Deducing Exon Connectivity by RNA-Templated DNA Ligation/Sequencing

Abstract

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.

We claim: 
     
         1 . A composition comprising a ligamer comprising a plurality of complementarity regions, wherein at least two of said plurality of complementary regions are separated by a central region, wherein said central region has an exon-specific barcode sequence comprising an identifier code for a specific exon, wherein said at least two of said plurality of complementarity regions are complementary to the flanking regions of said specific exon. 
     
     
         2 . The composition of  claim 1 , wherein each of said complementarity regions comprise a different nucleotide sequence. 
     
     
         3 . The composition of  claim 1 , wherein said composition further comprises a template oligonucleotide comprising a plurality of hybridization sites. 
     
     
         4 . The composition of  claim 3 , wherein said template oligonucleotide hybridization sites are complementary to said complementarity regions. 
     
     
         5 . The composition of  claim 3 , wherein said template oligonucleotide comprises an mRNA oligonucleotide. 
     
     
         6 . The composition of  claim 5 , wherein said mRNA oligonucleotide comprises said exon. 
     
     
         7 . The composition of  claim 6 , wherein at least one of said hybridization sites comprises a flanking region of said exon. 
     
     
         8 . The composition of  claim 6 , wherein said barcode sequence is non-complementary to said exon. 
     
     
         9 . The composition of  claim 1 , wherein said ligamer comprises between approximately 5-5,000 nt. 
     
     
         10 . The composition of  claim 1 , wherein said ligamer comprises between approximately 10-3,000 nt. 
     
     
         11 . The composition of  claim 1 , wherein said ligamer comprises between approximately 20-1,000 nt.