Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; Peña, José; Hysom, David A.; Borucki, Monica K.

Sequencing whole genomes of potentially heterogeneous or divergent viruses can be challenging from a small or complex sample with low viral concentrations. Deep sequencing to detect rare viral variants or metagenomic sequencing to genotype viruses from a complex background requires targeted viral amplification. Techniques such as consensus PCR, Ion Ampliseq (Life Technologies) [1] , TruSeq Amplicon (Illumina), and Haloplex (Agilent) [2] apply highly multiplexed PCR for target enrichment. Targeted enrichment should preferentially amplify the target virus over host or environmental DNA/RNA, in contrast to random amplification commonly used prior to whole genome sequencing. Primers designed to tile amplicons across a set of related viral genomes prior to sequencing can enrich whole viral genomes or large regions. However, high levels of intraspecific sequence variation combined with low virus concentrations mean that standard PCR primer design from a reference may fail due to mutations in the sample virus that prevent primer binding. To address this problem, we added a capability to the PriMux software distribution (http://sourceforge.net/projects/primux/) called run tiled primers that applies the PriMux software [3] to automate PCR primer design to achieve a near-minimal set of conserved, degenerate, multiplex-compatible primers designed to tile overlapping regions across multiple related whole genomes or regions.