Thesis Defense | Sushant Patil | Ph.D in Nanoscale Science
Defense Title: Applications of next-generation sequencing (NGS) platforms for multiplexed biosensing and aptamer discovery: G-quadruplex aptamers as a case study.
Candidate: Sushant Patil
Sponsoring Chair: Dr. Jennifer Weller
Committee: Dr. Jennifer Weller, Dr. Jerry Troutman, Dr. Joanna Krueger, Dr. Ed Stokes, Dr.Valery Grdzelishvili
Defense Date: April 5, 2016 – 2:30 PM
Location: BINF 105
Degree Program Name: Ph.D. in Nanoscale Science
Aptamers are single-stranded DNA or RNA molecules forming unique three dimensional structures that can bind to their targets (generally proteins or metabolites or other nucleic acids) with high specificity and affinity. Ever since the advent of high-throughput DNA sequencing technology platforms and chemistries, also called next-generation sequencing (NGS), there have been numerous efforts to find new NGS-based aptamer discovery methods that are much faster and cheaper than to conventional SELEX process of aptamer selection. A prominent feature of several of the best-characterized aptamers is a structure called G-quadruplex (GQ), wherein four guanines form a square planar structure called a tetrad; two or more such tetrads then stack on top of each other to form a three-dimensional structure. Such GQs are prevalent in many genomes as well and their involvement in modulating various cellular processes has been demonstrated. It has been noticed that strongly folded DNA secondary structure may cause DNA polymerases to stall in vitro. Do NGS platforms sequence through and accurately report the location and neighboring sequence of GQs? The question can be answered by comparing the performance of the two most popular NGS platforms, Ion Torrent and Illumina, in sequencing systematically varied GQ templates. Our results indicate that there are indeed sequencing errors in the presence of these templates, and that Illumina outperforms Ion Torrent in sequencing most of the GQ structure types tested, as determined by base-calling quality scores and % mismatches in sequencing. Each chemistry employs a different DNA polymerase, and the different paths adopted by each polymerase upon encountering GQs of different complexities have been highlighted.
To understand which characteristics make a GQ structure difficult to sequence, the sequencing output of systematically varied GQ structures was studied as a function of GQ melting temperature, Tm. The instrument’s operating temperature was found to be the determinant of its performance in sequencing such structures: for templates with Tms above the operating temperature, a negative correlation between the Tm and accuracy was noticed. The effectiveness of two compensatory modifications viz. E. Coli Single-stranded DNA Binding (SSB) protein and Ion PGM Hi-Q Kit were tested with respect to improving the Ion Torrent sequencing accuracy;neither were able to significantly alter the earlier results.
Finally, a universal, structure-independent protocol for multiplexed biosensing based on the principle of exonuclease I protection assay has been developed. As case studies, a thrombin and a lysozyme protein-binding aptamer were used, demonstrating such use of the platform is feasible. The method is validated for its sensitivity and accuracy in quantitative detection of the protein of interest.