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

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.

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.