Department of Forensic Medicine, The Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Bydgoszcz, Poland.
Department od Forensic Medicine, Medical University in Lublin, Poland.
Arch Med Sadowej Kryminol. 2023;73(2):131-138. doi: 10.4467/16891716AMSIK.23.011.18686.
Massively parallel sequencing of mitogenomes usually requires prior amplification. The PCR step may influence the quality of the data obtained, especially when low-level heteroplasmy detection is applied.
The aim of this study was to compare the reliability of two different DNA polymerases in detecting homoplasmic and heteroplasmic substitutions in human mitogenomes.
Mitogenomes of five samples were amplified with Long PCR Enzyme Mix from Fermentas or TaKaRa LA Taq DNA Polymerase from TaKaRa. Then, NexteraTM XT DNA libraries were sequenced on MiSeq FGx platform (Illumina). mtDNA substitutions were called for alternative variants above the 1% level.
All homoplasmic substitutions detected in amplicons generated with polymerases studied here and sequenced on MiSeq FGx system were consistently identified as homoplasmies with alternative sequencing methods. TaKaRa LA Taq DNA Polymerase was found to be less accurate in low-level heteroplasmy detection than Long PCR Enzyme Mix enzyme as more false negative and false positive results were observed for minority variants called above the 1% level. Nevertheless, both PCR systems studied can be successfully used to detect authentic mtDNA substitutions, for which minority variants exceed the 3.61% level assuming at least 10,000x coverage and sequencing Nextera XT DNA libraries on MiSeq FGx machine.
The accuracy and sensitivity of point heteroplasmy detection with the MiSeq FGx instrument varies on polymerase used for mtDNA amplification. Therefore, it is recommended to validate the laboratory protocols used for mtDNA substitution detection prior to their implementation for the forensic or medical genetics purposes.
对线粒体基因组进行大规模平行测序通常需要事先进行扩增。PCR 步骤可能会影响所获得数据的质量，尤其是在应用低水平异质性检测时。
本研究旨在比较两种不同 DNA 聚合酶在检测人类线粒体基因组中同质和异质替换的可靠性。
使用 Fermentas 的 Long PCR Enzyme Mix 或 TaKaRa 的 TaKaRa LA Taq DNA 聚合酶扩增五个样本的线粒体基因组。然后，在 MiSeq FGx 平台（Illumina）上对 NexteraTM XT DNA 文库进行测序。mtDNA 替换在替代变体超过 1%的水平上被调用。
使用本研究中研究的聚合酶生成并在 MiSeq FGx 系统上测序的所有同质替换均一致地被鉴定为同质体，具有替代测序方法。与 Long PCR Enzyme Mix 酶相比，TaKaRa LA Taq DNA 聚合酶在低水平异质性检测中准确性较低，因为对于调用的替代变体超过 1%的水平，观察到更多的假阴性和假阳性结果。然而，两种研究的 PCR 系统都可以成功地用于检测真实的 mtDNA 替换，假设至少有 10000x 的覆盖度，并且在 MiSeq FGx 机器上测序 Nextera XT DNA 文库，替代变体超过 3.61%。
使用 MiSeq FGx 仪器对点异质性检测的准确性和灵敏度取决于用于 mtDNA 扩增的聚合酶。因此，建议在将 mtDNA 替换检测的实验室方案用于法医或医学遗传学目的之前，对其进行验证。
