ANALYSIS OF NUCLEIC ACIDS
Method of haplotype analysis. In an exemplary method, an aqueous phase containing nucleic acid may be partitioned into a plurality of discrete volumes. At least one allele sequence may be amplified in the volumes from each of a first polymorphic locus and a second polymorphic locus that exhibit sequence variation in the nucleic acid. At least one measure of co-amplification of allele sequences from both loci in the same volumes may be determined. A haplotype of the first and second loci may be selected based on the at least one measure of co-amplification.
1 . A method of haplotype analysis, the method comprising:
partitioning an aqueous phase containing nucleic acid into a plurality of discrete volumes;
amplifying in the volumes at least one allele sequence from each of a first polymorphic locus and a second polymorphic locus that exhibit sequence variation in the nucleic acid;
determining at least one measure of co-amplification of allele sequences from both loci in the same volumes; and
selecting a haplotype of the first and second loci based on the at least one measure of co-amplification.
2 . The method of claim 1 , wherein the first and second loci are contained in a target region of the nucleic acid, and wherein the step of partitioning results in an average concentration of less than about five copies of the target region per volume.
3 . The method of claim 1 , wherein the step of determining at least one measure includes a step of determining at least one correlation coefficient for allele-specific amplification data of the first locus correlated with allele-specific amplification data of the second locus from the same volumes.
4 . The method of claim 1 , wherein the step of determining at least one measure includes a step of determining a first correlation coefficient and a second correlation coefficient for allele-specific amplification data of a first allele sequence and a second allele sequence of the first locus correlated respectively with allele-specific amplification data of the second locus from the same volumes, and wherein the step of selecting a haplotype is based on a step of comparing the first and second correlation coefficients with each other.
5 . The method of claim 1 , wherein the step of determining at least one measure includes a step of determining a number of volumes that exhibit co-amplification of a particular allele sequence of the first locus and a particular allele sequence of the second locus, and wherein the step of selecting a haplotype is based on the number of volumes.
6 . The method of claim 1 wherein the step of determining a number of volumes includes a step of determining a first number of volumes and a second number of volumes that exhibit respective co-amplification of a first allele sequence or a second allele sequence of the first locus with a particular allele sequence of the second locus, and wherein the step of selecting a haplotype is based on first and second numbers of volumes.
7 . The method of claim 1 , wherein the step of partitioning includes a step of forming an emulsion in which the volumes are droplets.
8 . A method of haplotype analysis, the method comprising:
partitioning an aqueous phase containing nucleic acid into a plurality of discrete volumes;
amplifying in the volumes at least one allele sequence from each of a first polymorphic locus and a second polymorphic locus contained in a target region of the nucleic acid;
collecting allele-specific amplification data for each of the loci from individual volumes;
correlating allele-specific amplification data for the first locus with allele-specific amplification data for the second locus from the same volumes; and
selecting a haplotype of the target region for the first and second loci based on the step of correlating.
9 . The method of claim 8 , wherein the step of partitioning includes a step of forming an emulsion, and wherein the step of collecting includes a step of collecting data from individual droplets of the emulsion.
10 . The method of claim 9 , wherein the step of forming an emulsion includes a step of passing the aqueous phase through an orifice such that monodisperse droplets of the aqueous phase are generated.
11 . The method of claim 8 , wherein the step of partitioning disposes an average of less than about one genome equivalent of the nucleic acid in each volume.
12 . The method of claim 8 , wherein the step of partitioning includes a step of forming at least about 1000 volumes.
13 . The method of claim 8 , wherein the step of partitioning includes a step of forming droplets that are about 10 to 1000 micrometers in diameter.
14 . The method of claim 8 , wherein the step of partitioning includes a step of partitioning an aqueous phase including optically distinguishable fluorescent probes capable of hybridizing specifically to each allele sequence amplified.
15 . The method of claim 8 , wherein the step of amplifying includes a step of amplifying a pair of different allele sequences from the first locus, and wherein the step of collecting data includes a step of collecting data that distinguishes amplification of each allele sequence of the pair in individual droplets.
16 . The method of claim 8 , wherein the step of correlating includes a step of separately correlating allele-specific amplification data for each allele sequence of the first locus with allele-specific amplification data for the allele sequence of the second locus.
17 . The method of claim 8 , wherein the step of selecting a haplotype is based on which allele-specific amplification data for the first locus exhibits a higher correlation with such allele-specific amplification data for the second locus.
18 . The method of claim 8 , further comprising a step of applying a threshold to the allele-specific amplification data to convert it to binary form, wherein the step of correlating is performed with the binary form of the data.
19 . The method of claim 8 , wherein the step of correlating includes a step of determining a number of volumes exhibiting co-amplification of a particular allele sequence from both loci.
20 . A method of haplotype analysis, the method comprising:
partitioning an aqueous phase including nucleic acid into a plurality of discrete volumes;
amplifying in the volumes an allele sequence from each of a first polymorphic locus and a second polymorphic locus in the nucleic acid;
collecting allele-specific amplification data for each allele sequence in individual volumes; and
selecting a haplotype of the nucleic acid based at least in part on whether the amplification data indicates a negative or a positive correlation for amplification of the allele sequences in the same volumes.