Dataset Information

RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS).

ABSTRACT: Fragmented and primer ligated dsRNA sequencing (FLDS) is a sequencing method applicable to long double-stranded RNA (dsRNA) that enables the complete genome sequencing of both double- and single-stranded RNA viruses. However, the application of this method on a low amount of dsRNA has been hindered by adaptor dimer formation during cDNA amplification and sequence library preparation. We herein developed FLDS ver. 3 by optimizing the terminal modification of an oligonucleotide adaptor and the conditions of adaptor ligation. We also examined the concentration of Mg2+ in the PCR reaction for cDNA amplification and the purification method of amplified cDNA. Fine sequence reads were successfully obtained from metagenomic shotgun sequencing libraries constructed from 10 and 100 pg dsRNA, and these libraries exhibited weaker detection sensitivity for low-abundance dsRNAs (viral genomes and genome segments) than that constructed from 1‍ ‍ng of dsRNA. We also report the utility of capillary electrophoresis for dsRNA quantification. The FLDS ver. 3 package expands the frontiers of our knowledge in RNA virus diversity and evolution.

SUBMITTER: Hirai M

PROVIDER: S-EPMC8209451 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS).

Hirai Miho M Takaki Yoshihiro Y Kondo Fumie F Horie Masayuki M Urayama Syun-Ichi SI Nunoura Takuro T

Microbes and environments 20210101 2

Fragmented and primer ligated dsRNA sequencing (FLDS) is a sequencing method applicable to long double-stranded RNA (dsRNA) that enables the complete genome sequencing of both double- and single-stranded RNA viruses. However, the application of this method on a low amount of dsRNA has been hindered by adaptor dimer formation during cDNA amplification and sequence library preparation. We herein developed FLDS ver. 3 by optimizing the terminal modification of an oligonucleotide adaptor and the con ...[more]

PMID: 33952860

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Project description:BackgroundIn a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally.ResultsWe have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute's (JCVI) high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates.ConclusionsAugmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus sequence that encapsulates the allelic variation of the targeted population and is a key step prior to designing degenerate primers is also formally described.

Dataset Information

RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS).

Publications

RNA Viral Metagenome Analysis of Subnanogram dsRNA Using Fragmented and Primer Ligated dsRNA Sequencing (FLDS).

Similar Datasets

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