Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of SARS-CoV-2 in real-time directly from environmental and clinical samples. Over 14 million SARS-CoV-2 genomes are now available on GISAID, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow, laborious, and costly. Here, we present Olivar, the first open-source computational tool capable of fully automating the design of tiled amplicons by integrating SNPs, non-specific amplification, etc. into a "risk score" for each single nucleotide of the target genome. Oli- var evaluates thousands sets of possible tiled amplicons and minimizes primer dimer in parallel. In a direct in-silico com- parison with PrimalScheme, we show that Olivar has fewer SNPs overlapping with primers and predicted PCR byproducts. We also compared Olivar head-to-head with ARTIC v4.1, the most widely used tiled amplicons for SARS-CoV-2 sequencing. We next tested Olivar on real wastewater samples and found that our automated approach had up to 3-fold higher map- ping rates compared to ARTIC v4.1 while retaining similar coverage. To the best of our knowledge, Olivar represents the first open-source, fully automated design tool that simultaneously evaluates and optimizes risks of known primer design issues for robust tiled amplicon sequencing. Olivar is available as a web application at https://olivar.rice.edu/. Olivar can also be installed locally as a command line tool with Bioconda. Source code, installation guide and usage are available at https: //gitlab.com/treangenlab/olivar.Competing Interest StatementThe authors have declared no competing interest....

Olivar: fully automated and variant aware primer design for multiplex tiled amplicon sequencing of pathogen genomes

10.1101/2023.02.11.528155