METHODS AND COMPOSITIONS FOR EFFICIENT BASE CALLING IN SEQUENCING REACTIONS

• US 20180346980A1
• Filed: 08/03/2018
• Published: 12/06/2018
• 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
• Granted Patent: US 10,662,473 B2
• Time in Patent Office: Days
• 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

1-21. -21. (canceled)

22. A method for determining which of four nucleotide bases is present at detection positions of a plurality of different polynucleotide templates, comprising:

(a) providing a nucleic acid array comprising spatially discrete sites on a surface, wherein single-stranded polynucleotide templates are disposed on the sites, and wherein individual sites each comprise a plurality of copies of a polynucleotide template sequence;

(b) annealing oligonucleotide primers to primer binding sites on the polynucleotide templates;

(c) performing multiple cycles of sequencing-by-extension reactions in which the array is contacted with a pool of nucleotides comprising

(1) first nucleotides comprising a first fluorescent label detectable at a first emission wavelength;

(2) second nucleotides comprising a second fluorescent label detectable at a second emission wavelength;

(3) third nucleotides wherein some of the third nucleotides comprise a fluorescent label detectable at the first emission wavelength but with different brightness than the first fluorescent label and some of the third nucleotides comprise a fluorescent label detectable at the second emission wavelength but with different brightness than the second fluorescent label; and

(4) fourth nucleotides that do not comprise fluorescent labels;

wherein the first, second, third and fourth nucleotides are different from each other and the annealed oligonucleotide primer at each site are extended by incorporating a first, second, third or fourth nucleotide;

(d) determining, for a plurality of the spatially discrete sites, which of the first, second, third and fourth nucleotides is present at a detection position of the polynucleotide templates disposed at the site, by detecting at the site a fluorescent signal at the first emission wavelength, a fluorescent signal at the second emission wavelength, or fluorescent signals at both the first and second emission wavelengths, or by detecting fluorescent signals at neither the first or second emission wavelengths.

23. The method of claim 22 wherein the fourth nucleotides are dGTP.

24. The method of claim 22 wherein the polynucleotide templates disposed at spatially discrete sites are amplicons that comprise adaptor arms.

25. The method of claim 24 wherein the amplicons are produced by PCR amplification primed from sites in the adaptor arms.