Compositions and methods for detecting rare sequence variants

• US 10,155,980 B2
• Filed: 11/01/2017
• Issued: 12/18/2018
• Est. Priority Date: 08/15/2016
• Status: Active Grant

• Current Assignee: Accuragen, Inc.
• Original Assignee: Accuragen, Inc.
• Inventors: Weng, Li, Sun, Zhaohui, Lin, Shengrong
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US15/800,558
• Publication Number:
  US 20180057871A1
• Time in Patent Office: 412 Days
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

In some aspects, the present disclosure provides methods for identifying sequence variants in a nucleic acid sample. In some embodiments, a method comprises identifying sequence differences between sequencing reads and a reference sequence, and calling a sequence difference that occurs in at least two different circular polynucleotides, such as two circular polynucleotides having different junctions, or two different sheared polynucleotides as the sequence variant. In some aspects, the present disclosure provides compositions and systems useful in the described methods.

Claims

1. A method of performing rolling circle amplification, the method comprising:

(a) in a reaction mixture, circularizing starting template polynucleotides in a plurality of starting template polynucleotides to form a plurality of circular polynucleotides using a ligase enzyme, wherein each polynucleotide of the plurality of polynucleotides has a 5′

end and a 3′

end prior to circularizing;

(b) adding a heat labile protease to said reaction mixture to degrade the ligase enzyme; and

(c) amplifying the circular polynucleotides after step (b) without isolating the circular polynucleotides from said reaction mixture between steps (a) and (c) to produce amplified polynucleotides,

wherein said method yields a higher level of recovery of the starting template polynucleotides as compared to performing a rolling circle amplification comprising steps (a) and (c) alone,

wherein the higher level of recovery is evidenced by an increased library complexity, and

wherein the increased library complexity is represented by an increased number of unique sequences amplified from said starting template polynucleotides.

2. The method of claim 1, further comprising degrading linear starting template polynucleotides prior to step (b) or step (c).

3. The method of claim 1, wherein the plurality of starting template polynucleotides are single-stranded.

4. The method of claim 1, wherein an individual circular polynucleotide has a junction that is distinct among the circularized polynucleotides.

5. The method of claim 1, wherein circularizing comprises the step of joining an adapter polynucleotide to the 5′

end, the 3′

end, or both the 5′

end and the 3′

end of a template polynucleotide in the plurality of template polynucleotides.

6. The method of claim 1, further comprising the step of heat inactivating the heat labile protease prior to step (c) but after step (b).

7. The method of claim 1, wherein amplifying comprises subjecting the circular polynucleotides to an amplification reaction comprising a plurality of primers, each of which specifically hybridizes to a different target sequence via sequence complementarity, said reaction being performed at about or above 30°

C.

8. The method of claim 1, wherein the sample is a sample from a subject.

9. The method of claim 8, wherein the sample is urine, stool, blood, saliva, tissue, or bodily fluid.

10. The method of claim 8, wherein the sample comprises tumor cells.

11. The method of claim 8, wherein the sample is a formalin-fixed paraffin embedded (FFPE) sample.

12. The method of claim 1, wherein the plurality of starting template polynucleotides comprises cell-free polynucleotides.

13. The method of claim 1, further comprising shearing the amplified nucleotides.

14. The method of claim 12, wherein the cell-free polynucleotides are cell-free DNA or RNA.

15. The method of claim 14, wherein the cell-free polynucleotides are cell-free tumor DNA or RNA.

16. The method of claim 1, further comprising sequencing the amplified polynucleotides to produce a plurality of sequencing reads.

17. The method of claim 16, further comprising identifying sequence differences between the sequencing reads and a reference sequence.

18. The method of claim 17, further comprising calling a sequence difference as a sequence variant in the plurality of starting template polynucleotides only when:

(i) the sequence difference occurs in a consensus sequence for a concatemer formed by rolling circle amplification;

(ii) the sequence difference is identified on both strands of a double-stranded input molecule; and

/or (iii) the sequence difference occurs in two different molecules.

19. The method of claim 18, wherein a sequence difference is identified as occurring in two different molecules when the sequence difference occurs in at least two circular polynucleotides having a different junction formed between the 5′

end and 3′

end.

20. The method of claim 18, wherein a sequence difference is identified as occurring in two different molecules when reads corresponding to the two different molecules have a different 5′

end and a different 3′

end.

21. The method of claim 18, wherein the sequence variant is a causal genetic variant.

22. The method of claim 18, wherein the sequence variant is associated with a type or stage of cancer.

23. The method of claim 1, wherein degrading the ligase enzyme in said reaction mixture is effectuated by adding the heat labile protease for about 10 minutes to about 60 minutes.

24. The method of claim 1, wherein the heat labile protease is inactivated at about 70°

C.

25. A method of generating a sequencing library, comprising:

(a) in a reaction mixture, circularizing individual starting template polynucleotides in a plurality of starting template polynucleotides to form a plurality of circular polynucleotides using a ligase enzyme, wherein each polynucleotide of the plurality of starting template polynucleotides has a 5′

end and a 3′

end prior to circularizing;

(b) adding a heat labile protease to said reaction mixture to degrade the ligase enzyme; and

(c) amplifying the circular polynucleotides after step (b) without isolating the circular polynucleotides from said reaction mixture between steps (a) and (c) to produce a sequencing library comprising amplified polynucleotides,

wherein the method generates a sequencing library with a complexity that is at least one-fold greater than a control library generated by performing a method comprising steps (a) and (c) alone, without performing step (b).

26. The method of claim 25, wherein the sequencing library generated has a complexity that is at least two-fold greater than that of the control library.

27. The method of claim 25, wherein the plurality of starting template polynucleotides comprises cell-free polynucleotides.

28. The method of claim 25, wherein the amplifying is performed at about or above 30°

C.

29. The method of claim 25, wherein the heat labile protease is inactivated at about 70°

C.

30. The method of claim 25, wherein degrading in (b) is effectuated by adding the heat labile protease for about 10 minutes to about 60 minutes.