Methods and compositions for efficient base calling in sequencing reactions

• US 10,662,473 B2
• Filed: 08/03/2018
• Issued: 05/26/2020
• 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 W
• Application Number:
  US16/054,968
• Publication Number:
  US 20180346980A1
• Time in Patent Office: 662 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 435 9151, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253
• 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 sequencing method for determining nucleotide sequences from a plurality of different polynucleotide templates, said different polynucleotide templates having different polynucleotide template sequences, comprising:

(a) providing a nucleic acid array comprising the plurality of different polynucleotide templates disposed at a plurality of spatially discrete sites on a surface, wherein each of the plurality of spatially discrete sites comprise a plurality of copies of one polynucleotide template of the plurality of different polynucleotide templates;

(b) annealing oligonucleotide primers to primer binding sites of the plurality of different polynucleotide templates disposed at the plurality of spatially discrete sites, thereby producing annealed oligonucleotide primers at each of the plurality of spatially discrete sites;

(c) after step (b),

(1) extending the annealed oligonucleotide primers at each of the individual spatially discrete sites by a sequencing-by-extension reaction in which each of the plurality of spatially discrete sites on the nucleic acid array are contacted with a pool of nucleotides and said pool of nucleotides comprises

(i) a first nucleotide comprising a first fluorescent dye that emits light detectable at a first wavelength,

(ii) a second nucleotide comprising a second fluorescent dye that emits light detectable at a second wavelength,

(iii) a third nucleotide, wherein the third nucleotide comprises a first type of the third nucleotide that comprises a fluorescent dye that emits light detectable at the first wavelength and a second type of the third nucleotide that comprises a fluorescent dye that emits light detectable at the second wavelength, wherein

the first nucleotide and the first type of the third nucleotide have different fluorescent intensities at the first wavelength when the first fluorescent dye of the first nucleotide and the fluorescent dye of the first type of the third nucleotide are excited, and the second nucleotide and the second type of the third nucleotide have different fluorescent intensities at the second wavelength when the second fluorescent dye of the second nucleotide and the fluorescent dye of the second type of the third nucleotide are excited, and

(iv) a fourth nucleotide that does not comprise a fluorescent dye;

wherein the first nucleotide, second nucleotide, third nucleotide and fourth nucleotide are different from each other and comprise different nucleotide bases selected from adenosine, guanine, thymidine and cytosine,

wherein the first wavelength and the second wavelength are different and wherein said extending the annealed oligonucleotide primers at each of the plurality of spatially discrete sites comprises incorporating one of the first nucleotide, the second nucleotide, the third nucleotide, or the fourth nucleotide to the annealed oligonucleotide primers, thereby producing extended primers at each of the plurality of spatially discrete sites, and then

(2) illuminating each of the plurality of the spatially discrete sites on the nucleic acid array;

(3) measuring the intensity of a fluorescence signal at the first wavelength and the intensity of a fluorescence signal at the second wavelength at each of the plurality of spatially discrete sites to determine which of the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide is incorporated into the extended primers at each of the plurality of spatially discrete sites wherein,

(i) detecting a fluorescent signal at the first wavelength and not detecting a fluorescent signal at the second wavelength at a first portion of the plurality of spatially discrete sites indicates that the first nucleotide is incorporated into the extended primers at the first portion of the plurality of spatially discrete sites;

(ii) detecting a fluorescent signal at the second wavelength and not detecting a fluorescent signal at the first wavelength at a second portion of the plurality of spatially discrete sites indicates that the second nucleotide is incorporated into the extended primers at the second portion of the plurality of spatially discrete sites;

(iii) detecting a fluorescent signal at the first wavelength and detecting a fluorescent signal at the second wavelength at a third portion of the plurality of spatially discrete sites indicate that the third nucleotide is incorporated into the extended primers at the third portion of the plurality of spatially discrete sites, and

(iv) not detecting a fluorescent signal at either the first wavelength or the second wavelength at fourth portion of the plurality of spatially discrete sites indicates that the fourth nucleotide is incorporated into the extended primers at the fourth portion of the plurality of spatially discrete sites;

(d) after step (c), carrying out multiple cycles of the sequencing-by-extension reaction, wherein each cycle of the multiple cycles of the sequencing-by-extension reaction is performed by contacting the plurality of spatially discrete sites on the nucleic acid array with the pool of nucleotides, thereby determining the nucleotide sequences from the plurality of different polynucleotide templates.

2. The method of claim 1, wherein the fourth nucleotide is dGTP.

3. The method of claim 1 wherein the plurality of different polynucleotide templates disposed at the plurality of spatially discrete sites are amplicons that comprise adaptor arms.

4. The method of claim 1, wherein the first nucleotide and the first type of the third nucleotide comprise different numbers of molecules of the first fluorescent dye and emit light detectable at the first wavelength with different intensities;

and the second nucleotide and the second type of the third nucleotide comprise different numbers of molecules of the second fluorescent dye and emit light detectable at the second wavelength at different intensities.

5. The method of claim 1, wherein the first nucleotide and the first type of the third nucleotide comprise different fluorescent dyes that emit light detectable at the first wavelength with different intensities and the second nucleotide and the second type of the third nucleotide comprise different fluorescent dyes that emit light detectable at the second wavelength with different intensities.