Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
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.
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 having (i) a unique target nucleic acid sequence that is the same for each population, the target nucleic acid sequence having two ends, and (ii) a pair of non-target oligonucleotide tag sequences at the two ends of each target nucleic acid sequence, such that in the pool of nucleic acid molecules, each oligonucleotide tag sequence comprises a unique portion for each target nucleic acid sequence; wherein the pool was prepared by tagging target nucleic acid molecules with a library of non-target oligonucleotide tag sequences that are present in a greater number than the target nucleic acid molecules;
(b) sequencing the nucleic acid molecules from both ends to obtain a paired end read; and
(c) sorting nucleic acid molecules having a predetermined sequence according to the identity of their corresponding paired end read.
2. The method of claim 1 further comprising amplifying the nucleic acid molecules having the predetermined sequence.
3. The method of claim 2 further comprising amplifying the nucleic acid molecules having the predetermined sequence using primers complementary to at least part of the oligonucleotide tag sequence.
4. The method of claim 1 wherein the target nucleic acid molecules comprises a first population of target nucleic acid molecules having the predetermined sequence and a second population of target nucleic acid molecules having a sequence different than the predetermined sequence.
5. The method of claim 4 further comprising assembling a plurality of target nucleic acid molecules onto a solid support and pooling the target nucleic acid molecules.
6. The method of claim 5 further comprising diluting the target nucleic acid molecules.
7. The method of claim 1 further comprising diluting the pool of nucleic acid molecules.
8. The method of claim 1 wherein the non-target oligonucleotide tag sequences are ligated to the two ends of each of the target nucleic acid molecules.
9. The method of claim 8 wherein the non-target oligonucleotide tag sequences are added to the target nucleic acid molecules by ligating the target nucleic acid molecules with one or more nucleic acid molecules comprising the non-target oligonucleotide tag sequences.
10. The method of claim 9 wherein the one or more nucleic acid molecules comprising the non-target oligonucleotide tag sequences are vectors.
11. The method of claim 1 wherein in the step of providing, the non-target oligonucleotide tag sequences are joined to the two ends of each of the target nucleic acid molecules by polymerase chain reaction.
12. The method of claim 1 wherein each non-target oligonucleotide tag sequence includes a degenerate nucleotide sequence, or a partially degenerate sequence.
13. The method of claim 12 wherein the degenerate nucleotide sequence 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.
14. The method of claim 2 wherein in the step of amplifying the primers are complementary to the pair of non-target oligonucleotide tag sequences.