Capitalizing on our group's experience on single molecule enzymology, we propose a novel method for multiplex sequencing of individual nucleic acid molecules using a sequencing-by-synthesis approach that employs fluorogenic nucleotide substrates. Upon incorporation of a non-fluorescent, terminal phosphate-labeled nucleotide substrate by a polymerase, a fluorogenic polyphosphate molecule is released, and subject to fast enzymatic digestion, yielding a single fluorophore, the color of which is dependent on the identity of the incorporated nucleotide. To facilitate single molecule fluorescence detection, an individual nucleic acid molecule is confined in a sealed sub-femtoliter nanoreactor, in which the sequencing reaction takes place continuously. Using conventional soft lithography, we fabricate an array of nanoreactors that allow simultaneous, real-time monitoring of thousands of isolated sequencing reactions with a fluorescence microscope and CCD camera. Our new approach offers low reagent cost, long read lengths, easy sample preparation, and high throughput at several megabases per minute. We also propose the integration of a massively parallel single molecule fluorogenic sequencer with microfluidic devices that process and deliver genetic material from a single cell.
This project will develop new methods of sequencing DNA at the single molecule level, providing a new path towards human genome sequencing for less than $1000. This ability to economically sequence full genomes will usher in a new era of personalized medicine.
|Huang, Lei; Ma, Fei; Chapman, Alec et al. (2015) Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications. Annu Rev Genomics Hum Genet 16:79-102|
|Shiroguchi, Katsuyuki; Jia, Tony Z; Sims, Peter A et al. (2012) Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proc Natl Acad Sci U S A 109:1347-52|
|Lu, Sijia; Zong, Chenghang; Fan, Wei et al. (2012) Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing. Science 338:1627-30|
|Zong, Chenghang; Lu, Sijia; Chapman, Alec R et al. (2012) Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338:1622-6|
|Sims, Peter A; Greenleaf, William J; Duan, Haifeng et al. (2011) Fluorogenic DNA sequencing in PDMS microreactors. Nat Methods 8:575-80|