Misu Masayasu, Yoshikawa Tomoki, Sugimoto Satoko, Takamatsu Yuki, Kurosu Takeshi, Ouji Yukiteru, Yoshikawa Masahide, Shimojima Masayuki, Ebihara Hideki, Saijo Masayuki
Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.
Department of Pathogen, Infection and Immunity, Nara Medical University, Nara, Japan.
Front Microbiol. 2023 Feb 23;14:1137086. doi: 10.3389/fmicb.2023.1137086. eCollection 2023.
RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.
RNA病毒是许多传染病的病原体。由于RNA病毒在基因组复制过程中容易出错，因此迫切需要快速、准确且经济的全RNA病毒基因组序列测定方法。下一代测序（NGS）技术因其高通量测序能力而可进行全病毒基因组测序。然而，NGS技术在样品制备方面负担较重。因为要实现完整的病毒基因组覆盖，需要通过使用病毒特异性引物的逆转录PCR或病毒颗粒浓缩来富集基因组核酸。此外，传统的NGS技术无法确定RNA病毒基因组的5'和3'末端序列。因此，需使用cDNA末端快速扩增（RACE）逐一确定末端序列。然而，由于一些RNA病毒具有分段基因组，使用RACE进行测定的负担与片段数量成正比。迄今为止，仅有一项研究尝试在不使用上述方法的情况下对多种RNA病毒进行全基因组测序，但与参考序列相比，所生成序列的准确性高达97%，由于读取深度较低，未达到100%。因此，我们建立了名为PCR-NGS和RCA-NGS的新方法，这些方法针对NGS仪器MinION进行了优化。这些方法不需要使用病毒特异性PCR引物进行核酸扩增、物理富集病毒颗粒以及RACE。这些方法通过结合以下技术实现全RNA病毒基因组测序：（1）通过核酸酶处理去除RNA病毒基因组以外不需要的DNA和RNA；（2）通过连接条形码接头确定病毒基因组序列的末端；（3）使用连接接头序列特异性PCR或等温DNA扩增技术（如滚环扩增（RCA））扩增病毒基因组cDNA。使用具有单链、双链、正链、负链、非分段或多分段基因组的分离RNA病毒对所建立的方法进行了评估。结果表明，至少使用其中一种方法，所有病毒基因组序列均可100%准确测定，且这些平均读取深度大于2500×。该方法应能实现准确RNA病毒基因组的简便且经济的测定。
