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1.  Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine 
Human Mutation  2015;36(12):1236-1247.
ABSTRACT
Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long‐range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.
doi:10.1002/humu.22905
PMCID: PMC5057296  PMID: 26387877
mitochondria; mtDNA; next‐generation sequencing; NGS; massively parallel sequencing; MPS
2.  Phy-Mer: a novel alignment-free and reference-independent mitochondrial haplogroup classifier 
Bioinformatics  2014;31(8):1310-1312.
Motivation: All current mitochondrial haplogroup classification tools require variants to be detected from an alignment with the reference sequence and to be properly named according to the canonical nomenclature standards for describing mitochondrial variants, before they can be compared with the haplogroup determining polymorphisms. With the emergence of high-throughput sequencing technologies and hence greater availability of mitochondrial genome sequences, there is a strong need for an automated haplogroup classification tool that is alignment-free and agnostic to reference sequence.
Results: We have developed a novel mitochondrial genome haplogroup-defining algorithm using a k-mer approach namely Phy-Mer. Phy-Mer performs equally well as the leading haplogroup classifier, HaploGrep, while avoiding the errors that may occur when preparing variants to required formats and notations. We have further expanded Phy-Mer functionality such that next-generation sequencing data can be used directly as input.
Availability and implementation: Phy-Mer is publicly available under the GNU Affero General Public License v3.0 on GitHub (https://github.com/danielnavarrogomez/phy-mer).
Contact: Xiaowu_Gai@meei.harvard.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btu825
PMCID: PMC4393525  PMID: 25505086
3.  Mitochondrial Disease Sequence Data Resource (MSeqDR): A global grass-roots consortium to facilitate deposition, curation, annotation, and integrated analysis of genomic data for the mitochondrial disease clinical and research communities 
Molecular genetics and metabolism  2014;114(3):388-396.
Success rates for genomic analyses of highly heterogeneous disorders can be greatly improved if a large cohort of patient data is assembled to enhance collective capabilities for accurate sequence variant annotation, analysis, and interpretation. Indeed, molecular diagnostics requires the establishment of robust data resources to enable data sharing that informs accurate understanding of genes, variants, and phenotypes. The “Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium” is a grass-roots effort facilitated by the United Mitochondrial Disease Foundation to identify and prioritize specific genomic data analysis needs of the global mitochondrial disease clinical and research community. A central Web portal (https://mseqdr.org) facilitates the coherent compilation, organization, annotation, and analysis of sequence data from both nuclear and mitochondrial genomes of individuals and families with suspected mitochondrial disease. This Web portal provides users with a flexible and expandable suite of resources to enable variant-, gene-, and exome-level sequence analysis in a secure, Web-based, and user-friendly fashion. Users can also elect to share data with other MSeqDR Consortium members, or even the general public, either by custom annotation tracks or through use of a convenient distributed annotation system (DAS) mechanism. A range of data visualization and analysis tools are provided to facilitate user interrogation and understanding of genomic, and ultimately phenotypic, data of relevance to mitochondrial biology and disease. Currently available tools for nuclear and mitochondrial gene analyses include an MSeqDR GBrowse instance that hosts optimized mitochondrial disease and mitochondrial DNA (mtDNA) specific annotation tracks, as well as an MSeqDR locus-specific database (LSDB) that curates variant data on more than 1,300 genes that have been implicated in mitochondrial disease and/or encode mitochondria-localized proteins. MSeqDR is integrated with a diverse array of mtDNA data analysis tools that are both freestanding and incorporated into an online exome-level dataset curation and analysis resource (GEM.app) that is being optimized to support needs of the MSeqDR community. In addition, MSeqDR supports mitochondrial disease phenotyping and ontology tools, and provides variant pathogenicity assessment features that enable community review, feedback, and integration with the public ClinVar variant annotation resource. A centralized Web-based informed consent process is being developed, with implementation of a Global Unique Identifier (GUID) system to integrate data deposited on a given individual from different sources. Community-based data deposition into MSeqDR has already begun. Future efforts will enhance capabilities to incorporate phenotypic data that enhance genomic data analyses. MSeqDR will fill the existing void in bioinformatics tools and centralized knowledge that are necessary to enable efficient nuclear and mtDNA genomic data interpretation by a range of shareholders across both clinical diagnostic and research settings. Ultimately, MSeqDR is focused on empowering the global mitochondrial disease community to better define and explore mitochondrial disease.
doi:10.1016/j.ymgme.2014.11.016
PMCID: PMC4512182  PMID: 25542617
4.  Fine Dissection of Human Mitochondrial DNA Haplogroup HV Lineages Reveals Paleolithic Signatures from European Glacial Refugia 
PLoS ONE  2015;10(12):e0144391.
Genetic signatures from the Paleolithic inhabitants of Eurasia can be traced from the early divergent mitochondrial DNA lineages still present in contemporary human populations. Previous studies already suggested a pre-Neolithic diffusion of mitochondrial haplogroup HV*(xH,V) lineages, a relatively rare class of mtDNA types that includes parallel branches mainly distributed across Europe and West Asia with a certain degree of structure. Up till now, variation within haplogroup HV was addressed mainly by analyzing sequence data from the mtDNA control region, except for specific sub-branches, such as HV4 or the widely distributed haplogroups H and V. In this study, we present a revised HV topology based on full mtDNA genome data, and we include a comprehensive dataset consisting of 316 complete mtDNA sequences including 60 new samples from the Italian peninsula, a previously underrepresented geographic area. We highlight points of instability in the particular topology of this haplogroup, reconstructed with BEAST-generated trees and networks. We also confirm a major lineage expansion that probably followed the Late Glacial Maximum and preceded Neolithic population movements. We finally observe that Italy harbors a reservoir of mtDNA diversity, with deep-rooting HV lineages often related to sequences present in the Caucasus and the Middle East. The resulting hypothesis of a glacial refugium in Southern Italy has implications for the understanding of late Paleolithic population movements and is discussed within the archaeological cultural shifts occurred over the entire continent.
doi:10.1371/journal.pone.0144391
PMCID: PMC4671665  PMID: 26640946
5.  Human genetics of the Kula Ring: Y-chromosome and mitochondrial DNA variation in the Massim of Papua New Guinea 
European Journal of Human Genetics  2014;22(12):1393-1403.
The island region at the southeastern-most tip of New Guinea and its inhabitants known as Massim are well known for a unique traditional inter-island trading system, called Kula or Kula Ring. To characterize the Massim genetically, and to evaluate the influence of the Kula Ring on patterns of human genetic variation, we analyzed paternally inherited Y-chromosome (NRY) and maternally inherited mitochondrial (mt) DNA polymorphisms in >400 individuals from this region. We found that the nearly exclusively Austronesian-speaking Massim people harbor genetic ancestry components of both Asian (AS) and Near Oceanian (NO) origin, with a proportionally larger NO NRY component versus a larger AS mtDNA component. This is similar to previous observations in other Austronesian-speaking populations from Near and Remote Oceania and suggests sex-biased genetic admixture between Asians and Near Oceanians before the occupation of Remote Oceania, in line with the Slow Boat from Asia hypothesis on the expansion of Austronesians into the Pacific. Contrary to linguistic expectations, Rossel Islanders, the only Papuan speakers of the Massim, showed a lower amount of NO genetic ancestry than their Austronesian-speaking Massim neighbors. For the islands traditionally involved in the Kula Ring, a significant correlation between inter-island travelling distances and genetic distances was observed for mtDNA, but not for NRY, suggesting more male- than female-mediated gene flow. As traditionally only males take part in the Kula voyages, this finding may indicate a genetic signature of the Kula Ring, serving as another example of how cultural tradition has shaped human genetic diversity.
doi:10.1038/ejhg.2014.38
PMCID: PMC4231407  PMID: 24619143
6.  Mitochondrial Mutations in Subjects with Psychiatric Disorders 
PLoS ONE  2015;10(5):e0127280.
A considerable body of evidence supports the role of mitochondrial dysfunction in psychiatric disorders and mitochondrial DNA (mtDNA) mutations are known to alter brain energy metabolism, neurotransmission, and cause neurodegenerative disorders. Genetic studies focusing on common nuclear genome variants associated with these disorders have produced genome wide significant results but those studies have not directly studied mtDNA variants. The purpose of this study is to investigate, using next generation sequencing, the involvement of mtDNA variation in bipolar disorder, schizophrenia, major depressive disorder, and methamphetamine use. MtDNA extracted from multiple brain regions and blood were sequenced (121 mtDNA samples with an average of 8,800x coverage) and compared to an electronic database containing 26,850 mtDNA genomes. We confirmed novel and rare variants, and confirmed next generation sequencing error hotspots by traditional sequencing and genotyping methods. We observed a significant increase of non-synonymous mutations found in individuals with schizophrenia. Novel and rare non-synonymous mutations were found in psychiatric cases in mtDNA genes: ND6, ATP6, CYTB, and ND2. We also observed mtDNA heteroplasmy in brain at a locus previously associated with schizophrenia (T16519C). Large differences in heteroplasmy levels across brain regions within subjects suggest that somatic mutations accumulate differentially in brain regions. Finally, multiplasmy, a heteroplasmic measure of repeat length, was observed in brain from selective cases at a higher frequency than controls. These results offer support for increased rates of mtDNA substitutions in schizophrenia shown in our prior results. The variable levels of heteroplasmic/multiplasmic somatic mutations that occur in brain may be indicators of genetic instability in mtDNA.
doi:10.1371/journal.pone.0127280
PMCID: PMC4444211  PMID: 26011537
7.  Neolithic mitochondrial haplogroup H genomes and the genetic origins of Europeans 
Nature communications  2013;4:1764.
Haplogroup (hg) H dominates present-day Western European mitochondrial (mt) DNA variability (>40%), yet was less common (~19%) amongst Early Neolithic farmers (~5450 BC) and virtually absent in Mesolithic hunter-gatherers. Here we investigate this major component of the maternal population history of modern Europeans and sequence 39 complete hg H mitochondrial genomes from ancient human remains. We then compare this ‘real-time’ genetic data with cultural changes taking place between the Early Neolithic (~5450 BC) and Bronze Age (~2200 BC) in Central Europe. Our results reveal that the current diversity and distribution of hg H were largely established by the Mid-Neolithic (~4000 BC), but with substantial genetic contributions from subsequent pan-European cultures such as the Bell Beakers expanding out of Iberia in the Late Neolithic (~2800 BC). Dated hg H genomes allow us to reconstruct the recent evolutionary history of hg H and reveal a mutation rate 45% higher than current estimates for human mitochondria.
doi:10.1038/ncomms2656
PMCID: PMC3978205  PMID: 23612305
8.  Concept for estimating mitochondrial DNA haplogroups using a maximum likelihood approach (EMMA)☆ 
The assignment of haplogroups to mitochondrial DNA haplotypes contributes substantial value for quality control, not only in forensic genetics but also in population and medical genetics. The availability of Phylotree, a widely accepted phylogenetic tree of human mitochondrial DNA lineages, led to the development of several (semi-)automated software solutions for haplogrouping. However, currently existing haplogrouping tools only make use of haplogroup-defining mutations, whereas private mutations (beyond the haplogroup level) can be additionally informative allowing for enhanced haplogroup assignment. This is especially relevant in the case of (partial) control region sequences, which are mainly used in forensics. The present study makes three major contributions toward a more reliable, semi-automated estimation of mitochondrial haplogroups. First, a quality-controlled database consisting of 14,990 full mtGenomes downloaded from GenBank was compiled. Together with Phylotree, these mtGenomes serve as a reference database for haplogroup estimates. Second, the concept of fluctuation rates, i.e. a maximum likelihood estimation of the stability of mutations based on 19,171 full control region haplotypes for which raw lane data is available, is presented. Finally, an algorithm for estimating the haplogroup of an mtDNA sequence based on the combined database of full mtGenomes and Phylotree, which also incorporates the empirically determined fluctuation rates, is brought forward. On the basis of examples from the literature and EMPOP, the algorithm is not only validated, but both the strength of this approach and its utility for quality control of mitochondrial haplotypes is also demonstrated.
doi:10.1016/j.fsigen.2013.07.005
PMCID: PMC3819997  PMID: 23948335
mtDNA; Haplogroup; EMPOP; Fluctuation rates; Phylotree
9.  Clinal distribution of human genomic diversity across the Netherlands despite archaeological evidence for genetic discontinuities in Dutch population history 
Background
The presence of a southeast to northwest gradient across Europe in human genetic diversity is a well-established observation and has recently been confirmed by genome-wide single nucleotide polymorphism (SNP) data. This pattern is traditionally explained by major prehistoric human migration events in Palaeolithic and Neolithic times. Here, we investigate whether (similar) spatial patterns in human genomic diversity also occur on a micro-geographic scale within Europe, such as in the Netherlands, and if so, whether these patterns could also be explained by more recent demographic events, such as those that occurred in Dutch population history.
Methods
We newly collected data on a total of 999 Dutch individuals sampled at 54 sites across the country at 443,816 autosomal SNPs using the Genome-Wide Human SNP Array 5.0 (Affymetrix). We studied the individual genetic relationships by means of classical multidimensional scaling (MDS) using different genetic distance matrices, spatial ancestry analysis (SPA), and ADMIXTURE software. We further performed dedicated analyses to search for spatial patterns in the genomic variation and conducted simulations (SPLATCHE2) to provide a historical interpretation of the observed spatial patterns.
Results
We detected a subtle but clearly noticeable genomic population substructure in the Dutch population, allowing differentiation of a north-eastern, central-western, central-northern and a southern group. Furthermore, we observed a statistically significant southeast to northwest cline in the distribution of genomic diversity across the Netherlands, similar to earlier findings from across Europe. Simulation analyses indicate that this genomic gradient could similarly be caused by ancient as well as by the more recent events in Dutch history.
Conclusions
Considering the strong archaeological evidence for genetic discontinuity in the Netherlands, we interpret the observed clinal pattern of genomic diversity as being caused by recent rather than ancient events in Dutch population history. We therefore suggest that future human population genetic studies pay more attention to recent demographic history in interpreting genetic clines. Furthermore, our study demonstrates that genetic population substructure is detectable on a small geographic scale in Europe despite recent demographic events, a finding we consider potentially relevant for future epidemiological and forensic studies.
doi:10.1186/2041-2223-4-9
PMCID: PMC3707805  PMID: 23687922
Population substructure; Genetic cline; Genome-wide diversity; SNP; Europe; Netherlands
10.  Mitochondrial Mutations and Polymorphisms in Psychiatric Disorders 
Frontiers in Genetics  2012;3:103.
Mitochondrial deficiencies with unknown causes have been observed in schizophrenia (SZ) and bipolar disorder (BD) in imaging and postmortem studies. Polymorphisms and somatic mutations in mitochondrial DNA (mtDNA) were investigated as potential causes with next generation sequencing of mtDNA (mtDNA-Seq) and genotyping arrays in subjects with SZ, BD, major depressive disorder (MDD), and controls. The common deletion of 4,977 bp in mtDNA was compared between SZ and controls in 11 different vulnerable brain regions and in blood samples, and in dorsolateral prefrontal cortex (DLPFC) of BD, SZ, and controls. In a separate analysis, association of mitochondria SNPs (mtSNPs) with SZ and BD in European ancestry individuals (n = 6,040) was tested using Genetic Association Information Network (GAIN) and Wellcome Trust Case Control Consortium 2 (WTCCC2) datasets. The common deletion levels were highly variable across brain regions, with a 40-fold increase in some regions (nucleus accumbens, caudate nucleus and amygdala), increased with age, and showed little change in blood samples from the same subjects. The common deletion levels were increased in the DLPFC for BD compared to controls, but not in SZ. Full mtDNA genome resequencing of 23 subjects, showed seven novel homoplasmic mutations, five were novel synonymous coding mutations. By logistic regression analysis there were no significant mtSNPs associated with BD or SZ after genome wide correction. However, nominal association of mtSNPs (p < 0.05) to SZ and BD were found in the hypervariable region of mtDNA to T195C and T16519C. The results confirm prior reports that certain brain regions accumulate somatic mutations at higher levels than blood. The study in mtDNA of common polymorphisms, somatic mutations, and rare mutations in larger populations may lead to a better understanding of the pathophysiology of psychiatric disorders.
doi:10.3389/fgene.2012.00103
PMCID: PMC3379031  PMID: 22723804
mitochondria; homoplasmy; common deletion; novel mutations; schizophrenia; bipolar disorder
11.  Population Genetic Structure in Indian Austroasiatic Speakers: The Role of Landscape Barriers and Sex-Specific Admixture 
Molecular biology and evolution  2010;28(2):1013-1024.
The geographic origin and time of dispersal of Austroasiatic (AA) speakers, presently settled in south and southeast Asia, remains disputed. Two rival hypotheses, both assuming a demic component to the language dispersal, have been proposed. The first of these places the origin of Austroasiatic speakers in southeast Asia with a later dispersal to south Asia during the Neolithic, whereas the second hypothesis advocates pre-Neolithic origins and dispersal of this language family from south Asia. To test the two alternative models, this study combines the analysis of uniparentally inherited markers with 610,000 common single nucleotide polymorphism loci from the nuclear genome. Indian AA speakers have high frequencies of Y chromosome haplogroup O2a; our results show that this haplogroup has significantly higher diversity and coalescent time (17–28 thousand years ago) in southeast Asia, strongly supporting the first of the two hypotheses. Nevertheless, the results of principal component and “structure-like” analyses on autosomal loci also show that the population history of AA speakers in India is more complex, being characterized by two ancestral components—one represented in the pattern of Y chromosomal and EDAR results and the other by mitochondrial DNA diversity and genomic structure. We propose that AA speakers in India today are derived from dispersal from southeast Asia, followed by extensive sex-specific admixture with local Indian populations.
doi:10.1093/molbev/msq288
PMCID: PMC3355372  PMID: 20978040
Austroasiatic; mtDNA; Y chromosome; autosomes; admixture
12.  An efficient multiplex genotyping approach for detecting the major worldwide human Y-chromosome haplogroups 
The Y chromosome is paternally inherited and therefore serves as an evolutionary marker of patrilineal descent. Worldwide DNA variation within the non-recombining portion of the Y chromosome can be represented as a monophyletic phylogenetic tree in which the branches (haplogroups) are defined by at least one SNP. Previous human population genetics research has produced a wealth of knowledge about the worldwide distribution of Y-SNP haplogroups. Here, we apply previous and very recent knowledge on the Y-SNP phylogeny and Y-haplogroup distribution by introducing two multiplex genotyping assays that allow for the hierarchical detection of 28 Y-SNPs defining the major worldwide Y haplogroups. PCR amplicons were kept small to make the method sensitive and thereby applicable to DNA of limited amount and/or quality such as in forensic settings. These Y-SNP assays thus form a valuable tool for researchers in the fields of forensic genetics and genetic anthropology to infer a man's patrilineal bio-geographic ancestry from DNA.
doi:10.1007/s00414-011-0605-2
PMCID: PMC3192277  PMID: 21785904
Y chromosome; Y-SNP; Haplogroup; Patrilineal ancestry; Bio-geographic ancestry; Multiplex SNaPshot
13.  Multiplex genotyping system for efficient inference of matrilineal genetic ancestry with continental resolution 
Background
In recent years, phylogeographic studies have produced detailed knowledge on the worldwide distribution of mitochondrial DNA (mtDNA) variants, linking specific clades of the mtDNA phylogeny with certain geographic areas. However, a multiplex genotyping system for the detection of the mtDNA haplogroups of major continental distribution that would be desirable for efficient DNA-based bio-geographic ancestry testing in various applications is still missing.
Results
Three multiplex genotyping assays, based on single-base primer extension technology, were developed targeting a total of 36 coding-region mtDNA variants that together differentiate 43 matrilineal haplo-/paragroups. These include the major diagnostic haplogroups for Africa, Western Eurasia, Eastern Eurasia and Native America. The assays show high sensitivity with respect to the amount of template DNA: successful amplification could still be obtained when using as little as 4 pg of genomic DNA and the technology is suitable for medium-throughput analyses.
Conclusions
We introduce an efficient and sensitive multiplex genotyping system for bio-geographic ancestry inference from mtDNA that provides resolution on the continental level. The method can be applied in forensics, to aid tracing unknown suspects, as well as in population studies, genealogy and personal ancestry testing. For more complete inferences of overall bio-geographic ancestry from DNA, the mtDNA system provided here can be combined with multiplex systems for suitable autosomal and, in the case of males, Y-chromosomal ancestry-sensitive DNA markers.
doi:10.1186/2041-2223-2-6
PMCID: PMC3078086  PMID: 21429198
14.  Evaluating Self-declared Ancestry of U.S. Americans with Autosomal, Y-chromosomal and Mitochondrial DNA 
Human Mutation  2010;31(12):E1875-E1893.
The current U.S. population represents an amalgam of individuals originating mainly from four continental regions (Africa, Europe, Asia and America). To study the genetic ancestry and compare with self-declared ancestry we have analyzed paternally, maternally and bi-parentally inherited DNA markers sensitive for indicating continental genetic ancestry in all four major U.S. American groups. We found that self-declared U.S. Hispanics and U.S. African Americans tend to show variable degrees of continental genetic admixture among the three genetic systems, with evidence for a marked sex-biased admixture history. Moreover, for these two groups we observed significant regional variation across the country in genetic admixture. In contrast, self-declared U.S. European and U.S. Asian Americans were genetically more homogeneous at the continental ancestry level. Two autosomal ancestry-sensitive markers located in skin pigmentation candidate genes showed significant differences in self-declared U.S. African Americans or U.S. European Americans, relative to their assumed parental populations from Africa or Europe. This provides genetic support for the importance of skin color in the complex process of ancestry identification. © 2010 Wiley-Liss, Inc.
doi:10.1002/humu.21366
PMCID: PMC3051415  PMID: 20886636
U.S. Americans; genetic ancestry; self-declared ancestry; ASM; AIM; Y-chromosome; NRY; mtDNA

Results 1-14 (14)