Scalable Characterization of Nucleic Acids by Parallel Sequencing

Abstract

A method for detecting the presence or absence of a target polynucleotide in a sample is described.

1 .- 33 . (canceled) 
     
     
         34 . A method of determining the presence or absence of a plurality of target polynucleotides in a sample, the method comprising the steps of:
 a) combining   i) a sample that may comprise one or more of said plurality of target polynucleotides, each of said target polynucleotides comprising a first target sequence and a second target sequence;   ii) a plurality of first complementary polynucleotides, each first complementary polynucleotide comprising a different first complementary sequence, wherein each first complementary sequence is complementary to a first target sequence of one of the plurality of target polynucleotides; and   iii) a plurality of second complementary polynucleotides, each comprising a different second complementary sequence, wherein each second complementary sequence is complementary to a second target sequence of the target polynucleotide, wherein when a plurality of first and second complementary sequences hybridize with a plurality of target sequences they have a Tm within about 10° C. of each other;   b) incubating the plurality of first and second complementary polynucleotides with the plurality of target polynucleotides under conditions that allow hybridization of complementary sequences, wherein when a first complementary polynucleotide and a second complementary polynucleotide are hybridized to the same target polynucleotide, the first complementary polynucleotide is joined to the second complementary polynucleotide to form one or more product polynucleotides; and   c) detecting the presence or absence of one or more product polynucleotides to determine the presence or absence of one or more of the plurality of target polynucleotides in the sample.   
     
     
         35 . The method of  claim 34 , wherein the first complementary polynucleotide is covalently or non-covalently joined to the second complementary polynucleotide when the first and second complementary polynucleotides are hybridized to the same target polynucleotide, wherein said joining occurs by chemical reaction, enzymatic reaction or non-enzymatic reaction. 
     
     
         36 . The method of  claim 35 , wherein the enzymatic reaction is ligation. 
     
     
         37 . The method of  claim 34 , further comprising an enriching step before the detecting step, wherein each of the first and second complementary sequences comprises a sequence complementary to one or more amplification primers, and the enriching step comprises amplification of the one or more product polynucleotides. 
     
     
         38 . The method of  claim 34 , further comprising an enriching step before the detecting step, wherein the enriching step comprises selecting the one or more product polynucleotides or removal or destruction of one or more non-product polynucleotides. 
     
     
         39 . The method of  claim 34 , wherein:
 i) each of the first target polynucleotides further comprises a polymorphic nucleotide or nucleotide sequence positioned between the first and second target sequence;   ii) each of the first complementary polynucleotides further comprises a first polymorphic nucleotide or nucleotide sequence, wherein the polymorphic nucleotide is complementary to the polymorphic nucleotide or nucleotide sequence of the first target polynucleotide; and further comprising an enriching step before the determining step, the enriching step comprising increasing the concentration of at least one product polynucleotide compared to a non-product polynucleotides.   
     
     
         40 . The method of  claim 34 , wherein the first complementary polynucleotide includes a polymorphism-specific tag or an allele-specific tag. 
     
     
         41 . The method of  claim 40 , wherein each of the first and/or second complementary polynucleotides comprises a locus-specific tag sequence, said locus-specific tag sequence corresponding to the identity of the locus and/or each of the first and/or second target sequences. 
     
     
         42 . The method of  claim 40 , wherein each of the plurality of first complementary polynucleotides comprises a polymorphism-specific tag, an allele-specific tag, a nucleotide-specific tag, a locus-specific tag, or combinations thereof. 
     
     
         43 . The method of  claim 37 , wherein at least one of the sequences complementary to an amplification primer comprises a sample-specific tag sequence, wherein said sample-specific tag is unique to the identity of the sample. 
     
     
         44 . The method of  claim 34 , wherein the inosine is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bases from the terminus of the first and/or second complementary polynucleotide. 
     
     
         45 . The method of  claim 34 , wherein the determining step is accomplished by sequencing all or part of one or more of the product polynucleotides or a complement thereof, wherein said one or more product polynucleotides are enriched prior to sequencing. 
     
     
         46 . The method of  claim 45 , further comprising comparing the quantity of read events for the first polynucleotide from a specific locus, and the quantity of read events for a second polynucleotide from the same specific locus to determine the presence or absence of heterozygosity or homozygosity. 
     
     
         47 . The method of  claim 37 , wherein the amplifying step comprises uracil incorporation. 
     
     
         48 . The method of  claim 34 , 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 10 to 10,000 samples or individuals. 
     
     
         49 . The method of  claim 34 , wherein one or more first and/or second complementary polynucleotides comprises inosine. 
     
     
         50 . The method of  claim 34 , wherein a first complementary polynucleotide and a second complementary polynucleotide are hybridized to the same target polynucleotide immediately adjacent one another. 
     
     
         51 . The method of  claim 40 , wherein said allele-specific tag is within the first complementary sequence.