Method for accurate sequencing of DNA
View Patent ↗DNA is sequenced by (a) independently sequencing first and second strands of a dsDNA to obtain corresponding first and second sequences; and (b) combining the first and second sequences to generate a consensus sequence of the dsDNA. By independently sequencing first and second strands the error probability of the consensus sequence approximates a multiplication of those of the first and second sequences.
1. A method of generating a sequencing library, comprising:
(a) digesting DNA with a dsDNA-specific nuclease to generate a plurality of DNA fragments;
(b) attaching sequencing adapters to the plurality of DNA fragments to form a plurality of adapter-DNA molecules; and
(c) amplifying the adapter-DNA molecules to generate copy strands of each adapter-DNA molecule;
wherein a chemical scavenger of hydrated electrons and oxygen radicals is added in one or more of steps (a)-(c) to reduce damage to the DNA.
2. The method of claim 1 , wherein the dsDNA-specific nuclease is shrimp nuclease or DFF40 nuclease.
3. The method of claim 1 , wherein the plurality of DNA fragments have blunt ends.
4. The method of claim 3 further comprising extending the blunt ends of the plurality of DNA fragments with dA before attaching sequencing adapters to the plurality of DNA fragments.
5. The method of claim 1 further comprising the step of removing nicks and abasic sites of the plurality of DNA fragments by a DNA repair enzyme before attaching sequencing adapters to the plurality of DNA fragments.
6. The method of claim 5 , wherein the DNA repair enzyme is formamidopyrimidine [fapy]-DNA glycosylase (Fpg), 8-oxoguanine DNA glycosylase, Uracil-DNA glycosylase, or a combination thereof.
7. The method of claim 5 , wherein the DNA repair enzyme is T7 ligase.
8. The method of claim 1 , wherein the chemical scavenger is selected from sodium iodide, sodium nitrate, or a combination thereof.
9. The method of claim 1 , wherein the chemical scavenger is in micromolar or milimolar concentrations.
10. The method of claim 1 , wherein the damage of the DNA is reduced by a factor of at least 10 or at least 100.
11. A method of sequencing DNA using a sequencer, comprising:
(a) generating a sequencing library comprising attaching sequencing adapters to a plurality of DNA fragments to form a plurality of adapter-DNA molecules;
(b) flowing the plurality of adapter-DNA molecules over a sequencer flow cell such that multiple adapter-DNA molecules bind to the flow cell;
(c) bridge amplifying the bound adapter-DNA molecules to form one or more polonies;
(d) sequencing the polonies to obtain a preliminary sequence read for each polony, wherein for each polony, sequencing includes:
(i) obtaining fluorescence intensity measurements for each nucleotide position of the polony; and
(ii) performing a first round of base calling by converting the fluorescence intensity measurements into a probable base call for each nucleotide position of the polony to provide preliminary sequence read, the preliminary sequence read having a preliminary error probability;
(e) grouping preliminary sequence reads from a particular adapter-DNA molecule;
(f) calculating a fluorescence intensity measurement average for each nucleotide position from grouped preliminary sequence reads; and
(g) performing a second round of base calling for each nucleotide position of the grouped preliminary sequence reads using the fluorescence intensity measurement averages to provide a sequence read;
wherein an error probability of the sequence read corresponding to the particular adapter-DNA molecule is lower than any one of the preliminary error probabilities of the individual preliminary sequence reads in the group.
12. The method of claim 11 , wherein the plurality of DNA fragments is generated by digesting the DNA with a dsDNA-specific nuclease.
13. The method of claim 12 , wherein the dsDNA-specific nuclease is shrimp nuclease or DFF40 nuclease.
14. The method of claim 11 , wherein a chemical scavenger of hydrated electrons and oxygen radicals is added in step (a) to reduce damage to the DNA.
15. The method of claim 14 , the chemical scavenger is selected from sodium iodide, sodium nitrate, or a combination thereof.
16. The method of claim 14 , wherein the chemical scavenger is in micromolar or milimolar concentrations.
17. The method of claim 11 , wherein step (a) further comprises amplifying the adapter-DNA molecules to generate clonal strands of each adapter-DNA molecule and performing steps (b)-(g) with the clonal strands of the adapter-DNA molecules.
18. The method of claim 11 , further comprising comparing one or more sequence reads to a reference sequence to identify a presence of a variant.
19. A method of sequencing DNA using improved base-calling, comprising:
(a) generating a sequencing library comprising the steps—
digesting DNA with a dsDNA-specific nuclease to generate a plurality of DNA fragments;
attaching sequencing adapters to the plurality of DNA fragments to form a plurality of adapter-DNA molecules; and
amplifying at least some of the adapter-DNA molecules to generate clonal strands;
wherein a chemical scavenger of hydrated electrons and oxygen radicals is added in each of the steps to reduce damage to the DNA;
(b) flowing a plurality of clonal strands over a sequencer flow cell such that multiple clonal strands from the sequencing library bind to the flow cell;
(c) bridge amplifying the bound clonal strands of the adapter-DNA molecules to form a plurality of polonies on the flow cell;
(d) sequencing the polonies to obtain a preliminary sequence read for each polony, wherein for each polony, sequencing comprises:
(i) obtaining fluorescence intensity measurements for each nucleotide position of the polony; and
(ii) performing a first round of base calling by converting the fluorescence intensity measurements into a probable base call for each nucleotide position of the polony to provide preliminary sequence read, the preliminary sequence read having a preliminary error probability;
(e) grouping preliminary sequence reads generated from the clonal strands derived from a particular adapter-DNA molecule;
(f) calculating a fluorescence intensity measurement average for each nucleotide position from grouped preliminary sequence reads;
(g) generating a genome-wide mapping of phasing errors; and
(h) performing a second round of base calling for a nucleotide position of the grouped preliminary sequence reads using the fluorescence intensity measurement averages to provide a sequence read;
wherein an error probability of the sequence read corresponding to the particular adapter-DNA molecule is lower than any one of the preliminary error probabilities of the individual preliminary sequence reads in the group.
20. The method of claim 19 , wherein the second round of base calling is limited to each nucleotide positions of the sequence regions with phasing errors.