Article

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

ABSTRACT 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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    • "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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    • "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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