Evaluation of analytical factors associated with targeted MEFV gene sequencing using long-range PCR/massively parallel sequencing of whole blood DNA for molecular diagnosis of Familial Mediterranean fever
Evaluation of analytical factors associated with targeted MEFV gene sequencing using long-range PCR/massively parallel sequencing of whole blood DNA for molecular diagnosis of Familial Mediterranean fever
复制标题
使用长程 PCR/全血 DNA 大规模平行测序评估与靶向 MEFV 基因测序相关的分析因素，以进行家族性地中海热的分子诊断
DOI：
10.1016/j.cca.2019.06.001
复制
发表时间：
2019
影响因子：
5
通讯作者：
Matsushita Kazuyuki
中科院分区：
文献类型：
--
作者：
Ishige Takayuki;Itoga Sakae;Kawasaki Kenji;Utsuno Emi;Beppu Minako;Sawai Setsu;Nishimura Motoi;Ichikawa Tomohiko;Nomura Fumio;Matsushita Kazuyuki
背景长程PCR（LR-PCR）用于富集基因组的目标区域。本研究旨在建立使用LR-PCR和大规模并行测序（MPS）的靶向基因测序流程。方法选择包含整个编码外显子的14kb长MEFV基因作为靶基因，并使用LR-PCR进行扩增。评估的分析因素如下：LR-PCR条件、三种类型的PCR后清理方法和两种类型的MPS文库制备方法。结果就LR-PCR条件而言，Tks Gflex DNA聚合酶在7分钟（30-s/kb）退火/延伸和100-ng基因组DNA输入时具有最高产量。关于PCR后纯化方法，基于磁珠的方法具有较高的回收率和纯度。在MPS文库制备方法中，基于连接的方法具有较高的目标碱基覆盖率（94.58%）、碱基覆盖均匀度（99.95%）以及目标碱基无链偏倚（97.40%）。通过基于连接和基于转座子的方法检测到桑格测序确定的外显子变异。结论评估了各种分析因素，并建立了使用LR-PCR和MPS进行靶向基因测序的流程。这些数据可以在临床实验室中使用 LR-PCR 和 MPS 优化目标基因测序。
BackgroundLong-range PCR (LR-PCR) is used to enrich the target regions of the genome. This study aimed to establish the pipeline of targeted gene sequencing using LR-PCR and massively parallel sequencing (MPS).MethodsThe 14-kb-longMEFVgene, including the entire coding exons, was selected as a target gene and amplified using LR-PCR. The evaluated analytical factors were as follows: LR-PCR conditions, three types of post-PCR cleanup methods, and two types of MPS library preparation methods.ResultsWith regard to LR-PCR conditions, Tks Gflex DNA polymerase at 7-min (30-s/kb) annealing/extension with 100-ng genomic DNA input had the highest yield. Regarding post-PCR purification methods, the magnetic beads-based method had high recovery and purity. In the MPS library preparation methods, the ligation-based method had a higher base coverage in the target (94.58%), uniformity of base coverage (99.95%), and target bases with no strand bias (97.40%). The exonic variants determined by Sanger sequencing were detected by both ligation- and transposon-based methods.ConclusionsVarious analytical factors were evaluated, and the pipeline of targeted gene sequencing using LR-PCR and MPS was established. These data can enable the optimization of targeted gene sequencing using LR-PCR and MPS in the clinical laboratory.