Entangled Mate Sequencing

• US 20120245039A1
• Filed: 09/22/2010
• Published: 09/27/2012
• Est. Priority Date: 09/22/2009
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Roth, Frederick P., Chee, Mark, Mellor, Joseph C., Lu, Yong
• Granted Patent: US 10,385,391 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Methods and compositions are provided for performing a set of N DNA sequencing reaction cycles whereby sequence information is obtained for approximately 2*N nucleotide bases.

Claims

1. A method of simultaneously determining nucleotide sequence identities of heterogeneous, tethered nucleic acid template sequences comprising the steps of:

providing a mosaic target nucleic acid sequence including a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are tethered;

determining a mixed nucleic acid sequence signature comprising the tethered first and second nucleic acid sequences; and

comparing the nucleic acid sequence signature to a reference collection of sequence signatures to determine the nucleotide sequence identity of each of the tethered first and second nucleic acid sequences.

2. The method of claim 1, wherein the heterogeneous, tethered nucleic acid sequences represent one or more of a genome, a proteome, a transcriptome or a cellular pathway.

3. A method of determining sequence identities of two tethered, heterogeneous nucleic acid sequences comprising the steps of:

providing a heterogeneous library of mosaic target nucleic acids, in which a plurality include a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are tethered;

determining a heterogeneous collection of mixed nucleic acid sequence signatures, each corresponding to a tethered pair of first and second nucleic acid sequences; and

comparing the nucleic acid sequence signature to a reference collection of sequence signatures to determine the nucleotide sequence identity of each of the tethered first and second nucleic acid sequences.

4. The method of claim 3, wherein the first nucleic acid sequences is a barcode sequence and the second nucleic acid sequence is a genomic sequence.

5. The method of claim 3, wherein the reference collection of sequence signatures is contained in a database.

6. The method of claim 3, wherein the mosaic target nucleic acid sequence includes a first nucleic acid sequence, a second nucleic acid sequence and a third nucleic acid sequence, wherein the first, second and third nucleic acid sequences are tethered;

and

wherein the identity of each set of three bases extended from the 3′

end of the first, second and third primers to determine a nucleic acid signature of the tightly co-localized linked first, second and third nucleic acid sequences is determined.

7. The method of claim 3, wherein the mosaic target nucleic acid sequence includes 3 or more tethered, nucleic acid sequences.

8. The method of claim 3, wherein the mosaic target nucleic acid sequence is present on an array.

9. A method of simultaneously determining nucleotide sequence identities of two tethered nucleic acid sequences comprising the steps of:

providing a heterogeneous library of mosaic target nucleic acids, in which a plurality include a first nucleic acid sequence and a second nucleic acid sequence, wherein the first and second nucleic acid sequences are tethered;

annealing a first primer sequence to a portion of the first nucleic acid sequence and a second primer sequence to a portion of the second nucleic acid sequence;

extending the annealed primers simultaneously;

sequentially determining the identity of each set of two bases extended from the 3′

ends of the first and second primers to determine a nucleic acid sequence signature of the tethered first and second nucleic acid sequences; and

comparing the nucleic acid signature of the first nucleic acid sequence to a reference collection of sequence signatures to determine the nucleotide sequence identity of each of the tethered first and second nucleic acid sequences.

10. The method of claim 9, wherein the second nucleic acid sequence is not present in the reference collection of sequence signatures.

11. The method of claim 9, wherein each set of two bases is selected from the group consisting of AA, CC, GG, TT, AC, AG, AT, CG, CT and GT.

12. The method of claim 9, wherein the first and second nucleic acid sequences are barcode sequences.

13. The method of claim 9, wherein the first and second nucleic acid sequences are genomic sequences.

14. The method of claim 9, wherein the first and second primer sequences have the same sequence identity.

15. The method of claim 9, wherein the first and second primer sequences have a different sequence identity.

16. A method of identifying a cell type having heterogeneous nucleic acid sequences comprising the steps of:

providing a cell having a mosaic target nucleic acid sequence including a first barcode sequence and a second barcode sequence, wherein the first and second barcode sequences are tethered;

annealing a first primer sequence to a portion of the first barcode and a second primer sequence to a portion of the second barcode;

extending the annealed primers simultaneously;

determining a nucleic acid sequence signature of the first and second barcodes; and

comparing the nucleic acid sequence signature to a reference collection of barcode sequence signatures to determine the identity of the cell.

17. The method of claim 16, wherein the cell is a yeast cell.

18. The method of claim 17, wherein the yeast cell contains two or more alterations.

19. The method of claim 16, wherein the heterogeneous nucleic acid sequences represent one or more of a genome, a proteome, a transcriptome or a cellular pathway.

20. The method of claim 16, wherein the heterogeneous nucleic acid sequences represent Homo sapiens nucleic acid sequences.