ICBR in collaboration with virologist Dr. Marco Salemi, Professor in the UF Department of Pathology, has begun to use high throughput DNA sequencing technology to help identify COVID19 variant spread within the state of Florida. The virus contains a single molecule of RNA that contains the genetic information to produce new progeny in infected cells. To determine the viral genome “code” viral RNA is isolated, and enzymatically converted to DNA. In a process called targeted sequencing, specific overlapping regions of the viral sequence are amplified using sequence-specific primers and PCR, and these DNA fragments are then analyzed using next generation DNA sequencing.
The complete viral genome can then be “assembled” from the overlapping DNA fragments. RNA isolated by Melanie Cash in the Salemi Lab is sent to the ICBR Gene Expression Core, where Dr. Yanping Zhang and her team have developed an automated procedure using a robotic liquid handler called the Mosquito® that automates the conversion of viral RNA into DNA. In response to the very large sample volume Dr. Zhang has modified a standard protocol that uses single test tube for each sample into a 384-well format. Each well of the plate contains a unique molecular bar code – a short, synthetic DNA sequence that gets incorporated into the DNA during the synthesis reaction. All the 384 samples are then combined into a “library” that is then sent to the ICBR NGS Sequencing Core led by Dr. David Moraga. Using the Illumina NovaSeq6000 instrument, Dr. Moraga can sequence 768 patient samples overnight – generating millions of short DNA “reads”. Using HiPerGATOR the “raw” DNA reads are then analyzed by Dr. Alberto Riva, Scientific Director of the ICBR Bioinformatics Core. Complete viral genomes are assembled from the millions of short DNA reads using the unique molecular bar code to identify which sequences are associated with each sample. Assembled genomes are then compared to a database of known COVID19 variants and each sample is assigned a variant type. Because the date and location of the sample collection are known, Dr. Salemi can use a custom-designed algorithm to build a phylogenetic relationship tree showing variant spread over time. This information will be shared within an international COVID19 genome sequence database and will help to understand the origins of variant spread in the state of Florida.
Source: University of Florida