LARGE-SCALE BIOMOLECULAR ANALYSIS WITH SSEQUENCE TAGS

• US 20150259734A1
• Filed: 06/27/2014
• Published: 09/17/2015
• Est. Priority Date: 07/01/2013
• Status: Active Grant

• Current Assignee: Adaptive Biotechnologies Corp.
• Original Assignee: Sequenta LLC
• Inventors: Asbury, Thomas, Faham, Malek, Moorhead, Martin, Weng, Li, Zheng, Jianbiao, Hervold, Kieran, Kotwaliwale, Chitra, Wittkop, Tobias
• Granted Patent: US 9,708,657 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16H 50/30 for calculating health indices; for individual health risk assessment
• CPC Class Codes: G16Z 99/00 Subject matter not provided for in other main groups of this subclass
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

The invention is directed to sequence-based profiling of populations of nucleic acids by multiplex amplification and attachment of one or more sequence tags to target nucleic acids anchor copies thereof followed by high-throughput sequencing of the amplification product. In some embodiments, the invention includes successive steps of primer extension, removal of unextended primers and addition of new primers either for amplification (for example by PRC) or for additional primer extension. Some embodiments of the invention are directed to minimal residual disease (MRD) analysis of patients being treated for cancer. Sequence tags incorporated into sequence reads provide an efficient means for determining clonotypes and at the same time provide a convenient means for detecting carry-over contamination from other samples of the same patient or from samples of a different patient which were tested in the same laboratory.

Claims

1. A method of generating clonotype profiles from a plurality of Mutt le cell receptor chains, the method comprising the steps of:

(a) combining in a reaction mixture under primer extension conditions a first set of primers with a sample of recombined nucleic acids from B-cells and/or T-cells and/or cell-free DNA, wherein each printer of the first set has a receptor-specific portion such that the receptor-specific portion anneals to a different recombined nucleic acid at a predetermined location and is extended to form a first extension product, and wherein each primer of the first set has a 5′

non-eomplementary end containing a first primer binding site

(b) removing from the reaction mixture non-extended primers of the first set;

(c) adding to the reaction mixture under primer extension conditions a second set of primers, wherein each primer of the second set has a receptor-specific portion such that the receptor-specific portion anneals to the first extension product at a predetermined location and has a 5′

-non-complementary end containing a second primer binding site, primers of the first set and/or primers of the second set comprising a sequence tag disposed between the receptor-specific portion and the first or second primer binding site, respectively, and wherein each primer of the second set is extended to form a second extension product, such that each second extension product comprises a first primer binding site, a second primer binding site, at least one sequence tag, and recombined nucleic acid encoding a portion or T cell receptor chain or a B cell receptor chain;

(d) performing a polymerase chain reaction in the reaction mixture to form an amplicon, the polymerase chain reaction using forward primers specific for the first primer binding site and reverse primers specific for the second primer binding site; and

(e) sequencing the nucleic acids or the amplicon to form a clonotype profile of a plurality of T cell receptor chains and/or B cell receptor chains.

2. The method of claim 1 wherein said plurality of T cell receptor chains and/or B cell receptor chains comprises TCRβ

, TCRδ and

TCRγ and

wherein said primers of said first set and said primer of said second set comprise primers that flank regions of said recombined nucleic acids encoding VDJ regions of TCRβ and

TCRδ and

primers that flank VJ regions of TCRγ

.

3. The method of claim 1 wherein said step of sequencing includes:

generating sequence reads each having an error rate and each comprising a tag sequence and a recombined nucleic acid sequence;

aligning sequence reads having like tag sequences to form groups of sequence rends having the same sequence tags;

coalescing sequence reads of groups to determine clonotypes, wherein. groups of sequence reads are coalesced into different recombined nucleic acid sequences whenever said groups of semtence reads are distinct with a likelihood of at least ninety-five percent; and

forming said clonotype profile from the clonotypes.

4. The method of claim 1 further including a.second:

step of removing after said second extension product is

5. The method of claim 1 wherein said annealing and extension of said primers of said first set is repeated after melting said first extension product.

6. The method of claim 1 wherein said annealing and extension of said primers of said second set is repeated after melting said second extension product.

7. The method of claim 1 wherein said step of removing includes adding an exonuclease I activity to said reaction mixture.

33. The method of claim 3 wherein said anneaiing and extension of said primers of said second set is repeated after melting said second extension product.

8. A method of generating clonotype profiles from a plurality of B cell receptor chains, the method comprising the steps of:

(a) combining in a reaction mixture tinder primer extension conditions a first set of primers with a sample of recombined nucleic acids from B-cells and/or cell-free DNA, wherein each primer of the first set has a receptor-specific portion such that the receptor-specific portion anneals to a different recombined nucleic acid at a predetermined tocition and is extended to form a lust extension product, and where ill each pt nn of the first set has a 5′

-non-complementary end containing it first primer binding site

(b) removing from the reaction mixture non-extended primers of the first set;

(c) adding to the reaction mixture under printer extension conditions a second set of primers, wherein each primer of the second sel has a receptor-specific put don such. that the .receptor-pecific portion anneals to the first extension product at a predetermined location and has a 5′

-non-complementary end containing is second primer binding site, primers of the first set and/or primers of the second set. comprising a sequence tag disposed between the receptor-specific portion and the first or second primer binding site, respectively, and wherein each primer of the second set is extended to form a second extension product, such that each second extension product comprises a first prhner binding site a seeond primer binding site, at least one sequence tag, and recombined nucleic acid encoding a portion of a B cell receptor chain;

(d) performing a polymerase chain reaction in the reaction mixture to form an amplicon, the polymerase chain reaction using forward primers specific for the first primer binding site and reverse primers specific for the second printer binding site and

(e) sequencing the nucleic acids of the amplicon to farm a clonotype profile of a plurality of B cell receptor chains.

9. The method of claim 8 wherein said plurality of B cell receptor chains comprises IgH and IgK and wherein said primers of said first set and said primers of said second set comprise primers that flank regions of said recombined nucleic xids encoding VDS regions of IGH, DJ regions of IgH and VJ regions of IgK.

10. The method of claim 9 wherein said primers of said first set or said second set include at least one nested set of primers specific fir a plurality of different primer binding sites in V regions of said inH chains.

12. The method of claim 8 further including a second step of removing after said second extension product is fonned.

13. The method of claim 8 wherein said annealing and extension of said primers of said first set is repeated after melting, said first extension product.

14. The method of claim 8 wherein said annealing and extension of said primers of said second set is repeated after melting said second extension product.

15. The method of claim 8 wherein said step of removing includes adding an exorinclease I activity to said reaction mixture.

11. The method of claim t vherein said step of sequencing includes:

generating sequence reads each haying an error rate and each comprising a lag sequence and a recombined nucleic acid sequence;

aligning sequence reads haying like tag sequences to form groups of sequence reads having the same sequence tags;

coalescing sequence reads of groups to determine clonotypes, wherein groups of sequence reads are coalesced into different recombined nucleic acid sequences whenever said groups of sequence reads are distract with a likelihood of at least ninety-five percent; and

forming said clonotype profile from the clonotypes.

16. A method of determining a clonotype profile of recombined nucleic acids encoding a plurality of immune receptor chains in a sample, the method comprising the steps of:

(a) obtaining a sample from an individual comprising T-cells and/or B-cells and/or cell-free DNA;

(b) attaching sequence tags to recombined nucleic acid molecules of T-cell receptor genes or immunoglobulin genes from the sample to form tag-nucleic acid conjugates, wherein at least one recombined nucleic acid or copies thereof;

have different sequence tags attached;

(c) amplifying the tag-nucleic acid conjugates;

(d) sequencing. a sample of the tag iucleic acid coniugates to provide sequence reads each having an error rate and each comprising a tag sequence and a recombined nucleic acid sequence;

(e) aligning sequence reads having like tag sequences to form groups of sequence reads having the same sequence tags;

(f) coalescing sequence reads of groups to determine clonotypes, wherein groups of sequence reads are coalesced into different recombined .nucleic acid sequences whenever said groups of sequence reads are distinct with a likelihood of at least ninety-five percent; and

(g) determining the clonotype profile of the sample by determining levels of the clonotypes.

17. The method of claim 16 wherein said steps of attaching and amplifying comprise:

combining in a reaction mixture under primer extension conditions a first set of primers with said sample, wherein each primer of the first set has a receptor-specific portion such that the receptor-specific portion anneals to a different recombined nucleic acid at a predetermined location and is extended to form a first extension product, and wherein each primer of the first set has a 5′

-non-complementary end containing a first primer binding site;

removing from the reaction mixture non extended primers of the first set;

adding to the reaction mixture under primer extension conditions a second set o primers, wherein each primer of the second set has a ;

receptor-specific portion such that the receptor-specific portion anneals to the first extension product at a predetermined location and has a 5′

-non-complementary end containing a second primer binding site, primers of the first set and/or primers of the second set comprising a sequence tag disposed between the receptor-specific portion and the first or second primer binding site, respectively, and wherein each primer of the second set is extended to form a second extension product, such that each second extension product comprises a first primer binding site, a second primer binding site, at least one sequence tag, and recombined nucleic acid encoding a portion of a T cell receptor chain or a B cell receptor chain; and

performing a polymerase chain reaction in the reaction mixture to form an amplicon, the polymerase chain reaction using forward primers specific for the first primer binding site and reverse primers specific for the second priater binding site.

18. The method of claim 17 further including a second step of removing after said second extension product is formed.

19. The method of claim 17 wherein said annealing and extension of said primers of said first set is repeated after melting said first extension product.

20. The method of claim 17 wherein said annealing and extension of said primers of said second set is repeated after melting said second extension product.

21. The method of claim 17 wherein said step of removing includes adding an exonuclease I activity to said reaction mixture.

22. The method of claim 17 wherein said steps of attaching and amplifying generate tag-nucleic acid conjugates wherein at least one said recombined nucleic acid and its respective copies have a plurality of different sequence tags attached.

23. The method of claim 22 wherein said recombined nucleic, acids comprise recombined nucleic acids encoding TCRβ

, TCRδ and

TCRγ

chains and wherein said primers of said first set and said primer of said second set comprise primers that flank regions of said recombined nucleic acids encoding VDJ regions of TCRβ and

TCRδ and

primers that flank VJ regions of TCRγ

.

24. The method of claim 22 wherein said recombined nucleic acids comprise recombined nucleic a ids encoding IgH and IgK and wherein said primers of said first set and said primers of said second set comprise primers that flank regions of said recombined nucleic acids encoding VDJ regions of IgH, DJ regions of IgH and VJ regions of IgK.

25. The method of claim 16 wherein said step of coalescing includes coalescing said groups of sequence reads into different recombined nucleic acid sequences whenever said groups of sequence reads are distinct with a likelihood of at least 99.9 percent.

26. A method of monitoring a minimal residual disease of a lymphoid proliferative disorder, the method comprising the steps of:

(a) obtaning from an individual a sample containing T-cells and/or B-cells and/or cell free DNA or RNA;

(b) attaching sequence tags to each of a plurality of recombined nucleic acids in the sample to form tag-nucleic acid conjugates, wherein at least one recombined nucleic acid or copies thereof have different sequence tags attached and are characteristic of a lymphoid proliferative disorder of the individual;

(c) amplifying the tag-nucleic acid conjugates;

(d) sequencing a sample of the tag-nucleic acid conjugates to provide sequence reads each comprising a tag sequence and a recombined nucleic acid sequence;

(e) aligning sequence reads having like tag squences to form groups of sequence reads having the same sequence tags;

(f) coalescing recombined nucleic acid seqtiences of groups to deteremine clonoltypes, wherein groups of sequence reads are coalesced into different clonotypes whenever said groups of recombined nucleic acid sequences are distinct with a likelihood of at least 99.9 percent; and

(g) detecting in a clonotype profile the presence, absence and/or level of clonotypes correlated with the lymphoid proliferative disorder of the individual.

27. The method of claim 26 wherein said steps of attaching, and amplifying comprise:

combining in a reaction mixture under primer extension conditions a first set of primers with a sample of recombined nucleic acids from B-cells and/or and/or cell-free DNA, wherein each primer of the first set has a receptor-specific portion such that the receptor-specific portion anneals to a different recombined nucleic acid at a predetermined location and is extended to form a first extension product, and wherein each primer of the first set has a 5′

-non-complementary end containing a first primer binding site;

removing from the reaction mixture non extended primers of the first set;

adding to the reaetion mixture under primer extension conditions a second set of primers, wherein each primer of the second set has a receptor-specific portion such that the receptor-specific portion anneals to the first extension product at a predetermined location and has a 5′

-non-complementary end containing a second primer binding site, primers of the first set and/or primers of the second set comprising a sequence tag disposed between the receptor-specific portion and the first or second primer binding site, respectively, and wherein each primer of the second set is extended to form a second extension product, such that each second extension product comprises a first primer binding site, a second primer binding site, at least one sequence tag, and recombined nucick acid encodim a portion or a T cell receptor chain or a B cell receptor chain;

performing a polymerase chain reaction in the reaction mixture to form an amplicon, the polymerase chain, reaction using forward primers specific for the first primer binding site and reverse primers specific for the second primer binding site.

28. The method of claim 27 wherein said recombined nucleic acids comprise recombined nucleic acids encoding TCRβ

, TCRδ and

TCRγ

chains and wherein said primers of said first set and said primer of said second set comprise primers that flank regions of said recombined nucleic acids encoding VDJ regions of TCRβ and

TCRδ and

primers that flank VJ regions of TCRγ

.

29. The method of claim 27 wherein said recombined nucleic acids comprise recombined nucleic acids encoding IgH and IgK and wherein said primers of said first set and said printers of said second set comprise primers that flank regions of said recombined nucleic acids encoding VDJ regions of IgH, DJ regions of IgH and VJ regions of IgK.

30. The method of claim 29 wherein said primers of said first set or said second set include at least one nested set of primers specific for a plurality of different primer binding sites in V regions of said IgH chains.

31. The method of claim 30 further including a second step of removing after said second extension product is formed.

32. The method of claim 30 wherein said annealing and extension of said primers of said first set is repeated after melting said first extension product.