Methods and compositions for efficient base calling in sequencing reactions

• US 9,222,132 B2
• Filed: 08/25/2014
• Issued: 12/29/2015
• 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): Mummert, Stephanie K
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US14/468,213
• Publication Number:
  US 20140378321A1
• Time in Patent Office: 491 Days
• Field of Search: None
• 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 method for determining identities of nucleotides at detection positions of a plurality of different nucleic acid templates by performing sequencing-by-extension reactions, the method comprising:

(a) providing an array comprising single-stranded nucleic acid templates disposed at positions on a surface;

(b) for each of a plurality of said single-stranded nucleic acid templates, determining the identity of nucleotides at detection positions in the nucleic acid template in multiple cycles of a sequencing-by-extension reaction, comprising;

i) binding a complementary nucleotide to a nucleotide at a detection position,

ii) detecting, at the position on the surface occupied by the nucleic acid template, the presence or absence of fluorescent signal(s) associated with the complementary nucleotide;

wherein

1) detecting a first fluorescent signal and not a second fluorescent signal at the position identifies the complementary nucleotide as a nucleotide selected from A, T, G and C;

2) detecting the second fluorescent signal and not the first fluorescent signal at the position identifies the complementary nucleotide as a nucleotide selected from A, T, G and C that is different from the nucleotide selected in (1);

3) detecting both the first fluorescent signal and the second fluorescent signal at the position identifies the complementary nucleotide as a nucleotide selected from A, T, G and C that is different from nucleotides selected in (1) and (2); and

4) detecting neither the first fluorescent signal nor the second fluorescent signal at the position identifies the complementary nucleotide as a nucleotide selected from A, T, G and C that is different from the nucleotides selected in (1), (2) and (3);

iii) deducing the identity of the nucleotide at the detection position in the nucleic acid template based on the identity of the complementary nucleotide.

2. The method of claim 1 wherein the nucleic acid templates comprise adaptors.

3. The method of claim 2 wherein the nucleic acid templates are formed from a plurality of genomic fragments.

4. The method of claim 3 wherein the genomic fragments are human genomic DNA.

5. A method for determining identities of nucleotides at detection positions of a plurality of different nucleic acid templates, said method comprising:

providing a plurality of nucleic acid templates each comprising an anchor site and, adjacent to the anchor site, a target nucleic acid sequence;

performing sequencing reactions on the plurality of different nucleic acid templates using two distinguishable fluorescent labels by hybridizing an anchor probe to the anchor site and extending individual anchor probes by one nucleotide per cycle in one or more cycles of sequencing-by-synthesis using a set of nucleotides that comprises;

(i) first nucleotides comprising a first label;

(ii) second nucleotides comprising a second label;

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

(iv) fourth nucleotides comprising neither the first label nor the second label, wherein the first label and the second label are distinguishable from each other; and

in each cycle of sequencing-by-synthesis, determining the identities of nucleotides at the detection positions by detecting the presence or absence of the first label and the presence or absence of the second label to determine the target nucleic acid sequences.

6. The method of claim 5 wherein the nucleic acid templates are immobilized on a surface.

7. The method of claim 5 wherein the nucleic acid templates comprise adaptors.

8. The method of claim 7 wherein the nucleic acid templates are formed from a plurality of genomic fragments.

9. The method of claim 8 wherein the genomic fragments are human genomic DNA.