Increasing Confidence of Allele Calls with Molecular Counting

• US 20130203047A1
• Filed: 03/28/2013
• Published: 08/08/2013
• 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 8,685,678 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 determining the presence of an allele, comprising:

a) amplifying a population of initial target DNA molecules from a tagged genomic sample to produce a population of amplified target DNA molecules, wherein the initial target DNA molecules comprise a polymorphic target region and wherein each of the initial target DNA molecules that comprises said polymorphic target region is tagged with a different degenerate base region (DBR) sequence, 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;

b) sequencing a plurality of the amplified target DNA molecules, thereby producing a plurality of sequence reads, wherein the sequencing step provides, for each of the amplified target DNA molecules that are sequenced;

(i) the nucleotide sequence of at least a portion of the polymorphic target region and (ii) a DBR sequence;

c) determining, for one allele of the polymorphic target region, the number of different DBR sequences that are associated with said allele;

d) determining, for said allele of the polymorphic target region, the number of sequence reads that comprise each of the different DBR sequences;

e) calculating the likelihood that said allele is present in said tagged genomic sample using the number of different sequences counted in step c) and the number of sequence reads counted in d); and

f) making an allele call based on the likelihood calculated in step e), wherein a higher likelihood increases the confidence of said allele call.

26. The method of claim 25, wherein steps (c)-(f) are done by a computer that is programmed to perform those steps.

27. The method of claim 25, wherein:

said determining step c) further comprises calculating the number of different DBR sequences that are associated with additional alleles of said polymorphic target region;

said determining step d) further comprises calculating the number of sequence reads that comprise each of the DBR sequences associated with each of the additional alleles counted in c);

said calculating step e) further comprises independently calculating the likelihood that each of the additional alleles is present in said tagged genomic sample using the number of different sequences counted for each of the additional alleles and the number of sequencing reads counted in d); and

said making an allele call step f) further comprises making an allele call for the additional alleles based on the likelihoods that said additional alleles are present in said tagged genomic sample.

28. The method of claim 25, wherein said population of initial target DNA molecules is made by ligating a set of adaptors that comprise said DBR sequence to an initial nucleic acid sample.

29. The method of claim 25, wherein said population of initial target DNA molecules is made by primer extension of a set of primers that comprises said DBR sequence, using an initial nucleic acid sample as a template.

30. The method of claim 25, wherein the method comprises, prior to said amplifying step (a), enriching for DNA molecules that comprises said polymorphic target region from an initial nucleic acid sample.

31. 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.

32. The method of claim 31, wherein the degenerate base region comprises from 3 to 20 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 said DBR sequence is error-correcting.

34. The method of claim 25, wherein the initial target DNA molecules are further tagged with a unique MID sequence that identifies the source of a nucleic acid molecule to which it is appended.

35. The method of claim 34, wherein said tagged 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.

36. The method of claim 35, wherein each of the sources is derived from a human subject.

37. The method of claim 35, wherein each of the sources is derived from different parts of a tumor.

38. The method of claim 35, wherein each of the sources is derived from a subject at different times.

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

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

41. The method of claim 25, wherein the tagged genomic sample comprises human genomic DNA and said polymorphic target region is a single nucleotide polymorphism of the human genome.

42. The method of claim 25, wherein said sequencing step b) comprises sequencing said plurality of amplified target DNA molecules on a next-generation sequencing platform.

43. The method of claim 25, wherein the amplifying is done by polymerase chain reaction.