Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants.

BioRxiv : the Preprint Server for Biology
Elizabeth JaworskiAndrew Routh

Abstract

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for Next-Generation Sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ' Tiled-ClickSeq '. Tiled-ClickSeq uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, obviating the need for a paired-primer. A sequencing adaptor containing a Unique Molecular ...Continue Reading

Datasets Mentioned

BETA
MW703487-MW703490
PRJNA707211

Methods Mentioned

BETA
PCR
Ligation-Sequencing
ClickSeq
electrophoresis
Illumina sequencing

Related Concepts

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