BMPR2-targeted MinION sequencing as a tool for genetic analysis in patients with pulmonary arterial hypertension Short title/Running head: BMPR2-targeted MinION sequencing in PAH. Homo sapiens

Background: Mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2) represent a major genetic cause of pulmonary arterial hypertension (PAH). Identification of BMPR2 mutations is crucial for the genetic diagnosis of PAH. MinION nanopore sequencer is a portable third-generation technology that enables long-read sequencing at a low-cost. This nanopore technology-based device has not been used previously for PAH diagnosis. This study aimed to determine the feasibility of using MinION nanopore sequencing for the genetic analysis of PAH patients, focused on BMPR2. Methods: We developed a protocol for the custom bioinformatics pipeline analysis of long reads generated by long-PCR. To evaluate the potential of using MinION sequencing in PAH, we analyzed five samples, including those of two idiopathic PAH patients and a family of three members with one affected patient. Sanger sequencing analysis was performed to validate the variants. Results: The median read length was around 3.4 kb and a good mean quality score of approximately 19 was obtained. The total number of reads generated was uniform among the cases and ranged from 2,268,263 to 3,126,719. The coverage was consistent across flow cells in which the average number of reads per base ranged from 80,375 to 135,603. We identified 2 polymorphic variants and 3 mutations in 4 out of 5 patients. Certain indel variant calling-related errors were observed, mostly outside coding sequences. Conclusion: We have shown the ability of this portable nanopore sequencer to detect BMPR2 mutations in patients with PAH. The MinION nanopore sequencer is a promising tool for screening BMPR2 mutations, especially in small laboratories and research groups.

背景：骨形态发生蛋白II型受体基因（bone morphogenetic protein receptor type 2 gene，BMPR2）的突变是肺动脉高压（pulmonary arterial hypertension，PAH）的主要遗传病因。对BMPR2突变的鉴定对于PAH的遗传诊断具有关键意义。MinION纳米孔测序仪是一种可实现低成本长读长测序的便携式第三代测序技术，此前尚未有将该纳米孔技术设备应用于PAH诊断的相关报道。本研究旨在评估以BMPR2为研究靶点、使用MinION纳米孔测序开展PAH患者遗传分析的可行性。方法：我们开发了一套针对长PCR扩增所得长读长数据的定制化生物信息学分析流程。为评估MinION测序应用于PAH检测的潜力，我们分析了5份样本，包括2例特发性PAH患者的样本，以及1个3口之家的样本（其中1名家庭成员患病）。采用桑格测序（Sanger sequencing）对检测到的变异进行验证。结果：本次测序得到的读长中位数约为3.4 kb，平均质量得分约为19，表现优异。所有样本的总读段数分布均匀，介于2268263至3126719之间。各测序流槽的覆盖度均一，每碱基对应的平均读段数介于80375至135603之间。我们在5例患者中的4例里检测到2个多态性变异与3处突变。研究中观察到部分与插入缺失变异（insertion-deletion，indel）检出相关的误差，且此类误差大多位于编码序列之外。结论：本研究证实，这款便携式纳米孔测序仪可在PAH患者中准确检测BMPR2突变。MinION纳米孔测序仪是一种颇具应用前景的BMPR2突变筛查工具，尤其适用于小型实验室与研究团队。