Methods for determining sequence variants using ultra-deep sequencing

• US 20060228721A1
• Filed: 04/12/2005
• Published: 10/12/2006
• Est. Priority Date: 04/12/2005
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 CORPORATION
• Inventors: Ronan, Michael Todd, Simons, Jan Fredrik, Drake, James, Lohman, Kenton, Desany, Brian, Lee, William Lun, Egholm, Michael, Rothberg, Jonathan, Leamon, John Harris
• Application Number:
  US11/104,781
• Publication Number:
  US 20060228721A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The claimed invention provides for new sample preparation methods enabling direct sequencing of PCR products using pyrophosphate sequencing techniques. The PCR products may be specific regions of a genome. The techniques provided in this disclosure allows for SNP (single nucleotide polymorphism) detection, classification, and assessment of individual allelic polymorphisms in one individual or a population of individuals. The results may be used for diagnostic and treatment of patients as well as assessment of viral and bacterial population identification.

Claims

1. A method for detecting one or more sequence variants in a nucleic acid population comprising the steps of:

(a) amplifying a DNA segment common to said nucleic acid population with a pair of nucleic acid primers that define a locus to produce a first population of amplicons each comprising said DNA segment;

(b) clonally amplifying each member of said first population of amplicons to produce a plurality of populations of second amplicons wherein each population of second amplicons derives from one member of said first population of amplicons;

(c) immobilizing said second amplicons to a plurality of mobile solid support such that each mobile solid support comprises one population of said second amplicons;

(d) determining a nucleic acid sequence for the second amplicons on each solid support to produce a population of nucleic acid sequences;

(e) determining an incidence of each type of nucleotide at each position of said DNA segment to detect the one or more sequence variant in said nucleic acid population.

2. The method of claim 1 wherein said primer is a bipartite primer comprising a 5′

region and a 3′

region, wherein said 3′

region is complementary to a region on said DNA segment and wherein said 5′

region is homologous to a sequencing primer or complement thereof.

3. The method of claim 2 wherein said 5′

region is homologous to a capture oligonucleotide or a complement thereof on said mobile solid support.

4. The method of claim 1 wherein said amplification is performed by polymerase chain reaction.

5. The method of claim 1 wherein said mobile solid support are beads with a diameter selected from the group consisting of between about 1 to about 500 microns, between about 5 to about 100 microns, between about 10 to about 30 microns and between about 15 to about 25 microns.

6. The method of claim 1 wherein said mobile solid support comprise an oligonucleotide which hybridizes and immobilize said first population of amplicons, second amplicons, or both.

7. The method of claim 1 wherein said step of determining a nucleic acid sequence is performed by delivering the plurality of mobile solid supports to an array of at least 10,000 reaction chambers on a planar surface, wherein a plurality of the reaction chambers comprise no more than a single mobile solid support;

and determining a nucleic acid sequence of the amplicons on each said mobile solid support.

8. The method of claim 1 wherein said step of determining a nucleic acid sequence is performed by pyrophosphate based sequencing.

9. The method of claim 1 wherein said sequence variant has a frequency selected from the group consisting of less than about 50%, less than about 10%, less than about 5%, less than about 2%, less than about 1%, less than about 0.5%, and less than about 0.2%.

10. The method of claim 1 wherein said sequence variant has a frequency of between 0.2 and 5%.

11. The method of claim 1 wherein said nucleic acid population comprises DNA, RNA, cDNA or a combination thereof.

12. The method of claim 1 wherein the nucleic acid population is derived from a plurality of organisms.

13. The method of claim 1 wherein the nucleic acid population is derived from one organism.

14. The method of claim 13 wherein said nucleic acid population is derived from multiple tissue samples of said organism.

15. The method of claim 13 wherein said nucleic acid population is derived from a single tissue of said organism.

16. The method of claim 1 wherein the nucleic acid population is from a diseased tissue.

17. The method of claim 16 wherein said diseased tissue comprises tumor tissue.

18. The method of claim 1 wherein said nucleic acid population is derived from a bacterial culture, viral culture, or environmental sample.

19. The method of claim 1 wherein the first population of amplicons is 30 to 500 bases in length.

20. The method of claim 1 wherein said first population of amplicons comprises more than 1000 amplicons, more than 5000 amplicons, or more than 10000 amplicons.

21. The method of claim 1 wherein each of said beads binds at least 10,000 members of said plurality of second amplicons.

22. The method of claim 1 wherein the nucleic acid sequence of said DNA segment is undetermined or partially undetermined before said method.

23. A method of identifying a population comprising a plurality of different individual organisms comprising the steps of:

(a) isolating a nucleic acid sample from said population;

(b) determining one or more sequence variant of a nucleic acid segment comprising a locus common to all organisms in said population using the method of claim 1, wherein each organism comprise a different nucleic acid sequence at said locus; and

(c) determining a distribution of organisms in said population based on said population of nucleic acid sequences.

24. The method of claim 23 wherein said population is a population of organisms selected from the group consisting of bacteria, viruses, unicellular organisms, plants and yeasts.

25. A method for determining a composition of a tissue sample comprising the steps of:

(a) isolating a nucleic acid sample from said tissue sample;

(b) detecting a sequence variant of a nucleic acid segment using the method of claim 1, wherein said segment comprises a locus common to all cells in said tissue sample and wherein each cell type comprises a different sequence variant at said locus; and

(c) determining the composition of said tissue sample from said nucleotide frequency.

26. An automated method for genotyping an organism comprising the steps of:

(a) isolating a nucleic acid from said organism;

(b) determining a nucleic acid sequence at one or more loci in said nucleic acid according to the method of claim 1 to produce the population of nucleic acid sequences at that one or more loci;

(c) determining a homozygosity or heterozygosity at said one or more loci from said population of nucleic acid sequences to determine the genotype of said organism.

27. The method of claim 26 further comprising the step of (d) comparing said population of nucleic acid sequence with the sequence of one or more reference genotypes to determine a genotype of said organism.

28. The method of claim 26 wherein said one or more loci comprises SNPs and wherein said genotype is a SNP genotype.