Methods and compositions for efficient base calling in sequencing reactions

• US 9,523,125 B2
• Filed: 12/02/2013
• Issued: 12/20/2016
• Est. Priority Date: 01/28/2008
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje
• Primary Examiner(s): Lu, Frank
• Application Number:
  US14/094,630
• Publication Number:
  US 20140356865A1
• Time in Patent Office: 1,114 Days
• Field of Search: 435/6.1, 435/6.11, 435/91.1, 435/91.2, 435/183, 435/89, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/26.7, 536/26.71, 536/26.72
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences. In particular, the present invention provides methods and compositions for improving the efficiency of sequencing reactions by using fewer labels to distinguish between nucleotides and by detecting nucleotides at multiple detection positions in a target sequence.

Claims

1. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:

(i) first dNTPs comprising a first label;

(ii) second dNTPs comprising a second label;

(iii) third dNTPs comprising both the first label and the second label; and

(iv) fourth dNTPs comprising neither the first label nor the second label;

wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and

wherein the first label and the second label are different labels and are distinguishable.

2. The composition of claim 1 wherein the first label and the second label are fluorescent dyes.

3. The composition of claim 2 wherein at least one of the first label and the second label comprises rhodamine.

4. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:

(i) first dNTPs comprising a first label;

(ii) second dNTPs comprising a second label;

(iii) third dNTPs wherein some of the third dNTPs comprise the first label but not the second label and some of the third dNTPs comprise the second label but not the first label; and

(iv) fourth dNTPs comprising neither the first label nor the second label;

wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and

wherein the first label and the second label are different labels and are distinguishable.

5. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:

(i) a plurality of first dNTPs labeled with a first label;

(ii) a plurality of second dNTPs comprising a second label;

(iii) a plurality of third dNTPs, wherein some of the third dNTPs are labeled with the first label but not the second label and some of the third dNTPs are labeled with the second label but not the first label; and

(iv) a plurality of fourth dNTPs labeled with neither the first label nor the second label;

wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and

wherein the first label and the second label are different labels and are distinguishable.

6. The composition of claim 5 wherein the first label and the second label are fluorescent dyes.

7. The composition of claim 6 wherein at least one of the first label and the second label comprises rhodamine.

8. A composition for determining the identity of a base in a target nucleic acid by sequencing-by-synthesis, the composition comprising a mixture of deoxyribonucleotide triphosphates (dNTPs) comprising:

(i) a plurality of first dNTPs labeled with a first label;

(ii) a plurality of second dNTPs comprising a second label;

(iii) a plurality of third dNTPs, wherein the third dNTPs are labeled with both the first label and the second label; and

(iv) a plurality of fourth dNTPs labeled with neither the first label nor the second label;

wherein each of the first, second, third, and fourth dNTPs is selected from the group consisting of dATP, dTTP, dCTP, and dGTP, and are different from each other; and

wherein the first label and the second label are different labels and are distinguishable.

9. The composition of claim 8 wherein the first label and the second label are fluorescent dyes.

10. The composition of claim 9 wherein at least one of the first label and the second label comprises rhodamine.