|CURRY, JOHN - Affymetrix, Inc|
|KOSHINSKY, HEATHER - Affymetrix, Inc|
|Thallman, Richard - Mark|
Submitted to: U.S. Patent and Trademark Office
Publication Type: Pct (patent Cooperation Treaty)
Publication Acceptance Date: 5/12/2020
Publication Date: 5/12/2020
Citation: Curry, J.D., Koshinsky, H., Lindholm-Perry, A.K., Thallman, R.M. 2020. Methods of determining the presence or absence of a plurality of target polynucleotides in a sample. U.S. Patent and Trademark Office. Patenet #10/648,030 B2.
Interpretive Summary: This invention makes possible the high-throughput, low cost genotyping of large numbers of DNA samples for targeted single-nucleotide polymorphisms (SNP) through next generation sequencing (NGS) technology. It works by applying "DNA barcodes" to the individual DNA samples so that the sample identification of each sequence read can be determined based on the barcode sequence in a specified location within the sequence of each read. Therefore, many samples can be pooled prior to constructing the final fragment library for sequencing (this is commonly done, but generally involves a library construction step costing >$200/sample and is generally limited to less than 100 individuals). This method uses the polymerase chain reaction (PCR) to add the barcodes to the DNA fragments in steps that cost < $1/sample, making it feasible to pool thousands or tens of thousands of samples within one NGS lane. Two independent barcodes can be combined at different locations within the DNA fragment to allow labelling many samples using relatively few oligonucleotide primers (e.g., 96 x 384 = 36,864 samples identified by 96 + 384 = 480 primers). In contrast with other applications of barcoding samples for NGS, the method produces sequencing reads only of specifically targeted SNP loci; therefore, sequencing reads are not wasted on uninformative loci and the number of samples (individuals) that can be evaluated in one NGS lane is much greater (tens of thousands). In the original proof-of-concept application (developed by ARS), the target SNP fragments were selected by highly multiplexed (96-fold) PCR. In the current implementation (developed by Eureka), the target SNP are selected by highly multiplexed (currently ~60-fold) ligation-mediated PCR, in which the multiplexing occurs at the ligation step, which may potentially be multiplexed to much higher fold levels than the PCR. The method uses the plateau effect of PCR to normalize the number of sequence reads with respect to both individuals and SNP loci.
Technical Abstract: The disclosure provides a method of determining the presence or absence of a plurality of target polynucleotides in a sample including combining a sample that may comprise one or more of the plurality of target polynucleotides with a plurality of sets of complementary polynucleotides; incubating the polynucleotides under conditions that allow hybridization of complementary sequences; joining the first and second complementary polynucleotides to form one or more product polynucleotides; and 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.