Method of tagging using a split DBR

• US 20130267424A1
• Filed: 03/29/2013
• Published: 10/10/2013
• 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 8,741,606 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 of sequencing, comprising:

a) appending a degenerate base region (DBR) sequence to both ends of a plurality of target nucleic acid molecules in a genomic sample, wherein said DBR sequence comprises at least one nucleotide base selected from;

R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein said appending produces a population of asymmetrically tagged nucleic acid molecules that each comprise;

(i) a target polynucleotide, (ii) a first DBR sequence at the 5′

end of the target polynucleotide and (iii) a second DBR sequence at the 3′

end of the target polynucleotide, wherein;

in each of said asymmetrically tagged nucleic acid molecules the first DBR sequence is different from the second DBR sequence; and

the different asymmetrically tagged nucleic acid molecules in said population have different first DBR sequences and different second DBR sequences, relative to one another;

b) amplifying at least some of said asymmetrically tagged nucleic acid molecules, thereby producing a population of amplified asymmetrically tagged nucleic acid molecules; and

c) sequencing a plurality of the amplified asymmetrically tagged nucleic acid molecules to produce a plurality of sequences, wherein the sequencing step provides, for each of the asymmetrically tagged nucleic acid molecules that are sequenced;

(i) the nucleotide sequence of at least a portion of a target polynucleotide;

(ii) the nucleotide sequence of a first DBR sequence; and

(iii) the nucleotide sequence of a second DBR sequence.

26. The method of claim 25, wherein said appending is done by ligating a set of adaptors that comprise said DBR sequence to said plurality of target nucleic acid molecules.

27. The method of claim 25, wherein said appending is done by extending a set of primers that comprises said DBR sequence, using an initial nucleic acid sample as a template.

28. The method of claim 25, wherein the method comprises, prior to said appending step (a), enriching for said target nucleic acid molecules from an initial nucleic acid sample.

29. The method of claim 25, wherein said DBR sequence 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.

30. The method of claim 29, wherein said DBR sequence 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.

31. The method of claim 25, wherein asymmetrically tagged nucleic acid molecules further comprise a unique MID sequence that identifies the source of a nucleic acid molecule to which it is appended.

32. The method of claim 31, wherein said genomic sample is a pooled sample comprising nucleic acid molecules from several different sources, where each of said sources is associated with a different MID sequence.

33. The method of claim 31, wherein each of the sources is derived from a human subject.

34. The method of claim 31, wherein each of the sources is derived from different sections of a tumor.

35. The method of claim 31, wherein each of the sources is derived from different tumors of a subject.

36. The method of claim 31, wherein each of the sources is derived from a subject at different times.

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

38. The method of claim 25, wherein the genomic sample comprises polynucleotides from a microorganism and/or a virus.

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

40. The method of claim 25, wherein said sequencing step c) comprises plurality of the amplified asymmetrically tagged nucleic acid molecules on a next-generation sequencing platform.

41. The method of claim 25, wherein the amplifying step (b) is done by polymerase chain reaction.