Efficient digital measurement of long nucleic acid fragments

Abstract

A method and system are provided for measuring the sizes of multiple nucleic acid molecules using a multiplexed digital amplification reaction. In various embodiments, the digital amplification reaction uses multiple pairs of separate forward and reverse primers to amplify a target region of a nucleic acid molecule, which is separated by a specific number of base pairs. In other embodiments, the digital amplification reaction uses multiple primer pairs that share a common pair to amplify overlapping target regions. The method and system are particularly useful for determining the size distribution of nucleic acid molecules or for classifying the pathology of which the nucleic acid molecules are sampled. Advantageously, the method and system can measure the size of long nucleic acid molecules without relying on the inefficient amplification of very long amplicons.

Claims

1. A method for analyzing a biological sample from a subject, wherein the method is Receiving the aforementioned biological sample containing multiple cell-free nucleic acid molecules, Distributing the aforementioned multiple cell-free nucleic acid molecules into multiple digital reactions, The addition of a reagent to each of the plurality of digital reactions, wherein the reagent for each of the plurality of reactions comprises a first primer set targeting a first region of a reference sequence, and a second primer set targeting a second region within a specific number of bases from the first region in the reference sequence, wherein the first primer set comprises a first forward primer, a first reverse primer, and a first probe, and the second primer set comprises a second forward primer, a second reverse primer, and a second probe, wherein the second forward primer is downstream of the first reverse primer in the reference sequence, and the specific number of bases is 5 kilobases or less. With respect to the first digital reaction among the plurality of digital reactions, the first signal from the first probe and the second signal from the second probe are detected. A method comprising determining, based on detecting the first signal and the second signal, that the first reaction includes a cell-free nucleic acid molecule from the plurality of cell-free nucleic acid molecules having a length of at least the specified number of bases and covering the first region and the second region.

2. The method according to claim 1, wherein the specified number of bases is 500 bases or more.

3. To detect a first number of the plurality of digital responses that are positive for only one of the first signal and the second signal, To detect a second number of the plurality of digital responses that are positive for both the first signal and the second signal, The method according to claim 1 or 2, further comprising determining a parameter using the first number and the second number, wherein the parameter measures a relative amount between the first number and the second number.

4. The method according to claim 3, further comprising determining the size distribution of the plurality of cell-free nucleic acid molecules using the parameters.

5. The method according to claim 3, further comprising determining a pathological classification for the subject using the parameters.

6. The method according to claim 5, wherein the pathology includes pre-eclampsia.

7. The reagent for each of the plurality of digital reactions further comprises a third set of primers targeting a third region of the reference sequence, the third set of primers comprising a third forward primer, a third reverse primer, and a third probe, wherein the third forward primer is downstream of the first reverse primer in the reference sequence, and the second forward primer is downstream of the third reverse primer in the reference sequence, and the method is The method according to claim 3, further comprising detecting the first signal, the second signal, and the third signal for each of the plurality of digital reactions, wherein the third signal is from the third probe.

8. The parameter is the first parameter, and the method is The third number of the plurality of digital responses that are positive for the third signal and for only one of the first signal and the second signal, The method according to claim 7, further comprising determining a second parameter using the first number and the third number, wherein the parameter measures and determines a relative amount between the first number and the third number.

9. The parameter is the first parameter, and the method is The third number of the plurality of digital responses that are positive for the third signal and for only one of the first signal and the second signal, The method according to claim 7, further comprising determining a second parameter using the second number and the third number, wherein the parameter measures and determines a relative amount between the second number and the third number.

10. The method according to claim 8 or 9, further comprising determining the size distribution of the plurality of cell-free nucleic acid molecules using the first and second parameters.

11. The method according to claim 8 or 9, further comprising determining the classification of the pathology of the subject using the first parameter and the second parameter.

12. The method according to claim 11, wherein the pathology includes pre-eclampsia.

13. The method according to any one of claims 7 to 12, wherein the third region has a length of less than 500 bp.

14. The method according to any one of claims 7 to 13, wherein the third signal includes a third fluorescence emission light.

15. The method according to claim 14, wherein the third probe includes a third fluorescent label.

16. The method according to any one of claims 1 to 15, wherein the first region and the second region each independently have a length of less than 500 bp.

17. The method according to any one of claims 1 to 16, wherein the plurality of digital reactions have an average of one cell-free nucleic acid molecule per digital reaction.

18. The method according to any one of claims 1 to 17, wherein the plurality of cell-free nucleic acid molecules consist of 100 to 500,000 cell-free nucleic acid molecules.

19. The method according to any one of claims 1 to 18, wherein each of the plurality of digital reactions is independently a droplet digital polymerase chain reaction (ddPCR).

20. The method according to any one of claims 1 to 19, wherein the reagent for each of the plurality of digital reactions further comprises a reverse transcriptase.

21. The method according to any one of claims 1 to 20, wherein the first signal includes a first fluorescence emission light, and the second signal includes a second fluorescence emission light.

22. The method according to claim 21, wherein the first probe includes a first fluorescent label, and the second probe includes a second fluorescent label.

23. A method for analyzing a biological sample from a subject who is pregnant with a fetus, wherein the method is Receiving the aforementioned biological sample containing multiple cell-free nucleic acid molecules, Distributing the aforementioned multiple cell-free nucleic acid molecules into multiple digital reactions, Adding a reagent to each of the plurality of digital reactions, wherein the reagent for each of the plurality of reactions comprises a first primer set targeting a first region, and a second primer set targeting a second region larger than the first region and including the first region, wherein the first primer set comprises a first forward primer, a first reverse primer, and a first probe, and the second primer set comprises a second probe and a second primer which is either a second forward primer or a second reverse primer, wherein the second primer set shares a common primer with the first primer set, and the second probe recognizes a portion of the second region that is not within the first region, To detect a first number of the plurality of digital responses that are positive only for the first signal from the first probe, Detecting a second number of the plurality of digital responses that are positive for both the first signal and the second signal, wherein the second signal is from the second probe, Determining a parameter using the first number and the second number, wherein the parameter measures and determines a relative amount between the first number and the second number. A method comprising determining the classification of pre-eclampsia for the subject using the aforementioned parameters.

24. The method according to claim 23, wherein the first region and the second region are each part of the same repeating region that appears at least 10 times in the reference human genome.

25. The method according to claim 23 or 24, wherein the first region has a length of 40 bp to 100 bp.

26. The method according to any one of claims 23 to 25, wherein the second region has a length of 100 bp to 1000 bp.

27. The reagent for each of the plurality of digital reactions further comprises a third primer set that targets a third region which is larger than the first region and includes the first region, wherein the third primer set comprises a third forward primer, a third reverse primer, and a third probe, wherein the third primer set shares a common primer with the first primer set, and the second region includes the third region, and the method, The method according to any one of claims 23 to 26, further comprising detecting a third number of the plurality of digital reactions that are not positive for the second signal and are positive for both the first signal and the third signal, wherein the third signal is from the third probe.

28. The method according to claim 27, wherein a second parameter is determined using the first number and the third number, further comprising determining that the second parameter measures a relative amount between the first number and the third number, and further using the second parameter to determine the classification of preeclampsia.

29. The method according to claim 27 or 28, wherein a third parameter is determined using the second number and the third number, the second parameter further comprising measuring and determining a relative amount between the second number and the third number, and the determination of the classification of preeclampsia further uses the third parameter.

30. The method according to any one of claims 27 to 29, wherein the third signal includes a third fluorescence emission light.

31. The method according to claim 30, wherein the third probe includes a third fluorescent label.

32. The method according to any one of claims 23 to 31, wherein the plurality of digital reactions have an average of one cell-free nucleic acid molecule per digital reaction.

33. The method according to any one of claims 23 to 32, wherein the plurality of cell-free nucleic acid molecules consist of 100 to 500,000 cell-free nucleic acid molecules.

34. The method according to any one of claims 23 to 33, wherein each of the plurality of digital reactions is independently a droplet digital polymerase chain reaction (ddPCR).

35. The method according to any one of claims 23 to 34, wherein the reagent for each of the plurality of digital reactions further comprises a reverse transcriptase.

36. The method according to any one of claims 23 to 35, wherein the first signal includes a first fluorescence emission light, and the second signal includes a second fluorescence emission light.

37. The method according to claim 36, wherein the first probe includes a first fluorescent label, and the second probe includes a second fluorescent label.

38. A computer product comprising a non-temporary computer-readable medium that stores a plurality of instructions, when executed, causing a computer system to perform the method described in any one of the prior claims.

39. It is a system, The computer product according to claim 38, A system comprising one or more processors for executing instructions stored in the computer-readable medium.

40. A system comprising means for carrying out any of the above methods.

41. A system comprising one or more processors configured to perform one of the methods described above.

42. A system comprising modules that perform one of the steps described above.

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