Rapid whole genome sequencing methods for RNA viruses.
Rapid whole genome sequencing methods for RNA viruses.
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RNA病毒的快速全基因组测序方法。
DOI：
10.3389/fmicb.2023.1137086
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发表时间：
2023
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
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作者：
RNA病毒是许多传染病的病原。由于RNA病毒在基因组复制过程中容易出错，因此需要快速、准确和经济的全RNA病毒基因组序列测定。下一代测序（NGS）技术由于其高通量测序能力而进行全病毒基因组测序。然而，NGS技术涉及到样品制备的重大负担。由于要产生完整的病毒基因组覆盖，需要通过使用病毒特异性引物的反转录PCR或病毒颗粒浓度来富集基因组核酸。此外，传统的NGS技术无法确定RNA病毒基因组的5 ‘和3 ’端序列。因此，使用cDNA末端快速扩增（RACE）逐个确定末端序列。然而，由于一些RNA病毒具有分段基因组，因此使用RACE测定的负担与片段数量成正比。迄今为止，只有一项研究没有使用上述方法对多种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，未能达到100%。因此，我们建立了新的方法，命名为PCR-NGS和RCA-NGS，并针对NGS机器MinION进行了优化。这些方法不需要用病毒特异性PCR引物进行核酸扩增，物理病毒颗粒富集和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不需要的DNA和RNA，而不是RNA病毒基因组；(2)利用条形码连接子确定病毒基因组序列的末端；(3)利用链结连接子序列特异性PCR或等温DNA扩增技术（如滚动圈扩增（RCA））扩增病毒基因组cDNA。采用单链、双链、正链、负链、非片段或多片段基因组分离的RNA病毒对建立的方法进行评估。结果，所有的病毒基因组序列都可以以100%的准确率确定，并且这些平均读取深度大于2500 ×，至少使用两种方法中的任何一种。这种方法可以方便、经济地测定准确的RNA病毒基因组。
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.