Amplicon sequencing of diverse viruses with 3’ anchored primer clouds. Amplicon sequencing of diverse viruses with 3’ anchored primer clouds

Amplicon sequencing is an important approach for virus genome sequencing, due to its high sensitivity and low cost. It has been widely used in the genomic surveillance of Ebola, Zika, SARS-CoV-2 and Mpox. However the design and synthesis of custom primers introduces delays in the early outbreak response, which could be avoided if pan-species schemes for potential threats were prepared in advance. There is also a necessity to include more primers in schemes to enable sequencing of larger genomes, or panels containing multiple targets. We have developed a new approach to amplicon sequencing which enables diverse virus species and is scalable to larger genomes. These features are implemented in a new primer design software, called PrimalScheme3. We have demonstrated the capabilities of this software by designing a Measles virus (MeV) scheme using hundreds of genome sequences available in online databases and using the scheme to completely sequence MeV isolates from a viral culture collection. We also show in silico validation of two scheme designs suitable for sequencing samples of Monkeypox virus (MPXV) of any clade; a long amplicon scheme for sequencing clinical isolates, and a short amplicon scheme for sequencing from wastewater.