METHODS, KITS, AND SYSTEMS FOR NUCLEIC ACID SEQUENCING BY HYBRIDIZATION

摘要

The present invention provides a DNA sequencing method based on hybridization of a universal panel of tiling probes. In various embodiments, millions of shotgun fragments are amplified in situ on a solid support using rolling circle amplification and then subjected to sequential hybridization with short fluorescent probes. Long reads ensure unique placement even in large genomes. The sequencing chemistry is simple, enzyme-free and consumes only dilute solutions of the probes, resulting in an order of magnitude reduction in sequencing cost, and a substantial increase in speed. As exemplified herein, a prototype instrument based on commonly available equipment was used to resequence the Bacteriophage ? and E. coli genomes to better than 99.9% accuracy at a raw throughput of 320 Mbp/day. The present invention further provides kits and systems for sequencing by hybridization.