System and method for nucleic acid library preparation via template switching mechanism

Abstract

The disclosure provides a composition comprising a double-stranded deoxyribonucleic acid (dsDNA) sequence comprising from 5′ to 3′, a sequence comprising a first adaptor sequence, a template sequence, and a second adaptor sequence, wherein the second adaptor sequence comprises a hybridization site for a template switching oligonucleotide (TSO). The disclosure provides methods for making the compositions of the disclosure using a template switching mechanism to add non-templated basepairs to the ends of a DNA molecule, hybridize a TSO to the non-templated basepairs, and then extend the sequence complementary to the TSO to add an adaptor.

1 - 57 . (canceled) 
     
     
         58 . A method of making a composition, wherein the composition comprises a double-stranded deoxyribonucleic acid (dsDNA) sequence comprising: (i) a sense strand comprising, from 5′ to 3′, a sequence comprising a first adaptor sequence, a template sequence, and a second adaptor sequence, and (ii) an anti-sense strand comprising a sequence comprising a sequence complementary to the sequence of the sense strand (i), wherein the second adaptor sequence comprises a hybridization site for a template switching oligonucleotide (TSO); wherein the method comprises the following steps:
 (a) contacting a template sequence and a polymerase under conditions sufficient to allow for terminal transferase activity, to produce an intermediate double-stranded deoxyribonucleic acid (dsDNA) sequence, wherein the intermediate dsDNA comprises the adaptor sequence at the 3′ end of the sense strand and the antisense strand; 
 (b) contacting the intermediate dsDNA, the polymerase and at least one template switching oligonucleotide (TSO) under conditions sufficient to allow for DNA-dependent DNA polymerase activity, to produce the composition. 
 
     
     
         59 . The method of  claim 58 , wherein the adaptor sequence at the 3′ end of the sense strand and the antisense strand comprises a poly(G) sequence or a poly(C) sequence. 
     
     
         60 . The method of  claim 58 , wherein the adaptor sequence at the 3′ end of the sense strand and the antisense strand comprises a poly(G) sequence. 
     
     
         61 . The method of  claim 58 , wherein the conditions sufficient to allow for terminal transferase activity or DNA-dependent DNA polymerase activity comprise a plurality of deoxynucleotides (dNTPs). 
     
     
         62 . The method of  claim 58 , wherein the conditions sufficient to allow for terminal transferase activity comprise a plurality of dCTPs, a plurality of dGTPs, or a combination thereof. 
     
     
         63 . (canceled) 
     
     
         64 . The method of  claim 58 , wherein the conditions sufficient to allow for DNA-dependent DNA polymerase activity comprise an incubation at temperatures from between 27° C. and 50° C., inclusive of the endpoints, for a period of between 2 and 20 minutes. 
     
     
         65 . (canceled) 
     
     
         66 . (canceled) 
     
     
         67 . The method of  claim 58 , wherein the polymerase comprises a reverse transcriptase. 
     
     
         68 . The method of  claim 67 , wherein the reverse transcriptase is a Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV) reverse transcriptase. 
     
     
         69 . The method of  claim 68 , wherein the conditions sufficient to allow for DNA-dependent DNA polymerase activity comprise the co-factor Mg 2+ . 
     
     
         70 . The method of  claim 69 , wherein the co-factor Mg 2+  is present at a concentration of between 20 and 40 mM. 
     
     
         71 . (canceled) 
     
     
         72 . The method of  claim 58 , wherein a concentration of template DNA in (a) is between 0.1 ng and 100 ng, inclusive of the endpoints. 
     
     
         73 - 170 . (canceled) 
     
     
         171 . The method of  claim 61 , wherein the conditions sufficient to allow for terminal transferase activity or DNA-dependent DNA polymerase activity comprise three dNTPs.