用不同聚合酶扩增并在 MiSeq FGx 平台上测序的人类有丝分裂基因组中的低水平点异质性检测

K. Skonieczna, Marzanna Ciesielka, Grzegorz Teresiński, Tomasz Grzybowski
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引用次数: 0

摘要

简介对有丝分裂基因组进行大规模平行测序通常需要事先进行扩增。目的:本研究旨在比较两种不同的 DNA 聚合酶检测人类有丝分裂基因组中同质和异质替换的可靠性:用Fermentas公司的Long PCR Enzyme Mix或TaKaRa公司的LA Taq DNA聚合酶扩增5个样本的有丝分裂基因组。然后,在MiSeq FGx平台(Illumina)上对NexteraTM XT DNA文库进行测序:结果:用本文研究的聚合酶生成的扩增子中检测到的所有同源变异,在 MiSeq FGx 系统上测序后,用其他测序方法也能一致鉴定为同源变异。研究发现,TaKaRa LA Taq DNA 聚合酶在低水平异质体检测方面的准确性不如 Long PCR Enzyme Mix 酶,因为在 1%水平以上的少数变异体中观察到更多的假阴性和假阳性结果。尽管如此,假定至少有 10,000 倍的覆盖率,并在 MiSeq FGx 机器上对 Nextera XT DNA 文库进行测序,所研究的这两种 PCR 系统都能成功用于检测真实的 mtDNA 替换,其少数变异超过 3.61% 的水平:结论:使用 MiSeq FGx 仪器检测点异质性的准确性和灵敏度因用于 mtDNA 扩增的聚合酶而异。因此,建议在用于法医或医学遗传学目的的 mtDNA 替换检测实验室方案实施前对其进行验证:作者声明无利益冲突
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Low-level point heteroplasmy detection in human mitogenomes amplified with different polymerases and sequenced on MiSeq FGx platform
Introduction: 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. Aim: The aim of this study was to compare the reliability of two different DNA polymerases in detecting homoplasmic and heteroplasmic substitutions in human mitogenomes. Materials and Methods: 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. Results: 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. Conclusions: 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. Conflicts of interest statement: The authors declare no conflict of interest
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