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INV-20100
Background
Accurate analysis of the precise order of nucleotides in deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules is fundamentally important to understanding the biology and function of all living organisms, including viruses. Since the complete sequence of nucleic acid was first reported in 1965, technologies for DNA sequencing have undergone a number of dramatic improvements. At present, next-generation sequencing (NGS), such as that offered by the Illumina platform and third-generation sequencing, the single-molecule real-time (SMRT) platform, or the nanopore platform, enables high-throughput sequencing (SMS) of DNA molecules. However, even with these advancements, two major limitations in conventional approaches to DNA sequencing exist:
- An inability to perform high-resolution reads of individual long molecules of DNA for single nucleotide variants (SNVs)
- Confounding data management and interpretation issues associated with nucleotide sequence errors introduced as artifacts by various sequencing technologies
Technology Overview
This invention provides detailed methods for the synthesis of continuous cDNA strands from individual long and ultralong RNA molecules through piecewise reverse transcription (pRT). This technological advancement over all existing methods of RT utilizes oligonucleotide primers complementary to and spaced along an entire RNA template irrespective of overall length, which then facilitates multiple and independent RT reactions in parallel with partial coverage. This produces a group of first-strand cDNA molecules spanning all areas of the RNA target that are then trimmed and ligated in sequence to generate a single first-strand cDNA covering the entire length of the desired RNA template with high efficiency and accuracy.
Benefits
- It can be used to distinguish individual long-molecule sequences within mixed or heterogeneous nucleic acid pools
- It enables high-resolution sequence analysis of these individual molecules
- It enables sequence analysis of continuous segments of DNA or RNA molecules
Applications
- The study of viral genomes, long and ultralong RNA molecules, microbial communities, mitochondrial genomes (mitochondrial DNA or mtDNA), and nuclear genomes (nuclear DNA)
- Can be used to characterize microbiomes in individual organisms and in the environment, detect and analyze mtDNA heteroplasmy
Opportunity
- License
- Partnering
- Research collaboration
