For all three populations, nearly complete resolution of the haplotypes was achieved with full mtGenome sequences: 90.3-98.8% of haplotypes were unique per population, an improvement of 7.7-29.2% over control region sequencing alone, and zero haplotypes overlapped between populations. Inferred maternal biogeographic ancestry frequencies for each population and heteroplasmy rates in the control region were generally consistent with published datasets. In the coding region, nearly 90% of individuals exhibited length heteroplasmy in the 12418-12425 adenine homopolymer; and despite a relatively high rate of point heteroplasmy (23.8% of individuals across the entire molecule), coding region point heteroplasmies shared by more than one individual were notably absent, and transversion-type heteroplasmies were extremely rare. The ratio of nonsynonymous to synonymous changes among point heteroplasmies in the protein-coding genes (1:1.3) and average pathogenicity scores in comparison to data reported for complete substitutions in previous studies seem to provide some additional support for the role of purifying selection in the evolution of the human mtGenome. Overall, these thoroughly vetted full mtGenome population reference data can serve as a standard against which the quality and features of future mtGenome datasets (especially those developed via massively parallel sequencing) may be evaluated, and will provide a solid foundation for the generation of complete mtGenome haplotype frequency estimates for forensic applications.

[1]  P. Forster,et al.  Natural radioactivity and human mitochondrial DNA mutations , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Walther Parson,et al.  EMPOP--a forensic mtDNA database. , 2007, Forensic science international. Genetics.

[3]  Rebecca Just,et al.  Short tandem repeat typing on the 454 platform: strategies and considerations for targeted sequencing of common forensic markers. , 2014, Forensic science international. Genetics.

[4]  D. Dressman,et al.  Heteroplasmic mitochondrial DNA mutations in normal and tumor cells , 2010, Nature.

[5]  Walther Parson,et al.  Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM)☆ , 2013, Forensic science international. Genetics.

[6]  Alfredo Coppa,et al.  The African diaspora: mitochondrial DNA and the Atlantic slave trade. , 2004, American journal of human genetics.

[7]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[8]  Mitchell M Holland,et al.  Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq. , 2014, Forensic science international. Genetics.

[9]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[10]  Mark Stoneking,et al.  Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes. , 2010, American journal of human genetics.

[11]  S. Ho,et al.  Ancient mitogenomics. , 2010, Mitochondrion.

[12]  Jacqueline Weber-Lehmann,et al.  Finding the needle in the haystack: differentiating "identical" twins in paternity testing and forensics by ultra-deep next generation sequencing. , 2014, Forensic science international. Genetics.

[13]  Charles H Brenner,et al.  Fundamental problem of forensic mathematics--the evidential value of a rare haplotype. , 2010, Forensic science international. Genetics.

[14]  E. S. Pearson,et al.  THE USE OF CONFIDENCE OR FIDUCIAL LIMITS ILLUSTRATED IN THE CASE OF THE BINOMIAL , 1934 .

[15]  T. Melton,et al.  Forensic mitochondrial DNA analysis: two years of commercial casework experience in the United States. , 2001, Croatian medical journal.

[16]  D. Turnbull,et al.  Comparative genomics and the evolution of human mitochondrial DNA: assessing the effects of selection. , 2004, American journal of human genetics.

[17]  M. Hofreiter,et al.  Mitogenomic analyses from ancient DNA. , 2013, Molecular phylogenetics and evolution.

[18]  Peter M Vallone,et al.  Evaluating Self-declared Ancestry of U.S. Americans with Autosomal, Y-chromosomal and Mitochondrial DNA , 2010, Human mutation.

[19]  Rebecca S. Just,et al.  Assessing the potential of next generation sequencing technologies for missing persons identification efforts , 2011 .

[20]  Mark R. Wilson,et al.  The mtDNA Population Database: An Integrated Software and Database Resource for Forensic Comparison , 2002 .

[21]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[22]  C. Chi,et al.  Somatic alterations in mitochondrial DNA and mitochondrial dysfunction in gastric cancer progression. , 2014, World journal of gastroenterology.

[23]  W. Parson,et al.  Consistent treatment of length variants in the human mtDNA control region: a reappraisal , 2006, International Journal of Legal Medicine.

[24]  W. Parson,et al.  Development of forensic-quality full mtGenome haplotypes: success rates with low template specimens. , 2014, Forensic science international. Genetics.

[25]  Á. Carracedo,et al.  Charting the ancestry of African Americans. , 2005, American journal of human genetics.

[26]  J. Salles,et al.  Detection and Quantification of the Age‐Related Point Mutation A189G in the Human Mitochondrial DNA , 2006, Journal of forensic sciences.

[27]  Eitan Rubin,et al.  Mitochondrial DNA heteroplasmy in diabetes and normal adults: role of acquired and inherited mutational patterns in twins. , 2012, Human molecular genetics.

[28]  M. Hofreiter,et al.  Next Generation Sequencing of Ancient DNA: Requirements, Strategies and Perspectives , 2010, Genes.

[29]  Walther Parson,et al.  A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA. , 2007, Forensic science international. Genetics.

[30]  Niels Morling,et al.  Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics. , 2014, Forensic science international. Genetics.

[31]  M. Holland,et al.  Second generation sequencing allows for mtDNA mixture deconvolution and high resolution detection of heteroplasmy , 2011, Croatian medical journal.

[32]  A. von Haeseler,et al.  Pattern of nucleotide substitution and rate heterogeneity in the hypervariable regions I and II of human mtDNA. , 1999, Genetics.

[33]  P. Radivojac,et al.  Evaluating Purifying Selection in the Mitochondrial DNA of Various Mammalian Species , 2013, PloS one.

[34]  T. Melton,et al.  Mitochondrial DNA Heteroplasmy. , 2004, Forensic science review.

[35]  V. Fofanov,et al.  Application of next generation sequencing technologies to the identification of highly degraded unknown soldiers’ remains , 2011 .

[36]  R. Just,et al.  A high-throughput Sanger strategy for human mitochondrial genome sequencing , 2013, BMC Genomics.

[37]  Cristina Santos,et al.  Frequency and Pattern of Heteroplasmy in the Control Region of Human Mitochondrial DNA , 2008, Journal of Molecular Evolution.

[38]  Ricardo Rocha,et al.  The diversity present in 5140 human mitochondrial genomes. , 2009, American journal of human genetics.

[39]  Gail P. Clement,et al.  A Twin Study of Mitochondrial DNA Polymorphisms Shows that Heteroplasmy at Multiple Sites Is Associated with mtDNA Variant 16093 but Not with Zygosity , 2011, PloS one.

[40]  B. Llamas,et al.  DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification , 2013, Investigative Genetics.

[41]  W R Mayr,et al.  DNA Commission of the International Society for Forensic Genetics: revised and extended guidelines for mitochondrial DNA typing. , 2014, Forensic science international. Genetics.

[42]  Myung Jin Park,et al.  Quantitative and qualitative profiling of mitochondrial DNA length heteroplasmy , 2004, Electrophoresis.

[43]  Walther Parson,et al.  Questioning the prevalence and reliability of human mitochondrial DNA heteroplasmy from massively parallel sequencing data , 2014, Proceedings of the National Academy of Sciences.

[44]  J. M. Ortega,et al.  Sex-biased gene flow in African Americans but not in American Caucasians. , 2007, Genetics and molecular research : GMR.

[45]  Ralf Bundschuh,et al.  Short-read, high-throughput sequencing technology for STR genotyping. , 2012, BioTechniques. Rapid dispatches.

[46]  F. Sanger,et al.  Sequence and organization of the human mitochondrial genome , 1981, Nature.

[47]  D. Turnbull,et al.  Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA , 1999, Nature Genetics.

[48]  Alfredo Coppa,et al.  The Role of Selection in the Evolution of Human Mitochondrial Genomes , 2006, Genetics.

[49]  Walther Parson,et al.  Development and expansion of high-quality control region databases to improve forensic mtDNA evidence interpretation. , 2007, Forensic science international. Genetics.

[50]  Walther Parson,et al.  Concept for estimating mitochondrial DNA haplogroups using a maximum likelihood approach (EMMA) , 2013, Forensic science international. Genetics.

[51]  Mark R. Wilson,et al.  Characterization of human control region sequences of the African American SWGDAM forensic mtDNA data set. , 2005, Forensic science international.

[52]  María del Mar González,et al.  Frequency and Pattern of Heteroplasmy in the Complete Human Mitochondrial Genome , 2013, PloS one.

[53]  Mark R. Wilson,et al.  A high observed substitution rate in the human mitochondrial DNA control region , 1997, Nature Genetics.

[54]  Manfred Kayser,et al.  Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation , 2009, Human mutation.

[55]  Jian Lu,et al.  Extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals , 2014, Proceedings of the National Academy of Sciences.

[56]  D. Deforce,et al.  Forensic STR analysis using massive parallel sequencing. , 2012, Forensic science international. Genetics.

[57]  Jill S. Barnholtz-Sloan,et al.  Dissecting the Within-Africa Ancestry of Populations of African Descent in the Americas , 2011, PloS one.

[58]  Mark R. Wilson,et al.  Evaluation of Variation in Control Region Sequences for Hispanic Individuals in the SWGDAM mtDNA Data Set , 2006, Journal of forensic sciences.

[59]  M. Wilson,et al.  Simultaneous Detection of Human Mitochondrial DNA and Nuclear‐Inserted Mitochondrial‐origin Sequences (NumtS) using Forensic mtDNA Amplification Strategies and Pyrosequencing Technology , 2014, Journal of forensic sciences.

[60]  R. Montiel,et al.  Understanding differences between phylogenetic and pedigree-derived mtDNA mutation rate: a model using families from the Azores Islands (Portugal). , 2005, Molecular biology and evolution.

[61]  Hans-Jürgen Bandelt,et al.  Extended guidelines for mtDNA typing of population data in forensic science. , 2007, Forensic science international. Genetics.

[62]  W. Anderson,et al.  The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age. , 2000, American journal of human genetics.

[63]  Hans-Jürgen Bandelt,et al.  Current next generation sequencing technology may not meet forensic standards. , 2012, Forensic science international. Genetics.

[64]  W. Parson,et al.  mtGenome reference population databases and the future of forensic mtDNA analysis. , 2011, Forensic science international. Genetics.

[65]  D. Turnbull,et al.  The pedigree rate of sequence divergence in the human mitochondrial genome: there is a difference between phylogenetic and pedigree rates. , 2003, American journal of human genetics.

[66]  Predrag Radivojac,et al.  Comparing phylogeny and the predicted pathogenicity of protein variations reveals equal purifying selection across the global human mtDNA diversity. , 2011, American journal of human genetics.

[67]  E. Willerslev,et al.  Application of full mitochondrial genome sequencing using 454 GS FLX pyrosequencing , 2009 .

[68]  T. Parsons,et al.  Mitochondrial control region sequences from an African American population sample. , 2009, Forensic science international. Genetics.

[69]  T. Parsons,et al.  Investigation of Heteroplasmy in the Human Mitochondrial DNA Control Region: A Synthesis of Observations from More Than 5000 Global Population Samples , 2009, Journal of Molecular Evolution.

[70]  M. Stoneking.  Hypervariable sites in the mtDNA control region are mutational hotspots. , 2000, American journal of human genetics.

[71]  Arne Röhl,et al.  Correcting for purifying selection: an improved human mitochondrial molecular clock. , 2009, American journal of human genetics.

[72]  M. Holland,et al.  A sensitive denaturing gradient-Gel electrophoresis assay reveals a high frequency of heteroplasmy in hypervariable region 1 of the human mtDNA control region. , 2000, American Journal of Human Genetics.

[73]  N. Morling,et al.  Characterization of mutations and sequence variants in the D21S11 locus by next generation sequencing. , 2014, Forensic science international. Genetics.

[74]  Predrag Radivojac,et al.  Automated inference of molecular mechanisms of disease from amino acid substitutions , 2009, Bioinform..

[75]  D. Turnbull,et al.  Relative rates of evolution in the coding and control regions of African mtDNAs. , 2007, Molecular biology and evolution.

[76]  T. Parsons,et al.  Mitochondrial control region sequences from a U.S. "Hispanic" population sample. , 2008, Forensic Science International: Genetics.

[77]  Ramesh Hariharan,et al.  Next-Generation Sequencing of Human Mitochondrial Reference Genomes Uncovers High Heteroplasmy Frequency , 2012, PLoS Comput. Biol..

[78]  Bruce Budowle,et al.  High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq. , 2014, Forensic science international. Genetics.