Fidelity of capture-enrichment for mtDNA genome sequencing: Influence of NUMTs

Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, D04103, Leipzig, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 05/2012; 40(18):e137. DOI: 10.1093/nar/gks499
Source: PubMed


Enriching target sequences in sequencing libraries via capture hybridization to bait/probes is an efficient means of leveraging
the capabilities of next-generation sequencing for obtaining sequence data from target regions of interest. However, homologous
sequences from non-target regions may also be enriched by such methods. Here we investigate the fidelity of capture enrichment
for complete mitochondrial DNA (mtDNA) genome sequencing by analyzing sequence data for nuclear copies of mtDNA (NUMTs). Using
capture-enriched sequencing data from a mitochondria-free cell line and the parental cell line, and from samples previously
sequenced from long-range PCR products, we demonstrate that NUMT alleles are indeed present in capture-enriched sequence data,
but at low enough levels to not influence calling the authentic mtDNA genome sequence. However, distinguishing NUMT alleles
from true low-level mutations (e.g. heteroplasmy) is more challenging. We develop here a computational method to distinguish
NUMT alleles from heteroplasmies, using sequence data from artificial mixtures to optimize the method.

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Available from: Roland Schröder, May 26, 2015
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    • "As a result, it may be more difficult to determine the phasing of reads and thus the overall haplotype of a low-level sequence – something that could otherwise assist in the identification of NUMT background. Though NUMT amplification does not pose a significant challenge with current mtDNA typing processes as applied to the CR even when extremely short amplicons are employed, the results of a few studies [64] [65] [66] suggest that NUMT detection should be considered in a MPS framework. Fortunately, and as with current Sanger sequence data, the identification of NUMTs as the source of very low-level mixed mtDNA data can almost certainly be addressed with laboratory-based, bioinformatic, and other data handling and review methods. "
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    ABSTRACT: Long an important and useful tool in forensic genetic investigations, mitochondrial DNA (mtDNA) typing continues to mature. Research in the last few years has demonstrated both that data from the entire molecule will have practical benefits in forensic DNA casework, and that massively parallel sequencing (MPS) methods will make full mitochondrial genome (mtGenome) sequencing of forensic specimens feasible and cost-effective. A spate of recent studies has employed these new technologies to assess intraindividual mtDNA variation. However, in several instances, contamination and other sources of mixed mtDNA data have been erroneously identified as heteroplasmy. Well vetted mtGenome datasets based on both Sanger and MPS sequences have found authentic point heteroplasmy in approximately 25% of individuals when minor component detection thresholds are in the range of 10-20%, along with positional distribution patterns in the coding region that differ from patterns of point heteroplasmy in the well-studied control region. A few recent studies that examined very low-level heteroplasmy are concordant with these observations when the data are examined at a common level of resolution. In this review we provide an overview of considerations related to the use of MPS technologies to detect mtDNA heteroplasmy. In addition, we examine published reports on point heteroplasmy to characterize features of the data that will assist in the evaluation of future mtGenome data developed by any typing method. Copyright © 2015 Z. Published by Elsevier Ireland Ltd.. All rights reserved.
    Full-text · Article · May 2015 · Forensic Science International: Genetics
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    • "The use of LR-PCR for massively parallel mitochondrial sequencing has proven to have the most sensitive heteroplasmy and large deletion detection [23]–[25]. This is largely due to LR-PCR's ability to deliver uniform coverage and to limit the amplification of similar NUMT sequences [26] found with methods that use hybridization capture techniques. Nevertheless, LR-PCR methods can be hindered by jumping PCR artifacts with NUMTs, meaning that often the heteroplasmy sensitivity is limited to allele frequencies of 1% or greater, despite the fact that sequencing techniques can deliver accurate allele frequencies far below this [26] with other templates. "
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    ABSTRACT: We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a "DNA diode", where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA.
    Full-text · Article · May 2014 · PLoS ONE
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    • "Chinese Liou et al. (2012) 64 vs 77 No association Tunisian Hsouna et al. (2013) "
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    ABSTRACT: The last 30years of research greatly contributed to shed light on the role of mitochondrial DNA (mtDNA) variability in aging, although contrasting results have been reported, mainly due to bias regarding the population size and stratification, and to the use of analysis methods (haplogroup classification) that resulted to be not sufficiently adequate to grasp the complexity of the phenomenon. A 5-years European study (the GEHA EU project) collected and analysed data on mtDNA variability on an unprecedented number of long-living subjects (enriched for longevity genes) and a comparable number of controls (matched for gender and ethnicity) in Europe. This very large study allowed a reappraisal of the role of both the inherited and the somatic mtDNA variability in aging, as an association with longevity emerged only when mtDNA variants in OXPHOS complexes co-occurred. Moreover, the availability of data from both nuclear and mitochondrial genomes on a large number of subjects paves the way for an evaluation at a very large scale of the epistatic interactions at a higher level of complexity. This scenario is expected to be even more clarified in the next future with the use of next generation sequencing (NGS) techniques, which are becoming applicable to evaluate mtDNA variability and, then, new mathematical/bioinformatic analysis methods are urgently needed. Recent advances of association studies on age-related diseases and mtDNA variability will be also discussed in this review, taking into account the bias hidden by population stratification. Finally very recent findings in terms of mtDNA heteroplasmy (i.e. the coexistence of wild type and mutated copies of mtDNA) and aging as well as mitochondrial epigenetic mechanisms will be also discussed.
    Full-text · Article · Apr 2014 · Experimental gerontology
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