Highly Accurate Chip-Based Resequencing of SARS-CoV-2 Clinical Samples.

SARS-CoV-2 has infected over 128 million people worldwide, and until a vaccine is developed and widely disseminated, vigilant testing and contact tracing are the most effective ways to slow the spread of COVID-19. Typical clinical testing only confirms the presence or absence of the virus, but rather, a simple and rapid testing procedure that sequences the entire genome would be impactful and allow for tracing the spread of the virus and variants, as well as the appearance of new variants. However, traditional short read sequencing methods are time consuming and expensive. Herein, we describe a tiled genome array that we developed for rapid and inexpensive full viral genome resequencing, and we have applied our SARS-CoV-2-specific genome tiling array to rapidly and accurately resequence the viral genome from eight clinical samples. We have resequenced eight samples acquired from patients in Wyoming that tested positive for SARS-CoV-2. We were ultimately able to sequence over 95% of the genome of each sample with greater than 99.9% average accuracy.

Fabrication of Inverted High-Density DNA Microarrays in a Hydrogel.

Current techniques for making high-resolution, photolithographic DNA microarrays suffer from the limitation that the 3' end of each sequence is anchored to a hard substrate and hence is unavailable for many potential enzymatic reactions. Here, we demonstrate a technique that inverts the entire microarray into a hydrogel. This method preserves the spatial fidelity of the original pattern while simultaneously removing incorrectly synthesized oligomers that are inherent to all other microarray fabrication strategies. First, a standard 5'-up microarray on a donor wafer is synthesized, in which each oligo is anchored with a cleavable linker at the 3' end and an Acrydite phosphoramidite at the 5' end. Following the synthesis of the array, an acrylamide monomer solution is applied to the donor wafer, and an acrylamide-silanized acceptor wafer is placed on top. As the polyacrylamide hydrogel forms between the two wafers, it covalently incorporates the acrydite-terminated sequences into the matrix. Finally, the oligos are released from the donor wafer upon immersing in an ammonia solution that cleaves the 3'-linkers, thus freeing the oligos at the 3' end. The array is now presented 3'-up on the surface of the gel-coated acceptor wafer. Various types of on-gel enzymatic reactions demonstrate a versatile and robust platform that can easily be constructed with far more molecular complexity than traditional photolithographic arrays by endowing the system with multiple enzymatic substrates. We produce a new generation of microarrays where highly ordered, purified oligos are inverted 3'-up, in a biocompatible soft hydrogel, and functional with respect to a wide variety of programable enzymatic reactions.