Error-free amplification of DNA for clonal sequencing

1. A method of providing low copy number circularized nucleic acid variants to reaction volumes, the method comprising:

(a) providing a population of clonal nucleic acids;

(b) generating a set of partially overlapping nucleic acid fragments from the population of clonal nucleic acids;

(c) circularizing the set of partially overlapping nucleic acid fragments to produce circularized nucleic acid variants of at least one member of the clonal population, the variants comprising partially overlapping subsequences of the member; and,

(d) aliquotting the circularized nucleic acid variants into low copy number reaction volumes.

2. The method of claim 1 , wherein (a) comprises:

producing a population of nucleic acid fragments of similar lengths;

attaching tags to ends of the nucleic acid fragments of similar lengths to produce tagged fragments; and,

amplifying the tagged fragments to generate the population of clonal nucleic acids.

3. The method of claim 2 , wherein producing the population of nucleic acid fragments of similar lengths comprises providing a population of nucleic acids whose lengths are within a 20% range of one another.

4. The method of claim 1 , wherein providing the population of clonal nucleic acids comprises providing a genomic DNA.

5. The method of claim 4 , wherein the genomic DNA has been denatured to produce a single-stranded DNA.

6. The method of claim 1 , wherein the population of clonal nucleic acids comprises a concatamer that comprises a plurality of tandem short expressed sequence tags (ESTs).

7. The method of claim 2 , wherein attaching tags to ends of nucleic acid fragments comprises hybridizing the tags to the ends of the nucleic acid fragments and extending the tags with a polymerase to produce tagged fragments.

8. The method of claim 2 , wherein attaching tags to ends of the nucleic acid fragments comprises ligating the tags to the ends of the nucleic acid fragments with a ligase to produce tagged fragments.

9. The method of claim 8 , wherein the ligase, which ligates the tags to the ends of the nucleic acid fragments, comprises an enzyme capable of catalyzing intermolecular ligation of single-stranded DNA molecules.

10. The method of claim 2 , wherein attaching tags to ends of the nucleic acid fragments comprises attaching tags comprising one or more moieties selected from: a biotinylated nucleotide, a phosphorylated nucleotide, a methylated nucleotide, a sequence capable of forming a hairpin secondary structure, an oligonucleotide hybridization site, a restriction site, a DNA promoter, an RNA promoter, a sample or library identification tag, and a cis regulatory sequence to the ends of the nucleic acid fragments, thereby forming tagged fragments.

11. The method of claim 2 , wherein amplifying the tagged fragments comprises performing: PCR, emulsion PCR, polony amplification, or surface amplification to produce the population of clonal nucleic acids.

12. The method of claim 2 , wherein amplifying the tagged fragments comprises amplifying the tagged fragments at an exponential rate to produce the population of clonal nucleic acids.

13. The method of claim 12 , wherein amplifying the tagged fragments comprises linear amplification of the tagged fragments prior to amplifying the tagged fragments at an exponential rate, which linear amplification results in a reduction of nucleotide misincorporation as compared to exponential amplification alone, to produce the population of clonal nucleic acids.

14. The method of claim 1 , wherein (b) comprises cleaving the population of clonal nucleic acids to produce the set of partially overlapping nucleic acid fragments.

15. The method of claim 14 , wherein cleaving the population of clonal nucleic acids comprises: enzymatic digestion, sonication, mechanical shearing, electrochemical cleavage, or nebulization to produce the set of partially overlapping nucleic acid fragments.

16. The method of claim 1 , wherein (b) comprises copying partially overlapping subsequences of the population of clonal nucleic acids to produce the set of partially overlapping nucleic acid fragments.

17. The method of claim 16 , wherein copying partially overlapping subsequences of the population of clonal nucleic acids comprises extending nested primers, which nested primers hybridize to interior sequences of the clonal nucleic acids, with a polymerase to produce the set of partially overlapping nucleic acid fragments.

18. The method of claim 1 , wherein (c) comprises ligating a first end of a fragment to a second end of the fragment with a ligase to produce circularized variants.

19. The method of claim 18 , wherein the ligase which ligates a first end of a fragment to a second end of the fragment to produce circularized variants comprises an enzyme capable of catalyzing intramolecular ligation of single-stranded DNA molecules.

20. The method of claim 1 , wherein aliquotting the circularized variants into low copy number reaction volumes comprises flowing or depositing the circularized variants onto zero-mode waveguides.

21. The method of claim 1 , wherein reaction volumes comprise one or more sequencing reagents selected from: a labeled nucleotide or nucleotide analog, a polymerase, and a divalent cation.

22. The method of claim 2 wherein attaching tags to ends of the nucleic acid fragments comprises attaching tags comprising at least one restriction site.

23. The method of claim 1 wherein (c) comprises ligation with a ligase to produce circularized variants.

24. The method of claim 1 , further comprising (e) determining the nucleic acid sequence of one or more of the circularized nucleic acid variants within the low copy number reaction volume.

25. The method of claim 24 wherein the sequence is determined using single-molecule sequencing.

26. The method of claim 1 wherein at least some of the low copy number volumes comprise a single circularized nucleic acid variant.