一般注記type:Thesis
RNA viruses are the etiological agents of many infectious diseases. Since RNAviruses are error-prone during genome replication, rapid, accurate and economicalwhole RNA viral genome sequence determination is highly demanded. Next generation sequencing (NGS) techniques perform whole viral genome sequencingdue to their high-throughput sequencing capacity. However, the NGS techniquesinvolve a significant burden for sample preparation. Since to generate completeviral genome coverage, genomic nucleic acid enrichment is required by reversetranscription 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 sequencesare determined one by one using rapid amplification of cDNA ends (RACE).However, since some RNA viruses have segmented genomes, the burden of thedetermination using RACE is proportional to the number of segments. To date,there is only one study attempting whole genome sequencing of multiple RNAviruses without using above mentioned methods, but the generated sequences’accuracy compared to the reference sequences was up to 97% and did not reach100% due to the low read depth. Hence, we established novel methods, namedPCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION.These methods do not require nucleic acid amplification with virus-specific PCRprimers, physical viral particle enrichment, and RACE. These methods enablewhole RNA viral genome sequencing by combining the following techniques: (1)removal of unwanted DNA and RNA other than the RNA viral genome by nucleasetreatment; (2) the terminal of viral genome sequence determination by barcodedlinkers ligation; (3) amplification of the viral genomic cDNA using ligated linkersequences-specific PCR or an isothermal DNA amplification technique, such asrolling circle amplification (RCA). The established method was evaluated usingisolated RNA viruses with single-stranded, double-stranded, positive-stranded,negative-stranded, non-segmented or multi-segmented genomes. As a result, allthe viral genome sequences could be determined with 100% accuracy, and thesemean read depths were greater than 2,500×, at least using either of the methods.This method should allow for easy and economical determination of accurateRNA viral genomes.
権利情報：© 2023 Misu, Yoshikawa, Sugimoto, Takamatsu, Kurosu, Ouji, Yoshikawa, Shimojima, Ebihara and Saijo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
identifier:Frontiers in Microbiology. 2023, vol.14, 1137086
identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/4205
identifier:Frontiers in Microbiology, 14: 1137086
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