Methods and compositions for efficient base calling in sequencing reactions

• US 20090263802A1
• Filed: 01/28/2009
• Published: 10/22/2009
• 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 8,617,811 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 of identifying a first nucleotide at a detection position of a target sequence comprising:

a) providing a surface comprising a plurality of concatemers, each concatemer comprising a plurality of monomers, each monomer comprising;

i) a first target domain of said target sequence;

ii) a first detection position;

iii) a first adapter adjacent to said first target domain, wherein said first adapter comprises a first anchor site;

b) providing a first sequencing set of sequencing probes comprising;

i) a first probe set comprising;

1. a first unique label;

2. a first probe domain complementary to said first target domain; and

3. a first unique nucleotide at a first interrogation position;

ii) a second probe set comprising;

1. a second unique label;

2. said first probe domain; and

3. a second unique nucleotide at said first interrogation position;

iii) a third probe set comprising;

1. said first label and said second label;

2. said first probe domain; and

3. a third unique nucleotide at said first interrogation position;

iv) a fourth probe set comprising;

1. said first probe domain; and

2. a fourth unique nucleotide at said first interrogation position;

(c) hybridizing an anchor probe to said first anchor site;

(d) applying said set of sequencing probes to said surface, wherein if a sequencing probe from said sequencing set has a unique nucleotide that is complementary to said first nucleotide, said sequencing probe hybridizes to said concatemer;

(e) ligating said hybridized sequencing probe to said hybridized anchor probe to form ligation products;

(f) determining the identity of said first nucleotide.

2. A method according to claim 1 wherein said fourth probe set comprises a fourth unique label.

3. A method according to claim 1, wherein said first unique label comprises a first fluorophore, said second unique label comprises a second fluorophore, and said third unique label comprises said first and said second fluorophore.

4. A method according to claim 2, wherein said third probe set comprises a plurality of third probes with said first fluorophore and a plurality of third probes with said second fluorophore.

5. A method according to claim 1 wherein said first unique label comprises a first fluorophore at a first intensity, said second unique label comprises said first fluorophore at a second intensity, and said third unique label comprises said first fluorophore at a third intensity.

6. A method according to claim 4 wherein said fourth probe set comprises a fourth unique label comprising said first fluorophore at a fourth intensity.

7. A method according to claim 4 or 5 wherein differences in intensity of said first fluorophore is created by changing the number of fluorophores per probe.

8. A method according to claim 4 or 5 wherein differences in intensity of said first fluorophore is created by changing the number of probes within each probe set comprising a fluorophore.

9. A method according to claim 1, wherein said method further comprises identifying a second nucleotide at a detection position of said target sequence, wherein each monomer further comprises:

i) a second target domain of said target sequence;

ii) a second detection position;

ii) a second adapter adjacent to said second target domain, wherein said second adapter comprises a second anchor site;

and wherein said method further comprises;

a) providing a second sequencing set of sequencing probes comprising;

i) a fifth probe set comprising;

1. a fifth unique label;

2. a second probe domain complementary to said second target domain; and

3. a first unique nucleotide at a second interrogation position;

ii) a sixth probe set comprising;

1. a sixth unique label;

2. a sixth probe domain complementary to said second target domain; and

3. a second unique nucleotide at said second interrogation position;

iii) a seventh probe set comprising;

1. said fifth label and said sixth label;

2. a seventh probe domain complementary to said second target domain; and

3. a third unique nucleotide at said second interrogation position;

iv) an eighth probe set comprising;

1. a eighth probe domain complementary to said second target domain; and

2. a fourth unique nucleotide at said second interrogation position;

(c) hybridizing an anchor probe to said second anchor site;

(d) applying said second set of sequencing probes to said surface, wherein if a sequencing probe from said second sequencing set has a unique nucleotide that is complementary to said second nucleotide, said sequencing probe hybridizes to said concatemer;

(e) ligating said hybridized second sequencing probes to said hybridized second anchor probes to form ligation products;

(f) determining the identity of said second nucleotide.

10. A method according to claim 9 wherein said first and said second adapters are the same.

11. A method according to claim 10 wherein said second target domain is adjacent to the opposite terminus of the adapter from the first target domain.

12. A method according to claim 9 wherein said fourth probe set comprises a fourth unique label.

13. A method according to claim 10, wherein said first unique label comprises a third fluorophore, said second unique label comprises a fourth fluorophore, and said third unique label comprises said third and said fourth fluorophores.

14. A method according to claim 13, wherein said third probe set comprises a plurality of third probes with said third fluorophore and a plurality of third probes with said fourth fluorophore.

15. A method according to claim 9 wherein said fifth unique label comprises a fluorophore at a first intensity, said sixth unique label comprises said fluorophore at a second intensity, and said seventh unique label comprises said fluorophore at a third intensity.

16. A method according to claim 15 wherein said fourth probe set comprises an eighth unique label comprising said fluorophore at a fourth intensity.

17. A method according to claim 15 or 16 wherein differences in intensity of said fluorophore is created by changing the number of fluorophores per probe.

18. A method according to claim 4 or 5 wherein differences in intensity of said fluorophore is created by changing the number of probes within each probe set comprising a fluorophore.

19. A method according to claim 1 or 9 wherein at least one of said labels is dissociable.

20. A method according to claim 19 comprising dissociating one of said labels after a first label measuring step and subsequently measuring again.

21. A method according to claim 1 or 9 wherein said anchor probe and said set of sequencing probes are applied simultaneously.