Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding.

Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.

Putative alternative translation start site-encoding nucleotides of CPR5 regulate growth and resistance.

BACKGROUND: The Arabidopsis CONSTITUTIVE EXPRESSER of PATHOGENESIS-RELATED GENES 5 (CPR5) has recently been shown to play a role in gating as part of the nuclear pore complex (NPC). Mutations in CPR5 cause multiple defects, including aberrant trichomes, reduced ploidy levels, reduced growth and enhanced resistance to bacterial and fungal pathogens. The pleiotropic nature of cpr5 mutations implicates that the CPR5 protein affects multiple pathways. However, little is known about the structural features that allow CPR5 to affect the different pathways. RESULTS: Our in silico studies suggest that in addition to three clusters of putative nuclear localization signals and four or five transmembrane domains, CPR5 contains two putative alternative translation start sites. To test the role of the methionine-encoding nucleotides implicated in those sites, metCPR5 cDNAs, in which the relevant nucleotides were changed to encode glutamine, were fused to the CPR5 native promoter and the constructs transformed to cpr5-2 plants to complement cpr5-compromised phenotypes. The control and metCPR5 constructs were able to complement all cpr5 phenotypes, although the extent of complementation depended on the specific complementing plant lines. Remarkably, plants transformed with metCPR5 constructs showed larger leaves and displayed reduced resistance when challenged to Pseudomonas syringae pv Pst DC3000, as compared to control plants. Thus, the methionine-encoding nucleotides regulate growth and resistance. We propose that structural features of the CPR5 N-terminus are implicated in selective gating of proteins involved in regulating the balance between growth and resistance. CONCLUSION: Plants need to carefully balance the amount of resources used for growth and resistance. The Arabidopsis CPR5 protein regulates plant growth and immunity. Here we show that N-terminal features of CPR5 are involved in the regulation of the balance between growth and resistance. These findings may benefit efforts to improve plant yield, while maintaining optimal levels of disease resistance.