Methods for Adding Adapters to Nucleic Acids and Compositions for Practicing the Same

Abstract

Provided are methods of adding adapters to nucleic acids. The methods include combining in a reaction mixture a template nucleic acid, a template switch oligonucleotide, a polymerase, and dNTPs. The reaction mixture components are combined under conditions sufficient to produce a product nucleic acid that includes the template nucleic acid and the template switch oligonucleotide each hybridized to adjacent regions of a single product nucleic acid including a region polymerized from the dNTPs by the polymerase. The methods further include attaching sequencing platform adapter constructs to ends of the product nucleic acid or a derivative thereof. Aspects of the invention further include compositions and kits.

1 - 39 . (canceled) 
     
     
         40 . A composition comprising:
 a template nucleic acid;   a template switch oligonucleotide; and   a sequencing platform adapter construct,   wherein the template nucleic acid and template switch oligonucleotide are each hybridized to adjacent regions of a nucleic acid strand.   
     
     
         41 . The composition of  claim 40 , wherein the sequencing platform adapter construct comprises a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof. 
     
     
         42 . The composition of  claim 40 , wherein the template switch oligonucleotide includes a 3′ hybridization domain. 
     
     
         43 . The composition of  claim 40 , wherein the composition is present in a reaction tube. 
     
     
         44 - 50 . (canceled) 
     
     
         51 . The composition of  claim 40 , wherein the sequencing platform adaptor construct is at a 5′ end of the nucleic acid strand. 
     
     
         52 . The composition of  claim 40 , wherein the template switch oligonucleotide comprises the sequencing platform adaptor construct. 
     
     
         53 . The composition of  claim 42 , wherein the 3′ hybridization domain comprises a homo-trinucleotide. 
     
     
         54 . The composition of  claim 42 , wherein the 3′ hybridization domain comprises a hetero-trinucleotide. 
     
     
         55 . The composition of  claim 40 , wherein the template switch oligonucleotide comprises a modification that prevents a polymerase from switching from the template switch oligonucleotide to a different template nucleic acid. 
     
     
         56 . The composition of  claim 55 , wherein the modification is selected from the group consisting of: an abasic lesion, a nucleotide adduct, an iso-nucleotide base, and combinations thereof. 
     
     
         57 . The composition of  claim 40 , wherein the template switch oligonucleotide comprises one or more modifications. 
     
     
         58 . The composition of  claim 40 , wherein the one or more modifications comprise nucleotide analogues. 
     
     
         59 . The composition of  claim 40 , wherein the template switch oligonucleotide comprises a linkage modification, an end modification, or both. 
     
     
         60 . The composition of  claim 40 , wherein the template switch oligonucleotide comprises one or more exo-sample nucleotides. 
     
     
         61 . The composition of  claim 40 , wherein the template nucleic acid is a template deoxyribonucleic acid (DNA). 
     
     
         62 . The composition of  claim 40 , wherein the template nucleic acid is a template ribonucleic acid (RNA). 
     
     
         63 . A double stranded nucleic acid comprising a template derived region flanked by first and second sequencing platform adaptor constructs, wherein the double stranded nucleic acid further comprises a template switch oligonucleotide domain positioned between the first sequencing platform adaptor construct and the template derived region. 
     
     
         64 . The double stranded nucleic acid of  claim 63 , wherein the template is a template deoxyribonucleic acid (DNA). 
     
     
         65 . The double stranded nucleic acid of  claim 63 , wherein the template a template deoxyribonucleic acid (RNA). 
     
     
         66 . The double stranded nucleic acid of  claim 63 , wherein the first and second sequencing platform adapter constructs each comprises a nucleic acid domain selected from the group consisting of: a domain that specifically binds to a surface-attached sequencing platform oligonucleotide, a sequencing primer binding domain, a barcode domain, a barcode sequencing primer binding domain, a molecular identification domain, and combinations thereof. 
     
     
         67 . The double stranded nucleic acid of  claim 63 , wherein the template switch oligonucleotide domain comprises a homo-trinucleotide. 
     
     
         68 . The double stranded nucleic acid of  claim 63 , wherein the template switch oligonucleotide domain comprises a hetero-trinucleotide.