A correctable decoding DNA sequencing with high accuracy and high throughput

Eliminating errors in next-generation sequencing has proven to be challenging. Here we present a novel strategy for DNA sequencing, called correctable two-color fluorogenic DNA decoding sequencing, which can significantly improve sequencing accuracy and throughput by employing a dual-nucleotide addition combined with fluorogenic sequencing-by-synthesis (SBS) chemistry. This sequencing method involves introducing a mixture of natural nucleotide X, labeled unblocked nucleotide X′, 3′ blocked nucleotide Y*, and labeled 3′ blocked nucleotide Y* into each reaction cycle. By cyclically interrogating a template twice with different nucleotide combinations, two sets of base-encoding are sequentially obtained, enabling accurate deduction of base sequence. We demonstrate the remarkable efficacy of this approach in detecting and correcting sequencing errors, achieving a theoretical error rate of 0.0005%, which is twice as accurate as Sanger sequencing. Furthermore, we show the capability to detect known mutation sites using information from only a single sequencing run. The correctable two-color fluorogenic DNA decoding sequencing approach should enable accurate identification of extremely rare genomic variations in diverse applications in biology and medicine.