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Hee-Sun Han

Merging microfluidicis and metagenomics for novel high throughput virus discovery

This work is a collaboration with Shelley K. Cockrell at the Pipas Lab (Upitt).

In this project, we are developing a platform for the detection, isolation and complete sequencing of single viral genomes from complex samples containing mixtures of viruses. Total virion populations are isolated from the sample, and deep sequencing and subsequent computational analysis detect the viral sequences present. Viruses of interest are selected, and PCR primers are designed based on the metagenomic sequence reads. Single virions are then placed in individual 1 picoliter droplets, merged with PCR primers and reagents, and passed one-at-a-time through a laser fluorescent detection system. PCR-positive droplets are diverted and collected for subsequent whole genome sequencing.

Drop based microfluidics system
Figure 1. Drop based microfluidics system. (A) Viruses are encapsulated in drops. Carrier oil flows from two side channels. Viruses in PCR mix flow through the central channel, and drops of water and viruses in oil form at the junction. (B) 106 drops (100 µL) stored in a vial prior to thermo cycling. (C) Bright field (left) and fluorescent (right) images of the drops after PCR. The template DNA was successfully amplified in drops that fluoresce. (D) Sorting mechanism. Single drops are passed through a beam that excites the fluorescent dye bound to amplified DNA. Fluorescent drops are directed to the lower branch for collection. The bright spot in the center of the image is the laser spot. Black lines are electrodes. All scale bars are 100 μm.

Our metagenomic studies of untreated wastewater have detected three viruses of possible significance to human health. The isolation and characterization of these viruses is of immediate importance. However, more importantly, the development of this microfluidic platform will break a rate-limiting logjam in our ability to discover and isolate novel viruses. While the power of metagenomics lies in the ability to detect potentially interesting novel viruses among a complex mixture containing many different viruses, microfluidics multiplies this power by isolating specific viral genomes of interest.