Method for accurately counting starting molecules

• US 8,715,967 B2
• Filed: 03/29/2013
• Issued: 05/06/2014
• 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
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/853,981
• Publication Number:
  US 20130224743A1
• Time in Patent Office: 403 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• 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. A method for estimating the number of initial target nucleic acid molecules in a sample, comprising:

a) amplifying a population of initial target nucleic acid molecules from a tagged sample thereby producing a population of amplified target DNA molecules, wherein the initial target nucleic acid molecules that comprise a target region are tagged with different degenerate base region (DBR) sequences, wherein said DBR sequences comprise at least one nucleotide base selected from;

R, Y, S, W, K, M, B, D, H, V, N and modified versions thereof and wherein the amplified target DNA molecules comprise said target region and an associated DBR sequence of said DBR sequences;

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, the nucleotide sequence of;

(i) at least a portion of the target region and (ii) an associated DBR sequence of said DBR sequences; and

c) estimating, using a computer, the number of initial target nucleic acid molecules that comprise said target region in said tagged sample based on;

(i) a determination of the number of said different DBR sequences that are associated with said target region in the sequence reads; and

(ii) a determination of the number of sequence reads that comprise each of the different DBR sequences that are associated with said target region.

2. The method of claim 1, wherein said estimating step c) is done using a maximum likelihood method.

3. The method of claim 1, wherein said population of initial target nucleic acid molecules is made by ligating a set of adaptors that comprise said DBR sequences to an initial nucleic acid sample.

4. The method of claim 3, wherein said initial nucleic acid sample comprises mRNA or cDNA.

5. The method of claim 3, wherein said initial nucleic acid sample comprises genomic DNA.

6. The method of claim 3, wherein said initial nucleic acid sample is an amplification product.

7. The method of claim 1, wherein said population of initial target nucleic acid molecules is made by extending a set of primers that comprises said DBR sequences, using an initial nucleic acid sample as a template.

8. The method of claim 7, wherein said initial nucleic acid sample comprises mRNA or cDNA.

9. The method of claim 7, wherein said initial nucleic acid sample comprises genomic DNA.

10. The method of claim 7, wherein said initial nucleic acid sample is an amplification product.

11. The method of claim 1, wherein the method comprises, prior to the amplifying step (a), enriching said population of initial target DNA molecules from an initial nucleic acid sample.

12. The method of claim 1, wherein said DBR sequences comprise 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.

13. The method of claim 1, wherein the DBR sequences comprise 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.

14. The method of claim 1, wherein said DBR sequences comprise an error-correcting code.

15. The method of claim 1, wherein the initial target nucleic acid molecules are further tagged with a unique multiplex identifier (MID) sequence that identifies the source of a nucleic acid molecule to which it is tagged.

16. The method of claim 15, wherein said tagged 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.

17. The method of claim 1, wherein the tagged sample comprises mammalian genomic DNA and said target region is a region that varies in copy number.

18. The method of claim 1, wherein the sequencing step b) comprises sequencing said plurality of amplified target DNA molecules on a next-generation sequencing platform.

19. The method of claim 1, wherein the amplifying step is done by polymerase chain reaction.

20. The method of claim 1, wherein said initial target nucleic acid molecules are cDNA molecules, and the estimating step c) provides an estimate of the abundance of cDNA molecules that comprise said target region.

21. The method of claim 1, wherein said initial target nucleic acid molecules are synthetic DNA molecules.

22. The method of claim 1, wherein the DBR sequences comprise 10 or more nucleotide bases, wherein each of the 10 or more nucleotide bases is selected from:

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