Nucleic acid constructs and methods of use
The present invention provides oligonucleotide constructs, sets of such oligonucleotide constructs, and methods of using such oligonucleotide constructs to provide validated sequences or sets of validated sequences corresponding to desired ROIs. Such validated ROIs and constructs containing these have a wide variety of uses, including in synthetic biology, quantitative nucleic acid analysis, polymorphism and/or mutation screening, and the like.
1. A method for analyzing sequences of nucleic acid target molecules, comprising:
a) tagging nucleic acid target molecules of a set of nucleic acid target molecules with identifiers from a set of identifiers to create a set of tagged constructs,
wherein each tagged construct comprises two or more identifiers, and
wherein the set of tagged constructs comprises a subset having different nucleic acid target molecules tagged with the same identifiers;
b) amplifying all or a subset of the tagged constructs to form a set of amplified constructs,
wherein amplified constructs that are descendants of a particular tagged construct comprise the same identifiers of the particular tagged construct;
c) sequencing a subset of the amplified constructs,
wherein the diversity of the identifiers of the set of identifiers is such that sequenced molecules that are descendants of different tagged constructs are distinguishable from one another using a combination of a sequence of the nucleic acid target molecule and the identifiers tagged thereto; and
d) comparing sequenced molecules that are descendants of the same tagged construct,
thereby identifying a sequence of the nucleic acid target molecule of the tagged construct.
2. The method of claim 1 , wherein sequenced molecules having different nucleic acid target molecules tagged with the same identifiers are identified as not descendants of the same tagged construct.
3. The method of claim 1 , wherein amplification sites are attached to the tagged constructs and the attached amplification sites flank the nucleic acid target molecule sequence and identifiers.
4. The method of claim 3 , wherein the attached amplification sites comprise universal primer binding sites.
5. The method of claim 1 , wherein the amplifying in step b) comprises performing a universal amplification.
6. The method of claim 1 , wherein the amplified constructs in step b) are enriched for the subset in step c).
7. The method of claim 6 , wherein the enrichment comprises hybridizing a labeled tag to the amplified constructs and affinity capture of the labeled tag.
8. The method of claim 1 , wherein the identifiers comprise a degenerate sequence.
9. The method of claim 1 , wherein the identifiers are selected from a diverse, pre-defined set of oligonucleotide sequences.
10. The method of claim 1 , wherein the tagging is performed by ligation.
11. The method of claim 1 , wherein the tagging is performed by PCR.
12. The method of claim 1 , wherein the amplifying comprises solid-phase amplification.
13. The method of claim 1 , wherein the amplifying comprises emulsion PCR.
14. The method of claim 13 , wherein the emulsion PCR comprises emulsion PCR with beads.
15. The method of claim 14 , wherein the beads are magnetic beads.
16. The method of claim 1 , wherein the sequencing comprises performing massively parallel next generation sequencing.
17. The method of claim 16 , wherein the next generation sequencing comprises a one pass sequencing method.
18. The method of claim 16 , wherein the next generation sequencing comprises paired-end sequencing.
19. The method of claim 16 , wherein the next generation sequencing comprises a hybridization-based method.
20. The method of claim 16 , wherein the next generation sequencing comprises a sequencing by synthesis method.
21. The method of claim 16 , wherein the next generation sequencing comprises a ligation-based method.
22. The method of claim 1 , further comprising inferring a starting number of nucleic acid target molecules in the set of nucleic acid target molecules.
23. The method of claim 1 , further comprising identifying a mutation in a target nucleic acid molecule, wherein the mutation is in sequenced molecules that are descendants of the same tagged construct and is not an error introduced in the manipulation of the nucleic acid target molecule.
24. The method of claim 1 , further comprising determining one or more sequence variants or allelic variants of a nucleic acid target molecule in the set of nucleic acid target molecules.
25. The method of claim 1 , wherein the identified sequence of the nucleic acid target molecule is an error-corrected sequence.
26. The method of claim 1 , wherein the identified sequence of the nucleic acid target molecule is an error-free sequence.