Senior Scientist, Biological Sciences Group Johns Hopkins University Applied Physics Laboratory
COVID-19 demonstrated an urgent need for pathogen genome sequencing approaches that can be deployed rapidly at scale. Our group has developed a highly optimized viral sequencing approach that integrates molecular indexing into one-step reverse-transcriptase PCR. Using influenza A virus, we demonstrate integrated-indexing multi-segment PCR (iiMS-PCR) using New England Biolab’s Lunascript Multiplex One-Step RT-PCR reagent set. Compared to standard approaches, iiMS-PCR provides a significant improvement in amplification of long influenza genome segments, ultimately reducing the amount of sequencing data required to produce a full consensus genome. Leveraging nanopore sequencing devices and deployable bioinformatics capabilities, we demonstrate sample-to-analysis timelines within a single 8-hour working day. This approach was deployed in several laboratories during development, including those responding to suspected human cases of highly pathogenic avian influenza. iiMS-PCR enabled near real-time evaluation of influenza A H5N1 virus genomes in a manner that directly informed local and global public health responses. Extension of the integrated indexing approach for additional pathogens of interest, including untargeted metagenomic sequencing, has promise to dramatically reduce pathogen genome production timelines in both traditional and automated laboratory workflows.
