Periodical titleFrontiers in Microbiology
Note (General)type:Thesis
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.
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
Related Material (DOI)10.3389/fmicb.2023.1137086
Data Provider (Database)国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）
Original Data Provider (Database)奈良県立医科大学 : 奈良県立医科大学機関リポジトリ GINMU