Deducing exon connectivity by RNA-templated DNA ligation/sequencing
A technology is described that is capable of generating high-throughput sequencing (HTS) read length DNA products to accurately and reliably provide exon connectivity information for alternatively spliced isoforms. The method is not limited by the initial size of the isoform as the technology removes the template oligonucleotide sequence and a newly formed full length ligated product provides an HTS-compatible read length sequence that comprises information that corresponds to the consecutive order of the exons in the original template oligonucleotide.
1. A method, comprising:
a) providing:
i) a plurality of ligamer subsets, wherein each of said ligamer subsets comprise a plurality of different complementary regions, wherein at least two of said plurality of different complementarity regions are separated by a central region, wherein said central region has a different exon-specific barcode sequence, wherein each said different barcode sequence comprises a different identifier code for a different specific exon, wherein said at least two of said different complementarity regions are complementary to the flanking regions of said different specific exon; and
ii) a template oligonucleotide, wherein said oligonucleotide comprises a plurality of hybridization sites;
b) mixing said plurality of ligamers with said template oligonucleotide under conditions such that said complementarity regions bind to said hybridization sites;
c) ligating said ligamers to create a full length ligated product (FLLP);
d) amplifying said FLLP under conditions to create a DNA product; and
e) identifying the consecutive order of said barcodes within said DNA product.
2. The method of claim 1 , wherein said template oligonucleotide comprises an mRNA oligonucleotide.
3. The method of claim 2 , wherein said mRNA oligonucleotide comprises a plurality of said different exons.
4. The method of claim 1 , wherein each of said barcodes corresponds to one of said different exons.
5. The method of claim 1 , wherein said identifying comprises nucleotide sequencing.
6. The method of claim 5 , wherein said sequencing comprises high throughput sequencing.
7. The method of claim 1 , wherein said template oligonucleotide ranges between approximately 100-100,000 nt.
8. The method of claim 1 , wherein said ligamer ranges between approximately 5-2,000 nt.
9. The method of claim 1 , wherein said DNA product ranges between approximately 100-1000 nt.
10. A kit, comprising:
a) a first container comprising a plurality of ligamer subsets, wherein each of said ligamer subsets comprise a plurality of different complementarity regions, wherein at least two of said plurality of different complementarity regions are separated by a central region, wherein said central region has a different exon-specific barcode sequence, wherein each different exon-specific barcode sequence comprises a different identifier code for a different specific exon, wherein said at least two of said different complementarity regions are complementary to the flanking regions of said different specific exon; and
b) a second container comprising a template oligonucleotide, wherein said oligonucleotide comprises a plurality of hybridization sites;
c) a third container comprising buffers and reagents for hybridizing said ligamers and template oligonucleotide;
d) a fourth container comprises a ligase and associated buffers and reagents; and,
e) a set of instructions for;
i) hybridizing said ligamers and said template oligonucleotide;
ii) ligating said ligamers to create a full length ligated product.