Method of adding a DBR by primer extension

• US 20130237458A1
• Filed: 03/29/2013
• Published: 09/12/2013
• Est. Priority Date: 09/21/2010
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Casbon, James, Brenner, Sydney, Osborne, Robert, Lichtenstein, Conrad, Claas, Andreas
• Granted Patent: US 8,728,766 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection

Abstract

Aspects of the present invention include methods and compositions for determining the number of individual polynucleotide molecules originating from the same genomic region of the same original sample that have been sequenced in a particular sequence analysis configuration or process. In these aspects of the invention, a degenerate base region (DBR) is attached to the starting polynucleotide molecules that are subsequently sequenced (e.g., after certain process steps are performed, e.g., amplification and/or enrichment). The number of different DBR sequences present in a sequencing run can be used to determine/estimate the number of different starting polynucleotides that have been sequenced. DBRs can be used to enhance numerous different nucleic acid sequence analysis applications, including allowing higher confidence allele call determinations in genotyping applications.

Claims

1-24. -24. (canceled)

25. A method for processing a DNA sample, comprising:

(a) hybridizing a genomic sample comprising a population of initial target DNA molecules with population of first primers that comprise;

i. a 3′

target-specific sequence that hybridizes to a sequence in said initial target DNA molecules and ii. a degenerate base region (DBR) that is 5′

to said target-specific sequence, wherein said DBR comprises at least one nucleotide base selected from;

R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof; and

iii. a generic primer sequence that is 5′

to said degenerate base region;

(b) subjecting the product of step (a) to one, two or three rounds of primer extension to produce tagged copies of the complements of said initial target DNA molecules, wherein each of said tagged copies comprises a different DBR sequence at its 5′

end;

(c) removing or inactivating any of said primers that have not been extended in step (b) from the product of step (b); and

(d) amplifying said copies of the complements of said target DNA molecules from the product of step (c) to produce multiple amplicons, wherein;

i. the amplifying is done by polymerase chain reaction (PCR) using a primer pair that comprises a generic primer that hybridizes to the complement of the generic primer sequence of the primers of (a);

ii. within each amplicon, each of the amplified target DNA molecules comprises the same DBR sequence; and

ii. the amplified target DNA molecules in different amplicons have different DBR sequences.

26. The method of claim 25, wherein step (b) comprises subjecting the product of step (a) to one round of primer extension prior to step (c).

27. The method of claim 25, wherein step (b) comprises subjecting the product of step (a) to two rounds of primer extension prior to step (c).

28. The method of claim 25, wherein said primers have an affinity tag, and said removing or inactivating step (c) is done by immobilizing said primers on a solid support.

29. The method of claim 25, wherein said removing or inactivating step (c) comprises inactivating said primers using an exonuclease.

30. The method of claim 25, wherein said removing or inactivating step (c) comprises enzymatically modifying said primers.

31. The method of claim 25, wherein said DBR comprises at least 2 nucleotide bases, wherein each of the at least 2 nucleotide bases are selected from:

R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.

32. The method of claim 31, wherein the DBR comprises from 3 to 10 nucleotide bases, wherein each of the 3 to 10 nucleotide bases is selected from:

R, Y, S, W, K, M, B, D, H, V, N, and modified versions thereof.

33. The method of claim 25, wherein the initial target DNA molecules are enriched from an initial genomic sample prior to said hybridizing.

34. The method of claim 25, wherein the genomic sample comprises polynucleotides from a tumor.

35. The method of claim 25, wherein the genomic sample comprises polynucleotides from a microorganism.

36. The method of claim 25, wherein the genomic sample comprises polynucleotides from a virus.

37. The method of claim 25, wherein the genomic sample comprises human genomic DNA.

38. The method of claim 25, wherein the DBR sequence is error correcting.