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Sensitivity of mitochondrial DNA heteroplasmy detection using Next Generation Sequencing

机译：使用下一代测序的线粒体DNA异质检测灵敏度

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Although the use of Next Generation Sequencing (NGS) in mitochondrial DNA (mtDNA) studies related to forensic and human genetics has contributed to the report of heteroplasmy at very low levels (lower than 1% and even 0.5%), their detection is not a straightforward process. Our purpose is to establish mitochondrial heteroplasmy detection limits, generating mixed bases at low frequencies by the PCR co-amplification of mtDNA and a nuclear insertion of mitochondrial origin (NUMT). A primer set that presents a perfect annealing with both mitochondrial and nuclear DNA was used to amplify the mitochondrial DNA region located between positions 6739 and 8910 and the corresponding region located inside a NUMT located in chromosome 1 (GRCh38.p12 Primary Assembly 631910-634079) that presents an identity of 98% with the corresponding region of mtDNA in two samples. Independent Nextera XT (R) (Illumina) NGS libraries were generated for each sample and sequenced in different MiSeq (Illumina) runs. Non-identical and identical positions between individual mtDNA and NUMT sequences were detected, and heteroplasmy detections limits were established: a) with a minor allele frequency < 1.5%, false positive and negative can occur, even with a depth higher than 3000X; b) with a minor allele frequency > 3%, no presence of false positive and negative were detected with a depth of similar to 1000X; and c) with a minor allele frequency between 1.5% and 3%, a minimal depth of 3000X was necessary to avoid false positives or negatives detection. Our results demonstrate an alternative strategy to establish a reliable limit of mitochondrial heteroplasmy detection.
机译：虽然使用与法医和人类遗传学相关的线粒体DNA（MTDNA）研究中的下一代测序（NGS）有助于异质在极低水平（低于1％甚至0.5％）的情况下，它们的检测不是一个直接进程。我们的目的是建立线粒体异质检测限制，通过PCR共同扩增的MTDNA和核插入线粒体来源（NUMT）在低频下产生混合碱基。用线粒体和核DNA呈现完美退火的引物组用于扩增位于位置6739和8910的线粒体DNA区域，以及位于位于染色体1的NUMT内的相应区域（GRCH38.P12主组件631910-634079）这与两个样品中的MTDNA相应区域呈现98％的同一性。为每个样品产生独立的Nextera XT（Illumina）NGS文库并在不同的Miseq（Illumina）运行中测序。检测单个MTDNA和NUMT序列之间的非相同且相同的位置，并建立了异质检测限制：a）较小的等位基因频率<1.5％，可能发生假阳性和阴性，即使深度高于3000x; b）具有次要等位基因频率> 3％，未检测到假阳性和阴性的存在，深度类似于1000x; C）在1.5％和3％之间的次要等位基因频率之间，最小的3000倍深度是必要的，以避免误报或底片检测。我们的结果证明了建立了对线粒体异质检测的可靠限制的替代策略。

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