Abstract 1207: Custom next-generation sequencing primer designs for targeted sequencing of multi strain viral targets

Abstract

Viruses are infectious agents that can cause many diseases including cancers. A viral species is comprised of many strains each having a unique pathology. For example, over 170 types of HPV have been described, of which only 2 strains, HPV16 and HPV18, are implicated in 70% of cervical cancer cases. Accurate identification of viral strains is often critical and also facilitates research into their association with prognosis or response and personalized therapies. Currently, sequencing virus genes or genome requires complex procedures that involve purifying the virus particle and its genomes from the host genome. Whole genome sequencing is inefficient since viral genomes are orders of magnitude smaller than human genomes, and human DNA is orders of magnitude more abundant than viral DNA. In contrast, targeted next-generation sequencing allows focusing of sequencing and analysis to the region or species of interest using PCR based enrichment. We developed a viral design pipeline that accepts a multiple sequence alignment of the viral genomes or genomic regions of interest to identify and design primer pairs in conserved regions flanking the variable regions. The presence/absence of a set of 1 or more amplicons is used to precisely differentiate the many strains of a viral species. For whole genome sequencing, the complete viral genomic sequence is used as target to design unique and highly specific amplicons covering the whole genome for enrichment of the entire virus. All primers pass through rigorous quality checks to meet our sensitivity, specificity, and primer quality standards. We have successfully designed multi-strain Ion Ampliseq custom viral panels for HCV, HIV, CMV, Measles and Pestivirus. Some of these panels were designed with the objective of targeting a specific region on a virus genome; other panels were designed to enrich and sequence the whole virus genome. The panel primers were highly specific to the region of interest in the viral genomes and did not amplify the host genome. In conclusion, our custom Ion AmpliSeq viral panels enable the enrichment and amplification of the unique regions of interest in the viral genomes or the whole virus genome, avoiding the massive excess of the host genome. These panels allow precise identification of the virus species and strains for many applications including research, phylogenomic classification and comparative genomics. For Research Use Only. Not for use in diagnostic procedures Citation Format: Ratnesh Singh, Na Li, Zunping Luo, Anna McGeachy, Antonio F. Martinez Alcantara, Aren Ewing, Fiona C. Hyland. Custom next-generation sequencing primer designs for targeted sequencing of multi strain viral targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1207.