Methods for sorting nucleic acids and multiplexed preparative in vitro cloning

• US 20140141982A1
• Filed: 04/24/2013
• Published: 05/22/2014
• Est. Priority Date: 04/24/2012
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Goldberg, Martin J., Kung, Li-yun A., Schindler, Daniel, Hudson, Michael E.
• Granted Patent: US 10,081,807 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups

Abstract

Methods and compositions relate to the sorting and cloning of high fidelity nucleic acids using high throughput sequencing. Specifically, nucleic acid molecules having the desired predetermined sequence can be sorted from a pool comprising a plurality of nucleic acids having correct and incorrect sequences.

Claims

1. A method of sorting nucleic acid molecules having a predetermined sequence, the method comprising:

(a) providing a pool of nucleic acid molecules comprising at least two populations of nucleic acid molecules, each population of nucleic acid molecule having a unique target nucleic acid sequence, the target nucleic acid sequence having a 5′

end and a 3′

end;

(b) tagging the 5′

end and the 3′

end of the target nucleic acid molecules with a non-target oligonucleotide tag sequence, wherein the oligonucleotide tag sequence comprises a unique nucleotide tag;

(c) diluting the tagged target nucleic acids;

(d) subjecting the tagged target nucleic acid molecules to a sequencing reaction from both ends to obtain a paired end read; and

(e) sorting the target nucleic acid molecules having the predetermined sequence according to the identity of their corresponding unique pair of oligonucleotide tags.

4. The method of claim 1 or claim 2 further comprising amplifying the nucleic acid molecules having the predetermined sequence.

6. The method of claim 1 wherein the step of providing comprises pooling a plurality of nucleic acid molecules to form the pool of nucleic acid molecules, wherein each plurality of nucleic acid molecules comprises a population of nucleic acid sequences having the predetermined sequence and a population of nucleic acid sequences having a sequence different than the predetermined sequence.

7. The method of claim 6 further comprising assembling a plurality of nucleic acid molecules onto a solid support prior to pooling the plurality of nucleic acid molecules.

8. The method of claim 6 further comprising diluting the plurality of nucleic acid molecules prior to the step of pooling or after the step of pooling.

9. The method of claim 1 or claim 2 wherein in the step of providing the pool of nucleic acid molecules is normalized.

10. The method of claim 1 or claim 3 wherein in the step of tagging, the non-target oligonucleotide tag sequences are ligated to the 5′

and 3′

end of the target nucleic acid molecules.

11. The method of claim 10 wherein in the step of tagging, the non-target oligonucleotide tag sequences are added to the target nucleic acid molecules by ligating the target nucleic acid molecules with a nucleic acid molecule comprising the oligonucleotide tag sequences.

12. The method of claim 10 wherein the nucleic acid molecules comprising the non-target oligonucleotide tag sequences are vectors.

13. The method of claim 1 wherein in the step of tagging, the non-target oligonucleotide tag sequences are joined to the 5′

and 3′

end of the target nucleic acid molecules by polymerase chain reaction.

14. The method of claim 1 wherein each target nucleic acid molecule comprises a 5′

end common adaptor sequence and 3′

end common adaptor sequence and wherein the oligonucleotide tag sequence further comprises a common adaptor sequence.

15. The method of claim 1 wherein each population of target nucleic acid molecules has a different desired nucleic acid sequence.

16. The method of claim 1 or claim 2 wherein the unique non-target oligonucleotide tag includes a degenerate nucleotide sequence, or a partially degenerate sequence.

17. The method of claim 16 wherein the sequence of non-target oligonucleotide tag is CCWSWDHSHDBVHDNNNNMM and/or CCSWSWHDSDHVBDHNNNNMM, wherein W represents A or T, S represents G or C, M represents A or C, B represents C, G or T, D represents A, G or T, H represents A, C or T, V represents A, C, or G and N represents any base A, C, G or T.

18. The method of claim 16 wherein each non-target oligonucleotide tag includes a unique sequence and a common sequence.

19. The method of claim 4 wherein in the step of amplifying the primers are complementary to the pair of non-target oligonucleotide tags.

2. A method of sorting nucleic acid molecules having a predetermined sequence, the method comprising:

(a) providing a pool of nucleic acid molecules comprising at least two populations of nucleic acid molecules, each population of nucleic acid molecule having (i) a unique target nucleic acid sequence, the target nucleic acid sequence having a 5′

end and a 3′

end, and (ii) a non-target oligonucleotide tag sequence at the 5′

end and the 3′

end, the oligonucleotide tag sequence comprising a unique oligonucleotide tag;

(b) subjecting the tagged target nucleic acid molecules to a sequencing reaction from both ends to obtain a paired end read; and

(c) sorting the target nucleic acid molecules having the predetermined sequence according to the identity of their corresponding unique pair of oligonucleotide tags.

3. The method of claim 2 wherein the step of providing comprises

pooling nucleic acid molecules, thereby forming a pool of nucleic acid molecules comprising at least two populations of nucleic acid molecules, each population of nucleic acid molecule having a unique target nucleic acid sequence, the nucleic acid sequence having a 5′

end and a 3′

end;

tagging the 5′

end and the 3′

end of the nucleic acid molecules with a non-target oligonucleotide tag sequence, wherein the oligonucleotide tag sequence comprises a unique nucleotide tag and a primer region.

5. The method of claim 5 further comprising amplifying the nucleic acid molecules using primers complementary to at least part of the oligonucleotide tag.

20. A method for designing a plurality of oligonucleotides for assembly into a nucleic acid sequence of interest having a predefined sequence, the method comprising:

(a) computationally dividing the sequence of each nucleic acid sequence of interest into partially overlapping construction oligonucleotide sequences;

(b) selecting a first plurality of construction oligonucleotide sequences such that every two adjacent construction oligonucleotide sequences overlap with each other by N bases, wherein each N-base sequence is at least 4 bases long;

(c) comparing the N-base sequences to one another so that one or more of the following constraints are met;

the N-base sequences differ to one another by at least 2 bases, or the N-base sequences differ to one another by at least one base in the last 3 bases of the 5′

end or 3′

end;

(d) identifying from the first plurality of construction oligonucleotide sequences, a second plurality of construction oligonucleotide sequences satisfying the constraints;

(e) determining the number of oligonucleotides in the second plurality of oligonucleotides;

(f) ranking the oligonucleotides from the second plurality of oligonucleotides that meet or exceed the constraints and based on the number of oligonucleotides;

(g) using the ranking to design a set of satisfactory partially overlapping construction oligonucleotides.

21. The method of claims 20 further comprising synthesizing the set of satisfactory partially overlapping construction oligonucleotides

22. The method of claim 20 wherein in the step of using the ranking, the set having the smaller number of oligonucleotides is selected, and/or the set having the higher number of base difference in the N-base sequence is selected.

23. The method of claim 20 further comprising computationally adding a non-target flanking sequence to the termini of at least a portion of said construction oligonucleotides.

24. The method of claim 23 wherein the non-target flanking sequences comprise a primer binding site.

25. The method of claim 21 wherein in the step of synthesizing the construction oligonucleotides are synthesized on a solid support.

26. The method of claim 20 wherein the construction oligonucleotides have an average length range between 98 and 104 base pairs.

27. The method of claim 21 further comprising assembling the construction oligonucleotides into the nucleic acid of interest.

28. A method of isolating a nucleic acid having a predefined sequence, the method comprising:

(a) providing at least one population of nucleic acid molecules;

(b) isolating a clonal population of nucleic acid molecules on a surface;

(c) determining the sequence of the clonal population of nucleic acid molecules;

(d) localizing the clonal population of nucleic acid molecules having the predefined sequence; and

(d) amplifying the nucleic acid molecule having the predefined sequence.

29. The method of claim 28 wherein the step of isolating is by dilution.

30. The method of claim 29 wherein in the step of isolating, the surface is a flow cell.

31. A method for isolating a nucleic acid having a predefined sequence, the method comprising:

(a) providing a pool of nucleic acid molecules comprising error-free and error-containing nucleic acid molecules;

(b) tagging the nucleic acid molecules;

(c) optionally fragmenting the nucleic acid molecules;

(d) determining the sequence of the nucleic acid molecules;

(e) localizing the error-free and error-containing nucleic acid molecules; and

(f) isolating the error-free nucleic acid molecules.

32. The method of claim 31 wherein the step of isolating comprises one or more of (a) ablating the error-containing nucleic acid molecules;

(b) amplifying the error-free nucleic acid molecules;

(c) immobilizing the error-free nucleic acid molecules onto a surface and separating the error-free nucleic acid molecules from the error-containing nucleic acid molecules.

33. The method of claim 32 wherein in the step of immobilizing, the surface is a bead.

34. The method of claim 33 wherein the pool of nucleic acid molecules comprises at least two populations of nucleic acids and wherein in the step of isolating each population of nucleic acids is onto a distinct population of beads.

35. The method of claim 34 further comprising sorting the distinct populations of beads.

36. A method of sorting nucleic acid molecules having a predetermined sequence, the method comprising:

(a) providing a pool of nucleic acid molecules comprising at least two populations of nucleic acid molecules, each population of nucleic acid molecule having a unique target nucleic acid sequence, the target nucleic acid sequence having a 5′

end and a 3′

end;

(b) tagging the 5′

end and the 3′

end of the target nucleic acid molecules with a pair of non-target oligonucleotide tag sequences, wherein each oligonucleotide tag sequence comprises a unique nucleotide tag;

(c) diluting the tagged target nucleic acids;

(d) amplifying the tagged nucleic acids;

(e) dividing the amplified tagged nucleic acids into two pools;

(f) subjecting a first pool comprising the tagged target nucleic acid molecules to a sequencing reaction from both ends to obtain a paired end read;

(g) subjecting a second pool comprising the tagged target nucleic acid molecules to ligation to form circular nucleic acid molecules thereby bringing the pair of tags in close proximity;

(h) sequencing the pair of tags; and

(i) sorting the target nucleic acid molecules having the predetermined sequence according to the identity of their corresponding unique pair of oligonucleotide tags.

37. The method of claim 37 wherein the pair of tags is amplified before being sequenced.

38. The method of claim 37 wherein the pair of tags is cleaved off before being sequenced.

39. The method of claim 38 wherein the pair of tags is cleaved of using a restriction enzyme.