Sequence analysis of complex amplicons

Patent metadata

Field	Value
Publication number	US-9523129-B2
Application number	US-201414173712-A
Country	US
Kind code	B2
Filing date	Feb 5, 2014
Priority date	Nov 7, 2008
Publication date	Dec 20, 2016
Grant date	Dec 20, 2016

Abstract

The invention is directed to methods of generating sequence profiles of populations of nucleic acids, whose member nucleic acids contain regions of high variability, such as populations of nucleic acids encoding T cell receptors or B cell receptors. In one aspect, the invention provides pluralities of sets of primers for generating nested sets of templates from nucleic acids in such populations, thereby insuring the production of at least one template from which sequence reads are generated, despite such variability, or despite limited lengths or quality of sequence reads. In another aspect, members of such populations are bidirectionally sequenced so that further sequence information is obtained by analyzing overlapping sequence reads in the zones of highest variability.

First claim

What is claimed is:

1. A method for determining a clonotype profile of T cell receptors and/or B cell receptors of a subject, the method comprising the steps of:
a) spatially isolating individual nucleic acid molecules, wherein the individual nucleic acid molecules are derived from nested sets of templates, wherein each of the nested sets of templates is generated from a nucleic acid in a sample comprising nucleic acids from T-cells and/or B-cells of the subject and contains a somatically rearranged region or a portion thereof, whereby each nested set of the nested sets of templates comprises a plurality of overlapping templates such that every template of the plurality of overlapping templates has a common end and a different end, and wherein each nested set is capable of producing a plurality of sequence reads each extending in the same direction and each starting from a different position on the nucleic acid from which the nested set was generated;
b) sequencing the spatially isolated individual nucleic acid molecules, wherein the sequencing produces sequence reads with a sequence length of at least 30 nucleotides, and wherein each nested set is represented by a plurality of sequence reads;
c) combining information from the plurality of sequence reads from each nested set to form a clonotype from each nested set, whereby the combining forms at least 1000 clonotypes from the nested sets of templates; and
d) determining abundances of each of clonotype from each nested set to generate the clonotype profile.

2. The method of claim 1 , wherein each of the somatically rearranged regions comprise a V region and a J region, and wherein each of the plurality of sequence reads starts from a different position in the V region and extends in the direction of its associated J region.

3. The method of claim 1 , wherein the step of sequencing comprises bidirectionally sequencing the spatially isolated individual nucleic acid molecules to produce at least one forward sequence read and at least one reverse sequence read.

4. The method of claim 3 , wherein at least one of the forward sequence reads and at least one of the reverse sequence reads have an overlap region, wherein bases of such overlap region are determined by a reverse complementary relationship between such sequence reads.

5. The method of claim 4 , wherein each of the somatically rearranged regions comprise a V region and a J region, and wherein the step of sequencing further includes determining a sequence of each of the individual nucleic acid molecules from at least one forward sequence read and at least one reverse sequence read starting from a position in a J region and extending in the direction of its associated V region.

6. The method of claim 1 , wherein the individual nucleic acid molecules comprise nucleic acids selected from the group consisting of complete IgH molecules, incomplete IgH molecules, complete IgK molecules, IgK inactive molecules, TCR.beta. molecules, TCR.gamma. molecules, complete TCR.delta. molecules, and incomplete TCR.delta. molecules.

7. The method of claim 1 , wherein the individual nucleic acid molecules comprise a repertoire of clonotypes present at a frequency of 0.01 percent or greater with a probability of ninety-nine percent.

8. The method of claim 1 , wherein the sample is obtained from T-cells and/or B-cells from peripheral blood or bone marrow of the subject.

9. The method of claim 1 , wherein the step of spatially isolating includes disposing the individual nucleic acid molecules on a solid surface and amplifying the individual nucleic acid molecules thereon to form isolated clonal populations thereof.

10. The method of claim 9 , wherein the amplifying is carried out by bridge PCR.

11. The method of claim 1 , wherein the plurality of sequence reads is generated by annealing a primer to a primer binding site on each template of the nested set of templates and extending the primer with a DNA polymerase.

12. The method of claim 11 , wherein at least one of the plurality of sequence reads overlaps at least one of the primer binding sites.

13. The method of claim 1 , wherein the step of sequencing comprises generating the sequence reads having monotonically decreasing quality scores.

14. The method of claim 1 , wherein the sample is from B cells of the subject.

15. The method of claim 1 , wherein the step of combining includes combining the sequence reads into different clonotypes whenever the sequence reads are different with a confidence of at least 99.9 percent.

16. The method of claim 15 , wherein the combining forms at least 10000 clonotypes.

17. The method of claim 13 , wherein the step of combining includes combining the one or more sequence reads based on copies of each sequence read, numbers of base differences, and quality scores of bases that differ.

18. The method of claim 13 , wherein the step of combining includes combining the one or more sequence reads based on average quality scores of the one or more sequence reads.

Assignees

• Adaptive Biotechnologies Corp

Inventors

• Faham Malek
• Moorhead Martin
• Willis Thomas

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/112

  Disease subtyping, staging or classification · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/6881

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title