Genetic assays
Provided herein are methods, compositions, systems, and kits for recombination assays, many of which involve amplification reactions such as PCR or droplet digital PCR.
1. A method of quantifying previously recombined nucleic acids containing a first sequence and a second sequence, the method comprising:
a. obtaining a sample comprising viral genomic nucleic acids, wherein the viral genomic nucleic acids comprise (i) first parental nucleic acids representing a first viral genome that includes the first sequence and not the second sequence, (ii) second parental nucleic acids representing a second viral genome that includes the second sequence and not the first sequence, and (iii) a plurality of previously recombined nucleic acids including, on a same strand, the first sequence and the second sequence;
b. partitioning the sample into more than about 1,000 compartments such that each compartment of only a subset of the more than about 1,000 compartments contains the first sequence, each compartment of only a subset of the more than about 1,000 compartments contains the second sequence, and each compartment of only a subset of the more than about 1,000 compartments contains both the first sequence and the second sequence;
c. performing amplification of the first sequence and amplification of the second sequence within the more than about 1,000 compartments;
d. detecting at least one amplification signal from the more than about 1,000 compartments indicating whether the first sequence is present in a given compartment and whether the second sequence is present in the given compartment;
e. enumerating compartments of the more than about 1,000 compartments that comprise the first sequence but not the second sequence to obtain a first value, the second sequence but not the first sequence to obtain a second value, both the first sequence and the second sequence to obtain a third value, and neither the first sequence nor the second sequence to obtain a fourth value, wherein the step of enumerating is performed using the at least one amplification signal detected from the more than about 1,000 compartments;
f. calculating an expected number of compartments of the more than about 1,000 compartments comprising both the first sequence and the second sequence by chance co-localization, using the first, second, and fourth values; and
g. adjusting the third value of step (e) using the expected number of step (f) in order to obtain a numerical value for the recombined nucleic acids.
2. The method of claim 1 , wherein the first parental nucleic acids comprise a genetic variant of the second sequence, and wherein the at least one amplification signal also indicates whether the genetic variant of the second sequence is present in a given compartment.
3. The method of claim 2 , wherein the second sequence and the genetic variant of the second sequence differ from one another by a genetic variation, and wherein the genetic variation is selected from the group consisting of single nucleotide polymorphisms, insertions, inversions, rearrangements, transversions, deletions, indels, microsatellite repeats, minisatellite repeats, short tandem repeats, transposable elements, large scale structural variants, and combinations thereof.
4. The method of claim 2 , wherein each of the more than about 1,000 compartments comprises a probe including a polynucleotide attached to a fluorophore, wherein the polynucleotide of the probe is capable of hybridizing to the second sequence, and wherein the polynucleotide of the probe is capable of hybridizing to the genetic variant of the second sequence.
5. The method of claim 2 , wherein the step of enumerating compartments includes a step of enumerating compartments that comprise both the first sequence and the genetic variant of the second sequence.
6. The method of claim 2 , wherein the second parental nucleic acids include a genetic variant of the first sequence, and wherein the at least one amplification signal also indicates whether the genetic variant of the first sequence is present in a given compartment.
7. The method of claim 6 , wherein the step of enumerating compartments includes a step of enumerating compartments of the more than about 1,000 compartments that comprise both the second sequence and the genetic variant of the first sequence, and a step of enumerating compartments of the more than about 1,000 compartments that comprise both the first sequence and the genetic variant of the second sequence.
8. The method of claim 2 , wherein amplification of the second sequence and amplification of the variant of the second sequence are detected via probes that emit light of a same color as one another.
9. The method of claim 1 , wherein the viral genomic nucleic acids comprise RNA.
10. The method of claim 1 , wherein the first viral genome is selected from the group consisting of Adenovirus, Coxsackievirus, Epstein-Barr virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Herpes simplex virus, type 2, Cytomegalovirus, Human herpesvirus, type 8, Human immunodeficiency virus, Influenza virus, Measles virus, Mumps virus, Human papillomavirus, Parainfluenza virus, Poliovirus, Rabies virus, Respiratory syncytial virus, Varicella-zoster virus, and a variant strain thereof.
11. The method of claim 1 , wherein the first viral genome is from an influenza strain, and wherein the second viral genome is from an influenza strain.
12. The method of claim 11 , wherein the influenza strain of the first viral genome is H1N1, H5N1, H3N2, H7N9, or H1N2, or a recombinant strain thereof, and wherein the influenza strain of the second viral genome is H1N1, H5N1, H3N2, H7N9, or H1N2, or a recombinant strain thereof.
13. The method of claim 1 , wherein the first and second viral genomes are Human immunodeficiency virus genomes.
14. The method of claim 1 , wherein the sample is combined with a label prior to step (c).
15. The method of claim 14 , wherein amplification of the first sequence and amplification of the second sequence are detected via labels that emit light of different color from one another.
16. The method of claim 1 , wherein a distance between the first and second sequences in the previously recombined nucleic acids is less than about 20 kilobases.
17. The method of claim 1 , wherein the sample is partitioned into more than about 10,000,000 compartments.
18. The method of claim 1 , wherein the more than about 1,000 compartments include a first probe that binds to the first sequence and a second probe that binds to the second sequence.
19. The method of claim 1 , wherein the first and second parental nucleic acids represent at least 80% of viral genomic nucleic acids in the sample that contain the first sequence and/or the second sequence.
20. The method of claim 1 , wherein the first parental nucleic acids and the second parental nucleic acids are provided by different strains of a same species of virus.
21. The method of claim 1 , wherein the first parental nucleic acids and the second parental nucleic acids are provided by different virus species.
22. The method of claim 1 , wherein amplification of the first sequence and amplification of the second sequence are detected via probes that emit light of a same color as one another.
23. The method of claim 1 , wherein the step of calculating includes a step of calculating the expected number of compartments using the following:
Expected
number
=
(
first
value
)
×
(
second
value
)
fourth
value
.
24. The method of claim 1 , wherein the step of adjusting includes a step of subtracting the expected number of compartments from the third value.