Methods of determining the presence or absence of a plurality of target polynucleotides in a sample

1. A method of determining the allele frequency of one or more target polynucleotides of a plurality of target polynucleotides in a sample, the method comprising:

a) combining a sample comprising one or more of the plurality of target polynucleotides with a plurality of sets of complementary polynucleotides, said one or more of the plurality of target polynucleotides suspected to have a site of a single nucleotide polymorphism (SNP) containing a polymorphic nucleotide;

wherein each of the plurality of sets of complementary polynucleotides comprises:

(i) a first complementary polynucleotide comprising a complementary sequence to a first target sequence of a target polynucleotide, a first allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a first polymorphic nucleotide,

(ii) a second complementary polynucleotide comprising a complementary sequence to the first target sequence, a second allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a second polymorphic nucleotide; and

(iii) a third complementary polynucleotide comprising a complementary sequence to a second target sequence of the target polynucleotide,

wherein the allele-specific barcodes are not complementary to the first target sequence, and

wherein the allele-specific barcodes allow up to 4 sequencing errors while still allowing identification of the specific allele ;

b) incubating the plurality of sets of complementary polynucleotides with the plurality of target polynucleotides under conditions that allow hybridization of complementary sequences;

c) joining a pair of the first and third complementary polynucleotides and/or joining a pair of the second and third complementary polynucleotides by a ligation reaction when both complementary polynucleotides of each pair are hybridized to the target polynucleotide to form one or more product polynucleotides; and

d) detecting the presence of one or more product polynucleotides to determine the allele frequency of the target polynucleotide, wherein the detecting step is accomplished by sequencing all or part of one or more of the product polynucleotides or a complement thereof;

e) dividing the number of sequence reads of one polymorphism by the total number of sequence reads for the target polynucleotide; and

f) determining the target polynucleotide is homozygous if the frequency of one allele is 0.7 or greater, or 0.3 or lower, and determining the target polynucleotide is heterozygous if the frequency of one allele is between 0.3 and 0.7.

2. The method of claim 1 , further comprising an enriching step before the detecting step, wherein each of the complementary polynucleotides comprises a sequence complementary to an amplification primer, and the enriching step comprises amplification of the one or more product polynucleotides.

3. The method of claim 2 , wherein the enriching step comprises selecting the one or more product polynucleotides or removal or destruction of the one or more non-product polynucleotides.

4. The method of claim 1 , wherein the allele-specific barcode in the first complementary polynucleotide is a locus-allele- specific barcode that identifies both the locus and the allele.

5. The method of claim 1 , wherein the allele-specific barcode in the second complementary polynucleotide is a locus-allele- specific barcode that identifies both the locus and the allele.

6. The method of claim 2 , wherein at least one of the sequences complementary to an amplification primer comprises a sample-specific tag sequence corresponding to the identity of the sample.

7. The method of claim 2 , wherein the enriching step comprises uracil incorporation.

8. The method of claim 1 , wherein at least steps a-c are conducted from, (i) 2 to 10 times on 2 to 10 samples or individuals, (ii) 10 to 100 times on 10 to 100 samples or individuals, (iii) 10 to 1000 times on 10 to 1000 samples or individuals, or (iv) 10 to 10,000 times on 10to 10,000 samples or individuals.

9. The method of claim 1 , wherein when a plurality of first, second, and third complementary polynucleotides hybridize with a plurality of target polynucleotides, they have a Tm within about 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. of each other.

10. The method of claim 1 , wherein the complementary polynucleotides in each pair are hybridized to the same target polynucleotide immediately adjacent one another.

11. A method of determining the allele frequency of one or more target polynucleotides of a plurality of target polynucleotides in a sample, the method comprising:

a) combining a sample comprising one or more of the plurality of target polynucleotides with a plurality of sets of complementary polynucleotides, said one or more of the plurality of target polynucleotides suspected to have a site of a single nucleotide polymorphism (SNP) containing a polymorphic nucleotide;

wherein each of the plurality of sets of complementary polynucleotides comprises:

(i) a first complementary polynucleotide comprising a complementary sequence to a first target sequence of a target polynucleotide, a first locus-allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a first polymorphic nucleotide;

(ii) a second complementary polynucleotide comprising a complementary sequence to the first target sequence, a second locus-allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a second polymorphic nucleotide; and

(iii) a third complementary polynucleotide comprising a complementary sequence to a second target sequence of the target polynucleotide,

wherein the locus-allele-specific barcodes are not complementary to the first target sequence,

wherein the locus-allele-specific barcode identifies both the locus and the allele, and

wherein at least one of the sets of complementary polynucleotides in the plurality of sets of complementary polynucleotides comprises at least one inosine within the first complementary polynucleotide, the second complementary polynucleotide and/or the third complementary polynucleotide;

b) incubating the plurality of sets of complementary polynucleotides with the plurality of target polynucleotides under conditions that allow hybridization of complementary sequences;

c) joining a pair of the first and third complementary polynucleotides and/or joining a pair of the second and third complementary polynucleotides by a ligation reaction when both complementary polynucleotides of each pair are hybridized to the target polynucleotide to form one or more product polynucleotides; and

d) detecting the presence of one or more product polynucleotides to determine the allele frequency of the target polynucleotide, wherein the detecting step is accomplished by sequencing all or part of one or more of the product polynucleotides or a complement thereof;

e)dividing the number of sequence reads of one polymorphism by the total number of sequence reads for the target polynucleotide; and

f) determining the target polynucleotide is homozygous if the frequency of one allele is 0.7 or greater, or 0.3 or lower, and determining the target polynucleotide is heterozygous if the frequency of one allele is between 0.3 and 0.7.

12. A method of determining the allele frequency of one or more target polynucleotides of a plurality of target polynucleotides in a sample, the method comprising:

a) combining a sample comprising one or more of the plurality of target polynucleotides with a plurality of sets of complementary polynucleotides, said one or more of the plurality of target polynucleotides suspected to have a site of a single nucleotide polymorphism (SNP) containing a polymorphic nucleotide;

wherein each of the plurality of sets of complementary polynucleotides comprises;

(i) a first complementary polynucleotide comprising a complementary sequence to a first target sequence of a target polynucleotide, a first allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a first polymorphic nucleotide;

(ii) a second complementary polynucleotide comprising a complementary sequence to the first target sequence, a second allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a second polymorphic nucleotide; and

(iii) a third complementary polynucleotide comprising a complementary sequence to a second target sequence of the target polynucleotide,

wherein the allele-specific barcodes are not complementary to the first target sequence and are 5to 15 nucleotides in length, and

wherein the allele-specific barcodes allow up to 4 sequencing errors while still allowing identification of the specific allele;

b) incubating the plurality of sets of complementary polynucleotides with the plurality of target polynucleotides under conditions that allow hybridization of complementary sequences;

c) joining a pair of the first and third complementary polynucleotides and/or joining a pair of the second and third complementary polynucleotides by a ligation reaction when both complementary polynucleotides of each pair are hybridized to the target polynucleotide to form one or more product polynucleotides;

d) amplifying the one or more product polynucleotides, wherein each of the first, second, and third complementary polynucleotides comprises a sequence complementary to an amplification primer bound by a primer for the amplifying, wherein at least one of the sequences complementary to an amplification primer comprises a sample-specific tag sequence corresponding to the identity of the sample; and

e) detecting the presence of one or more product polynucleotides to determine the allele frequency of the target nucleotide, wherein the detecting step is accomplished by sequencing all or part of one or more of the product polynucleotides or a complement thereof by next generation sequencing, wherein the sequencing is performed without sequencing the SNP position;

f)dividing the number of sequence reads of one polymorphism by the total number of sequence reads for the target polynucleotide; and

g) determining the target polynucleotide is homozygous if the frequency of one allele is 0.7 or greater, or 0.3 or lower, and determining the target polynucleotide is heterozygous if the frequency of one allele is between 0.3 and 0.7, wherein steps b to d are carried out in one tube.

13. The method of claim 1 , wherein the sequencing is performed by next generation sequencing.

14. The method of claim 13 , wherein the sequencing is performed without sequencing the SNP position.

15. The method of claim 11 , wherein the sequencing is performed by next generation sequencing, and wherein the sequencing is performed without sequencing the SNP position.

16. The method of claim 15 , wherein the allele-specific barcode is a locus-allele-specific barcode that identifies both the locus and the allele.

17. The method of claim 11 , wherein the first and second complementary polynucleotide comprising at least one inosine further comprises a 3′ polymorphic nucleotide and/or the third complementary polynucleotide comprising at least one inosine further comprises a 5′ polymorphic nucleotide.

18. The method of claim 11 , wherein the inosine is 2-10 bases from the 3′ end of the first complementary polynucleotide.

19. The method of claim 11 , wherein the inosine is 2-10 bases from the 5′ end of the second complementary polynucleotide.

20. The method of claim 11 , wherein the inosine is positioned within a first or second complementary polynucleotide of a set to aid in preventing or reducing joining of the first or second complementary polynucleotide to the third complementary polynucleotide of the set when the polymorphic polynucleotide of the first or second complementary polynucleotide is not complementary to a polymorphic nucleotide of the target polynucleotide.