Methods for synchronizing nucleic acid molecules

• US 10,584,378 B2
• Filed: 08/12/2016
• Issued: 03/10/2020
• Est. Priority Date: 08/13/2015
• Status: Active Grant

• Current Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Original Assignee: Centrillion Technology Holdings Corporation (Centrillion Technology, Inc.)
• Inventors: Crnogorac, Filip, Zhou, Wei
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/235,668
• Publication Number:
  US 20170044601A1
• Time in Patent Office: 1,306 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

The methods, systems, kits and reagents provided herein relate to the synchronization of dephased/prephased nucleic acid molecules during sequencing reactions. The methods, systems, kits and reagents provided herein can be utilized to improve the efficiency and accuracy of sequencing technologies.

Claims

1. A method of synchronizing one or more unsynchronized sequencing products, comprising:

(a) hybridizing a plurality of sequencing primers to a plurality of target nucleic acids;

(b) performing 50 or more successive rounds of sequencing, thereby producing a plurality of sequencing products comprising said one or more unsynchronized sequencing products;

wherein each round of said 50 or more successive rounds of sequencing comprising;

extending said plurality of sequencing primers in the presence of one labeled nucleotide, and

(c) after said 50 or more successive rounds of sequencing, performing one or more synchronization steps on said plurality of sequencing products comprising said one or more unsynchronized sequencing products, wherein each of said one or more synchronization steps comprise;

(i) contacting said plurality of sequencing products comprising said one or more unsynchronized sequencing products with a first set of three different unlabeled nucleotides selected from the group consisting of;

dATP, dTTP, dCTP, dGTP and dUTP;

(ii) extending said plurality of sequencing products comprising said one or more unsynchronized sequencing products with a DNA polymerase to produce a plurality of extended sequencing products; and

(iii) optionally, removing said first set of three different nucleotides, thereby synchronizing said one or more unsynchronized sequencing products.

2. The method of claim 1, wherein said one or more synchronization steps further comprise:

contacting said plurality of extended sequencing products with a second set of three different unlabeled nucleotides selected from the group consisting of;

dATP, dTTP, dCTP, dGTP and dUTP, wherein said second set of unlabeled nucleotides is different from said first set of three different unlabeled nucleotides.

3. The method of claim 1, wherein said first set of three different unlabeled nucleotides comprises a reversible terminator nucleotide.

4. The method of claim 3, wherein after each synchronization step of said one or more synchronization steps, said reversible terminator nucleotide is deblocked and said plurality of sequencing products are made ready for further extension.

5. The method of claim 1, wherein, after performing said one or more synchronization steps, at least 95% of said plurality of extended sequencing products are synchronized.

6. The method of claim 1, wherein, after performing said one or more synchronization steps, said plurality of extended sequencing products having a chastity score of at least 0.85.

7. The method of claim 1, further comprising, removing said first set of three different nucleotides (i) by washing or (ii) by the use of a nucleotide degrading enzyme.

8. A method of synchronizing one or more unsynchronized sequencing products, comprising:

performing one or more synchronization steps on a plurality of sequencing products, said plurality of sequencing products comprising said one or more unsynchronized sequencing products, wherein each of said one or more synchronization steps comprises;

(i) contacting said plurality of sequencing products with a set of up to three different nucleotides selected from the group consisting of;

dATP, dTTP, dCTP, dGTP and dUTP;

(ii) extending said plurality of sequencing products with a DNA polymerase; and

(iii) optionally, removing said set of up to three different nucleotides,

thereby synchronizing said one or more unsynchronized sequencing products;

wherein said one or more synchronization steps comprises eight consecutive synchronization steps, said eight consecutive synchronization steps comprising;

contacting said plurality of sequencing products with dCTP, dGTP and dTTP;

contacting said plurality of sequencing products with dATP, dTTP, and dGTP;

contacting said plurality of sequencing products with dATP, dCTP, and dTTP;

contacting said plurality of sequencing products with dATP, dCTP and dGTP;

contacting said plurality of sequencing products with dATP, dCTP, and dTTP;

contacting said plurality of sequencing products with dATP, dTTP, and dGTP;

contacting said plurality of sequencing products with dCTP, dGTP and dTTP; and

contacting said plurality of sequencing products with dATP, dCTP and dGTP.

9. The method of claim 8, further comprising, prior to performing said one or more synchronization steps, performing one or more successive rounds of sequencing.

10. The method of claim 9, wherein said one or more successive rounds of sequencing comprise 50 or more successive rounds of sequencing.

11. The method of claim 9, wherein each of said one or more successive rounds of sequencing comprises (i) extending a plurality of sequencing primers using a plurality of target nucleic acids as template in the presence of one or more labeled nucleotides to generate said plurality of sequencing products;

and (ii) determining nucleic acid sequences of said plurality of sequencing products.

12. The method of claim 11, wherein said one or more labeled nucleotides comprise fluorescent labels.

13. The method of claim 11, wherein said plurality of target nucleic acids comprises DNA, RNA or modified nucleic acids.

14. The method of claim 11, wherein said plurality of target nucleic acids comprises at least 100,000 target nucleic acids.

15. The method of claim 8, further comprising, after performing said one or more synchronization steps, repeating, one or more times, one or more rounds of sequencing followed by one or more synchronization steps.

16. The method of claim 8, wherein said set of up to three different nucleotides comprises native nucleotides.

17. The method of claim 8, wherein said set of up to three different nucleotides comprises unlabeled nucleotides.