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 dsR

SUBMITTER: Hirai M 

PROVIDER: S-EPMC8209451 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature