MULTIBASE DELIVERY FOR LONG READS IN SEQUENCING BY SYNTHESIS PROTOCOLS

• US 20120295262A1
• Filed: 07/10/2012
• Published: 11/22/2012
• Est. Priority Date: 12/23/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Ronaghi, Mostafa, Eltoukhy, Helmy A.
• Granted Patent: US 8,476,022 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• 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 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The present technology relates to molecular sciences, such as genomics. More particularly, the present technology relates to methods for obtaining long lengths of sequencing data.

Claims

1. A method for obtaining nucleic acid sequence information, said method comprising the steps of:

(a) providing a first low resolution sequence representation for a target nucleic acid, wherein said first low resolution sequence representation is degenerate with respect to two or more nucleotide types,

(b) providing a second low resolution sequence representation for said target nucleic acid, wherein said second low resolution sequence representation is degenerate with respect to two or more nucleotide types, wherein at least one of said two or more nucleotide types in the first low resolution sequence representation is different from at least one of said two or more nucleotide types in said second low resolution sequence representation; and

(c) comparing said first low resolution sequence representation and said second low resolution sequence representation to determine the actual sequence of said target nucleic acid at single nucleotide resolution.

2. The method of claim 1, wherein said first low resolution sequence representation is degenerate with respect to a pair of nucleotide types.

3. The method of claim 1, wherein said first low resolution sequence representation is degenerate with respect to two pairs of nucleotide types.

4. The method of claim 3, wherein said first low resolution sequence representation is degenerate with respect to A and T at particular positions in said actual sequence of said target nucleic acid and wherein said first low resolution sequence representation is degenerate with respect to G and C at particular positions in said actual sequence of said target nucleic acid.

5. The method of claim 4, wherein said second low resolution sequence representation is degenerate with respect to A and C at particular positions in said actual sequence of said target nucleic acid and wherein said second low resolution sequence representation is degenerate with respect to G and T at particular positions in said actual sequence of said target nucleic acid.

6. The method of claim 4, wherein said second low resolution sequence representation is degenerate with respect to A and G at particular positions in said actual sequence of said target nucleic acid and wherein said second low resolution sequence representation is degenerate with respect to C and T at particular positions in said actual sequence of said target nucleic acid.

7. The method of claim 1, wherein said first low resolution sequence representation degenerate with respect to a triplet of nucleotide types.

8. The method of claims 1, wherein said first low resolution sequence representation comprises a string of symbols and the number of different symbol types in the string is less than the number of different nucleotide types in said actual sequence of said target nucleic acid.

9. The method of claim 1, wherein said first low resolution sequence representation is obtained from a sequencing reaction comprising repeated steps of:

(a) detecting the incorporation of a first sequencing reagent into a polynucleotide strand complementary to at least a portion of said target nucleic acid, wherein said first sequencing reagent comprises at least two different nucleotide monomers; and

(b) detecting the incorporation of a second sequencing reagent into said polynucleotide strand, wherein said second sequencing reagent comprises one or more nucleotide monomers, at least one of said one or more nucleotide monomers being different from the nucleotide monomers present in said first sequencing reagent.

10. The method of claim 1, wherein pattern recognition methods are used to determine said actual sequence of said target nucleic acid at single nucleotide resolution.

11. The method of claim 1, wherein said comparing is carried out by alignment of said first low resolution sequence representation and said second low resolution sequence to reference sequences in a database, wherein said reference sequences comprise said actual sequence of said target nucleic acid.

12. A method for determining the presence or absence of a target nucleic acid, said method comprising the steps of:

(a) providing a first low resolution sequence representation for a target nucleic acid, wherein said target nucleic acid is obtained from a target sample, wherein said first low resolution sequence representation is degenerate with respect to two or more nucleotide types,

(b) providing a second low resolution sequence representation for said target nucleic acid, wherein said target nucleic acid is obtained from a reference sample, wherein the second low resolution sequence representation is degenerate with respect to two or more nucleotide types; and

(c) comparing said first low resolution sequence representation and said second low resolution sequence representation to determine the presence or absence of said target nucleic acid in said target sample.

13. The method of claim 12, wherein said two or more nucleotide types in said first low resolution sequence representation are the same as said two or more nucleotide types in said second low resolution sequence representation.

14. The method of claim 12, wherein a first plurality of low resolution sequence representations for a plurality of nucleic acids in said target sample are provided and a second plurality of low resolution sequence representations for a plurality of nucleic acids in said reference sample are provided.

15. The method of claim 14, wherein said first low resolution sequence representation for the target nucleic acid and said second low resolution sequence representation for said target nucleic acid are distinguished from low resolution sequence representations in said first plurality and in the second plurality.

16. The method of claim 14, further comprising quantifying the amount of the target nucleic acid in said target sample relative to the amount of the target nucleic acid in said reference sample.

17. The method of claim 16, wherein said target nucleic acid is an mRNA and said amount is indicative of an expression level for said mRNA.

18. The method of claim 14, wherein said first and second low resolution sequence representations have a known correlation with said actual sequence of said target nucleic acid at single nucleotide resolution.

19. The method of claim 14, wherein said first low resolution sequence representation and said second low resolution sequence representation are the same.