Identification of Low-Abundance RNA Viruses with Circular Sequencin
CirSeq accurately identifies ultra-rare and low-frequency genetic variants in RNA viruses. The method uses a unique step for circularization of fragmented viral RNAs, followed by rolling-circle RT (Acevedo et al., 2014) (Acevedo et al., 2014). CirSeq corrects for mutations introduced during its amplification steps by aligning the tandem-repeat sequences with each other and excluding those reads using informatics tools.
First, single-stranded RNAs are fragmented using Zn2+ and size-selected to no more than one-third of the sequencing read length. Next, they are circularized and reverse-transcribed using random primers. Rolling-circle RT is used to generate tandem-repeat cDNA strands. The first-strand cDNAs are amplified, generating double-stranded cDNAs, followed by end repair, poly(A) tailing, and adapter ligation. The cDNA libraries are ready for sequencing.
- Detects ultra-rare and low-frequency genetic variants in RNA viruses
- Rolling-circle RT creates tandem-repeat cDNAs that can be used to correct artificial mutations
- Error rates reported are far below those observed using standard RNA virus sequencing methods
- Whole process takes ~5 days
- Unsuitable for sequencing clinical isolates, because it needs large quantities of purified viral RNAs
- Not applicable for de novo sequencing of viral RNAs
Illumina Library prep and Array Kit Selector
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