Method of sequence determination using sequence tags

• US 9,506,119 B2
• Filed: 03/15/2013
• Issued: 11/29/2016
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Adaptive Biotechnologies Corp.
• Inventors: Zheng, Jianbiao, Faham, Malek, Moorhead, Martin, Willis, Thomas
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US13/835,093
• Publication Number:
  US 20130236895A1
• Time in Patent Office: 1,355 Days
• Field of Search: 435/6.12, 435/91.2, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The invention is directed to the use of sequence tags to improve sequence determination of amplicons of related sequences, particularly large and complex amplicons, such as those comprising recombined nucleic acids encoding immune receptor molecules. In one aspect, sequence reads having the same sequence tags are aligned after which final base calls are determined from a (possibly weighted) average base call from sequence read base calls at each position. Similarly, in another aspect, sequence reads comprising series of incorporation signals are aligned by common sequence tags and base calls in homopolymer regions are made as a function incorporation signal values at each “flow” position.

Claims

1. A method of determining clonotypes of an immune repertoire, the method comprising the steps:

(a) attaching sequence tags to molecules of recombined nucleic acids of I-cell receptor genes or immunoglobulin genes of T-cells and/or B-cells from a sample comprising cells and/or B-cells to form tag-molecule conjugates, wherein substantially every molecule of the tag-molecule conjugates has a unique sequence tag;

(b) amplifying the tag-molecule conjugates;

(c) sequencing by synthesis the tag-molecule conjugates to produce sequence reads comprising at least 1000 clonotypes per run, wherein each sequence read comprises a sequence tag portion and a clonotype portion; and

(d) aligning like sequence tag portions of the sequence reads to determine a clonotype sequence from corresponding clonotype portions of the aligned sequence reads.

2. The method of claim 1, wherein the aligning further includes determining a nucleotide sequence of each of the clonotype portion by determining a majority nucleotide at each nucleotide position of the clonotype portions aligned by the like sequence tag portions.

3. The method of claim 1, wherein the step of attaching includes labeling by sampling the molecules of recombined nucleic acids.

4. The method of claim 3, wherein each of the sequence tags attached to the molecules differs in sequence from every other such sequence tag by at least 25 percent of its nucleotides.

5. The method of claim 4, wherein the sequence tags are incorporated into primers specific for the molecules of recombined nucleic acids.

6. The method of claim 5, wherein the sequence tags are mosaic tags.

10. A method of detecting clonotype carry over contamination in a patient being monitored for minimal residual disease, the method comprising the steps of:

monitoring a patient for minimal residual disease by periodically measuring a clonotype profile of the patient in accordance with the method of claim 1;

recording said sequence of each of the sequence tags of each measurement of clonotype profiles; and

detecting clonotype carry over contamination if a sequence tag of any prior clonotype profile is detected in a subsequent clonotype profile.

7. A method of determining clonotypes of an immune repertoire, the method comprising the steps:

(a) labeling by sampling molecules from T-cells and/or B-cells from a sample comprising T-cells and/or B-cells to form tag-molecule conjugates, wherein each tag has a sequence and each molecule comprises a clonotype comprising a recombined nucleic acid from a T-cell receptor gene or an immunoglobulin gene;

(b) sequencing by synthesis the tag-molecule conjugates to produce sequence reads comprising at least 1000 clonotypes per run, wherein each sequence read comprises a sequence tag portion and a clonotype portion;

(c) grouping the sequence reads having like tags to align sequence reads from like clonotypes; and

(d) determining a nucleotide sequence of each clonotype from the aligned sequence reads of like tags.

8. The method of claim 7, wherein the step of aligning further includes determining a nucleotide sequence of each of the clonotype of each of the tag-molecule conjugate by determining a majority nucleotide at each nucleotide position of the clonotypes having the like sequence tags.

9. The method of claim 7, wherein the step of labeling by sampling is implemented in a reaction mixture having a population of distinct the tags, the population of distinct the tags having a size, and a population of the molecules of recombined nucleic acid, the population of the molecules of recombined nucleic acid having a size, such that the size of the population of distinct the tags is at least 100 times the size of the population of the molecules of recombined nucleic acid.

11. A method of determining nucleotide sequences of one or more polynucleotides in one or more sequencing-by-synthesis reactions, the method comprising the steps:

(a) attaching a sequence tag to each of the one or more polynucleotides to form tag-polynucleotide conjugates, wherein substantially every polynucleotide of the tag-polynucleotide conjugates has a unique sequence tag;

(b) amplifying the tag-polynucleotide conjugates;

(c) sequencing by synthesis amplified tag-polynucleotide conjugates to produce sequence reads comprising at least 1000 clonotypes per run, wherein sequencing by synthesis comprises exposing templates of amplified tag-polynucleotide conjugates to at least one dNTP flow to produce a sequence of incorporation signals for each template;

(d) aligning sequences of incorporation signals from like tag-polynucleotide conjugates by their sequence tags; and

(e) determining for each tag-polynucleotide conjugate having the same sequence tag a number of nucleotide incorporations for each dNTP flow as a function of measured incorporation signals for each such dNTP flow.

12. The method of claim 11, wherein the one or more polynucleotides are a plurality of polynucleotides.

13. The method of claim 11, wherein the plurality is at least 10⁴.

14. The method of claim 11, wherein said number of nucleotide incorporations for each of said dNTP flows is a whole number closest to an average of said measured incorporation signals.

15. The method of claim 11, wherein the step of sequencing by synthesis comprises the steps of:

(a) forming for each of the tag-polynucleotide conjugates a complex comprising a sequencing primer, a nucleic acid polymerase, and a tag-polynucleotide conjugate under conditions that permit annealing of the sequencing primer to such tag-polynucleotide conjugate and extension of such sequencing primer along the tag-polynucleotide conjugate in the presence of nucleoside triphosphates by the nucleic acid polymerase;

(b) introducing a dNTP to the complex by a dNTP flow;

(c) measuring incorporation signals;

(d) washing the complex; and

(e) repeating steps (b) through (d).

16. The method of claim 15, wherein the dNTPs are extension-blocked dNTPs and wherein the step of washing further includes a step of de-blocking incorporated extension-blocked dNTPs so that in a subsequent step of introducing a further extension-blocked dNTP may be incorporated.

17. The method of claim 11, wherein each different tag-polynucleotide conjugate is in a different reaction confinement region.

18. The method of claim 11, wherein the step of attaching includes labeling by sampling the one or more polynucleotides to form the tag-polynucleotide conjugates.

19. The method of claim 11, wherein the sequence tags are mosaic tags having variable regions with lengths in the range of from 1 to 5 nucleotides.

20. The method of claim 11, wherein the sequence tags comprise alternating regions comprising nucleotides selected from disjoint subsets of A, C, G and T, such that each alternating region has a length in the range of from 1 to 5 nucleotides.

21. The method of claim 11, wherein the step of sequencing comprises sequencing a sample of the amplified tag-polynucleotide conjugates.

22. The method of claim 11, wherein the one or more polynucleotides are molecules of recombined nucleic acids of T-cell receptor genes or immunoglobulin genes.