INCREASING CONFIDENCE OF ALLELE CALLS WITH MOLECULAR COUNTING

• US 20120071331A1
• Filed: 09/20/2011
• Published: 03/22/2012
• Est. Priority Date: 09/21/2010
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney, Osborne, Robert, Casbon, James, Lichtenstein, Conrad
• Granted Patent: US 8,481,292 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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 of estimating the number of starting polynucleotide molecules sequenced from multiple samples, the method comprising:

attaching an adapter to starting polynucleotide molecules in multiple different samples, wherein the adapter for each sample comprises;

a unique MID specific for the sample; and

a degenerate base region (DBR) comprising at least one nucleotide base selected from;

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

pooling the multiple different adapter-attached samples to generate a pooled sample;

amplifying the adapter-attached polynucleotides in the pooled sample;

sequencing a plurality of the amplified adapter-attached polynucleotides, wherein the sequence of the MID, the DBR and at least a portion of the polynucleotide is obtained for each of the plurality of adapter-attached polynucleotides; and

determining the number of distinct DBR sequences present in the plurality of sequenced adapter-attached polynucleotides from each sample to determine the number of starting polynucleotides from each sample that were sequenced in the sequencing step.

2. The method of claim 1, wherein the adapter further comprises a sequencing primer site.

3. The method of claim 1, wherein the adapter further comprises one or more of:

a reflex sequence, and a promoter site.

4. The method of claim 1, wherein the 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, and N.

5. The method of claim 4, wherein the DBR 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, and N.

6. The method of claim 1, wherein the determined number of polynucleotides from each sample that were sequenced in the sequencing step is employed in a method for allele calling.

7. The method of claim 1, wherein the sequencing step comprises performing a next-generation sequencing process.

8. The method of claim 1, wherein each of the multiple samples is a genomic DNA sample.

9. The method of claim 8, wherein each of the multiple different genomic DNA samples is derived from a different subject.

10. The method of claim 9, wherein the subject is a human.

11. The method of claim 1, further wherein the sample is enriched for a region of interest prior to the attaching step.

12. The method of claim 1, further comprising enriching the adapter-attached polynucleotides for a region of interest.

13. The method of claim 4, wherein the adapter is an asymmetric adapter, wherein a first adapter domain is present at a first end of the polynucleotide and a second adapter domain is present at a second end of the polynucleotide, wherein the DBR is a split DBR comprising one or more of the at least 2 nucleotide bases in the first adapter domain and one or more of the at least 2 nucleotide bases in the second adapter domain.

14. The method of claim 1, wherein the adapter is attached to the polynucleotide molecules in an amplification reaction, wherein the DBR is present in a synthesis primer employed in the amplification reaction.

15. The method of claim 14, wherein the amplification reaction is a PCR.

16. The method of claim 14, wherein the method further comprises determining the number of starting polynucleotides amplified in the PCR reaction.

17. The method of claim 1, wherein the method is drawn to determining the genetic heterogeneity of the polynucleotides in the sample.

18. The method of claim 17, wherein the sample comprises polynucleotides derived from tumor tissue.

19. The method of claim 17, wherein the sample comprises polynucleotides derived from microorganisms and/or viruses.

20. The method of claim 1, wherein the method is drawn to determining the genetic heterogeneity of the polynucleotides present in the multiple different samples.

21. The method of claim 20, wherein the multiple different samples are derived from different sections of a tumor.

22. The method of claim 20, wherein the multiple different samples are derived from different tumors from a subject.

23. The method of claim 22, wherein the multiple different samples are derived from a subject over time.

24. The method of claim 23, wherein the subject has an infection, wherein the genetic heterogeneity of the infectious agent or agents over time is determined.