Increasing confidence of allele calls with molecular counting

• US 8,685,678 B2
• Filed: 03/28/2013
• Issued: 04/01/2014
• 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:
  US13/852,887
• Publication Number:
  US 20130203047A1
• Time in Patent Office: 369 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, 536/1, 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 assessing the presence of an allele of a polymorphic target sequence in a tagged genomic sample, comprising:

a) amplifying a population of different initial target DNA molecules from a tagged genomic sample thereby producing a population of amplified target DNA molecules, wherein the different initial target DNA molecules that comprise a polymorphic target sequence are tagged with different degenerate base region (DBR) sequences, wherein said DBR sequences 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 the amplified target DNA molecules comprise said polymorphic target sequence 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;

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

c) assessing, using a computer, the presence of said allele in said tagged genomic sample, based on;

(i) a determination of the number of said different DBR sequences that are associated with said allele; and

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

2. The method of claim 1, wherein:

said assessing step c) further comprises independently assessing the presence of an additional allele of the polymorphic target sequence in said tagged genomic sample based on;

(i) a determination of the number of said different DBR sequences that are associated with the additional allele of said polymorphic target sequence; and

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

3. The method of claim 1, wherein said population of different initial target DNA 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 is an amplification product.

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

6. The method of claim 5, wherein said initial nucleic acid sample is an amplification product.

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

8. 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.

9. The method of claim 8, wherein the DBR sequences comprise 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.

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

11. The method of claim 1, wherein the different initial target DNA 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.

12. The method of claim 11, wherein said tagged genomic sample is a pooled sample comprising nucleic acid molecules from several different sources, wherein each of said sources is associated with a unique MID sequence that identifies the source of the nucleic acid molecule to which it is tagged.

13. The method of claim 12, wherein each of the sources is derived from a human subject.

14. The method of claim 12, wherein each of the sources is derived from different parts of a tumor.

15. The method of claim 12, wherein each of the sources is derived from a subject at different times.

16. The method of claim 1, wherein the tagged genomic sample comprises polynucleotides from a tumor.

17. The method of claim 1, wherein the tagged genomic sample comprises polynucleotides from a microorganism and/or a virus.

18. The method of claim 1, wherein the tagged genomic sample comprises human genomic DNA and said polymorphic target sequence comprises a single nucleotide polymorphism of a human genome.

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

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

21. The method of claim 1, wherein the method further assessing step c) comprises performing a maximum likelihood analysis.

22. The method of claim 1, wherein the method further comprises determining the amount of the allele in the tagged genomic sample.