Methods of lowering the error rate of massively parallel DNA sequencing using duplex consensus sequencing
View Patent ↗Next Generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of approximately 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, a method Duplex Consensus Sequencing (DCS) is provided. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors will result in errors in only one strand.
1. A method of generating an error-corrected sequence read of a double stranded target nucleic acid molecule, comprising
a) ligating the double-stranded target nucleic acid molecule to at least one adapter molecule, to form an adaptor-target nucleic acid complex, wherein the at least one adaptor molecule comprises:
i. a degenerate or semi-degenerate single molecule identifier (SMI) sequence that alone or in combination with the target nucleic acid shear points uniquely labels the double stranded target nucleic acid molecule; and
ii. a nucleotide sequence that tags each strand of the adaptor-target nucleic acid complex such that each strand of the adaptor-target nucleic acid complex has a distinctly identifiable nucleotide sequence relative to its complementary strand,
b) amplifying each strand of the adaptor-target nucleic acid complex to produce a plurality of first strand adaptor-target nucleic acid complex amplicons and a plurality of second strand adaptor-target nucleic acid complex amplicons;
c) sequencing the adaptor-target nucleic acid complex amplicons to produce a plurality of first strand sequence reads and a plurality of second strand sequence reads; and
d) comparing at least one sequence read from the plurality of first strand sequence reads with at least one sequence read from the plurality of second strand sequence reads and generating an error corrected sequence read of the double stranded target nucleic acid molecule by discounting nucleotide positions that do not agree.
2. The method of claim 1 , wherein the double-stranded target nucleic acid molecule is a DNA or an RNA molecule.
3. The method of claim 1 , wherein the adaptor molecule-nucleic acid complex comprises at least two primer binding sites.
4. The method of claim 1 , wherein the adaptor molecule-nucleic acid complex comprises a Y-shape, a U-shape, or a combination thereof.
5. The method of claim 1 , wherein the adaptor molecule-nucleic acid complex comprises an SMI sequence in each of its strands.
6. The method of claim 1 , wherein the adaptor molecule-nucleic acid complex comprises an SMI sequence at each terminus.
7. The method of claim 5 , wherein the adaptor molecule-nucleic acid complex comprises (i) a first degenerate or semi-degenerate sequence and (ii) a second degenerate or semi-degenerate sequence that is complementary to the first degenerate or semi-degenerate sequence.
8. The method of claim 7 , wherein the first and second degenerate or semi-degenerate sequence comprises from 3 to 20 nucleotides.