Library characterization by digital assay

• US 8,663,920 B2
• Filed: 07/30/2012
• Issued: 03/04/2014
• Est. Priority Date: 07/29/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Saxonov, Serge, Tzonev, Svilen S., Lucero, Michael Y., Hindson, Benjamin J., Koehler, Ryan T.
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US13/562,198
• Publication Number:
  US 20130045875A1
• Time in Patent Office: 582 Days
• Field of Search: 506/7, 435/6.1
• US Class Current: 435/6.1
• 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 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes

Abstract

Methods of characterizing a nucleic acid library by digital assay.

Claims

1. A method of library characterization, comprising:

obtaining a nucleic acid library including members each having a first adapter region and a second adapter region, wherein at least a subset of the members have an insert disposed between the first and second adapter regions;

forming partitions containing members of the library;

performing a digital assay on the partitions with an adapter region probe to generate data collected from a plurality of the partitions while members of the library are contained within the plurality of partitions, the data indicating whether a library member is present in each of the plurality of partitions; and

determining a characteristic of the library based on the data,

wherein the probe binds selectively to empty members of the library having no insert between the adapter regions, relative to members of the library having an insert.

2. The method of claim 1, wherein the step of forming partitions includes a step of forming droplets containing members of the library.

3. The method of claim 1, wherein the step of performing a digital assay includes a step of amplifying members of the library in the partitions with one or more primers that bind to the first adapter region and the second adapter region.

4. The method of claim 1, wherein the step of determining a characteristic includes a step of determining a level of empty members of the library having no insert between the adapter regions.

5. The method of claim 4, wherein the step of determining a characteristic includes a step of determining a level of members of the library that include an insert between the adapter regions.

6. The method of claim 4, wherein the probe binds selectively to a junction sequence produced by direct attachment of the first and second adapter regions to one another without an insert.

7. A method of library characterization, comprising:

obtaining a nucleic acid library including members each having a first adapter region and a second adapter region, wherein at least a subset of the members have an insert disposed between the first and second adapter regions;

forming partitions containing members of the library;

performing a digital assay on the partitions with an adapter region probe to generate data collected from a plurality of the partitions while members of the library are contained within the plurality of partitions, the data indicating whether a library member is present in each of the plurality of partitions; and

determining a characteristic of the library based on the data,

wherein the step of determining a characteristic includes a step of determining a level of empty members of the library having no insert between the adapter regions.

8. The method of claim 7, wherein the step of obtaining a nucleic acid library includes a step of attaching at least one type of adapter to opposing ends of nucleic acid fragments corresponding to the insert.

9. The method of claim 7, wherein the partitions formed are uniform in size, and wherein some of the partitions formed contain no library members and others of the partitions contain only one library member.

10. The method of claim 7, wherein the step of forming partitions includes a step of forming droplets containing members of the library.

11. The method of claim 7, wherein the step of performing a digital assay includes a step of detecting luminescence from a luminophore of the probe while the luminophore is contained by the plurality of partitions.

12. The method of claim 7, wherein the step of determining a characteristic includes a step of determining a level of members of the library that include an insert between the adapter regions.

13. The method of claim 7, wherein the step of performing a digital assay uses a first probe that binds to the first adapter region and a distinct second probe that binds to the second adapter region.

14. The method of claim 13, wherein the step of determining a characteristic includes a step of determining a level of library members containing both the first adapter region and the second adapter region.

15. The method of claim 14, wherein the level determined accounts for a presence of the first and second adapter regions in the same partitions without being linked to one another.

16. A method of library characterization, comprising:

obtaining a nucleic acid library including members each having a first adapter region and a second adapter region, wherein at least a subset of the members have an insert disposed between the first and second adapter regions;

forming partitions containing members of the library;

performing a digital assay on the partitions to generate data collected from a plurality of the partitions while members of the library are contained within the plurality of partitions, the data indicating whether a library member is present in each of the plurality of partitions; and

determining a characteristic of the library based on the data,

wherein the step of performing a digital assay uses a first probe that binds to the first adapter region and a distinct second probe that binds to the second adapter region,

wherein the step of determining a characteristic includes a step of determining a level of library members containing both the first adapter region and the second adapter region, and

wherein the step of determining a characteristic also includes a step of determining a level of library members containing the first adapter region and not the second adapter region and a level of library members containing the second adapter region and not the first adapter region.

17. The method of claim 16, wherein the partitions formed are uniform in size, and wherein some of the partitions formed contain no library members and others of the partitions contain only one library member.

18. The method of claim 16, wherein the step of forming partitions includes a step of forming droplets containing members of the library.

19. The method of claim 16, wherein the step of performing a digital assay includes a step of amplifying members of the library in the partitions with one or more primers that bind to the first adapter region and the second adapter region.

20. The method of claim 16, wherein the step of performing a digital assay includes a step of detecting luminescence from at least one luminophore of at least one of the probes while the at least one luminophore is contained by the plurality of partitions.

21. The method of claim 16, wherein the step of determining a characteristic includes a step of determining a level of empty members of the library having no insert between the adapter regions.

22. The method of claim 21, wherein the step of determining a characteristic includes a step of determining a level of members of the library that include an insert between the adapter regions.

23. A method of library characterization, comprising:

obtaining a nucleic acid library including members each having a first adapter region and a second adapter region, wherein at least a subset of the members have an insert disposed between the first and second adapter regions;

forming partitions containing members of the library;

performing a digital assay on the partitions to generate data collected from a plurality of the partitions while members of the library are contained within the plurality of partitions, the data indicating whether a library member is present in each of the plurality of partitions; and

determining a characteristic of the library based on the data,

wherein the step of performing a digital assay uses a first probe that binds to the first adapter region and a distinct second probe that binds to the second adapter region, and

wherein the step of determining a characteristic includes a step of determining a level of library members containing both the first adapter region and the second adapter region and substantially excluding empty members, and a step of determining a level of empty members from the data.

24. The method of claim 23, wherein the step of obtaining a nucleic acid library includes a step of attaching at least one type of adapter to opposing ends of nucleic acid fragments corresponding to the insert.

25. The method of claim 23, wherein the partitions formed are uniform in size, and wherein some of the partitions formed contain no library members and others of the partitions contain only one library member.

26. The method of claim 23, wherein the step of forming partitions includes a step of forming droplets containing members of the library.

27. The method of claim 23, wherein the step of performing a digital assay includes a step of amplifying members of the library in the partitions with one or more primers that bind to the first adapter region and the second adapter region.

28. The method of claim 23, wherein the step of performing a digital assay includes a step of detecting luminescence from at least one luminophore of at least one of the probes while the at least one luminophore is contained by the plurality of partitions.

29. The method of claim 23, further comprising a step of selecting an amount of the library for use in a sequencing protocol based on the characteristic.

30. The method of claim 23, wherein at least one of the probes includes a luminophore and is present in the partitions when the partitions are formed.

31. A method of library characterization, comprising:

obtaining a nucleic acid library including members each having a first constant region and a second constant region, wherein at least a subset of the members have a variable region disposed between the first and second constant regions;

forming droplets containing members of the library at limiting dilution;

amplifying members of the library in the droplets using a primer for each constant region;

detecting light emitted from the droplets; and

determining a level of empty members of the library based on the light emitted.

32. The method of claim 31, further comprising a step of determining a level of members of the library having both constant regions based on the light emitted.

33. The method of claim 31, wherein a constant region probe includes a luminophore and is present in the droplets when the droplets are formed.