Compositions and methods for directional nucleic acid amplification and sequencing

Abstract

The invention provides methods and compositions, including kits, for directional nucleic acid amplification and sequencing. The invention further provides methods and compositions for the construction of directional cDNA libraries.

What is claimed is: 
     
         1 . A method for construction of a directional cDNA library, the method comprising:
 a. reverse transcribing an RNA in an RNA sample to generate a first strand cDNA;   b. generating a second strand cDNA from the first strand cDNA, wherein the generating the second strand cDNA comprises incorporating a modified dNTP during the generating the second strand cDNA, thereby generating a double-stranded cDNA;   c. performing end repair on the double-stranded cDNA;   d. ligating a stem-loop adaptor to the double-stranded cDNA, wherein the stem-loop adaptor comprises a modified dNTP, thereby generating a ligation product;   e. selectively cleaving the second strand cDNA and the portion of the stem-loop adaptor that has the modified dNTP in the ligation product; and   f. amplifying the first strand resulting from step (e), thereby generating a directional cDNA library comprising the first strand cDNA.   
     
     
         2 . The method of  claim 1 , wherein the RNA in the RNA sample comprises RNA fragments. 
     
     
         3 . The method of  claim 1 , wherein the reverse transcribing the RNA sample is performed using a primer comprising a random sequence. 
     
     
         4 . The method of  claim 3 , wherein the primer is a random hexamer primer. 
     
     
         5 . The method of  claim 1 , wherein the modified dNTP comprises dUTP. 
     
     
         6 . The method of  claim 1 , wherein the method further comprises purifying the double-stranded cDNA prior to step (c). 
     
     
         7 . The method of  claim 1 , wherein the end repair comprises adding a dA nucleotide to the double-stranded cDNA. 
     
     
         8 . The method of  claim 1 , wherein the stem loop adaptor comprises a dT overhang. 
     
     
         9 . The method of  claim 1 , wherein the amplifying the first strand cDNA is performed using one or more tailed primers. 
     
     
         10 . The method of  claim 1 , wherein the amplified product of step (f) comprises a barcode sequence. 
     
     
         11 . The method of  claim 1 , wherein the amplification comprises polymerase chain reaction (PCR). 
     
     
         12 . The method of  claim 1 , further comprising sequencing the amplified product of step (f). 
     
     
         13 . The method of  claim 12 , wherein the sequencing comprises massively parallel sequencing. 
     
     
         14 . The method of  claim 1 , wherein step (e) comprises selectively cleaving a base portion of the modified dNTP, thereby forming an a basic site. 
     
     
         15 . The method of  claim 14 , wherein the selectively cleaving the base portion of the modified dNTP comprises use of an enzyme. 
     
     
         16 . The method of  claim 15 , wherein the enzyme comprises a glycosylase. 
     
     
         17 . The method of  claim 16 , wherein the glycosylase is uracil DNA glycosylase (UDG). 
     
     
         18 . The method of  claim 14 , wherein step (e) comprises cleaving a phosphodiester backbone at the a basic site. 
     
     
         19 . The method of  claim 18 , wherein the cleaving the phosphodiester backbone at the a basic site comprises use of an enzyme. 
     
     
         20 . The method of  claim 19 , wherein the enzyme comprises an endonuclease. 
     
     
         21 . The method of  claim 1 , wherein step (e) comprises use of a glycosylase and an endonuclease. 
     
     
         22 . The method of  claim 1 , wherein the reverse transcribing is performed using a reverse transcriptase, wherein the reverse transcriptase is moloney murine leukemia virus (M-MLV) reverse transcriptase. 
     
     
         23 . The method of  claim 1 , further comprising purifying the ligation product of step (d). 
     
     
         24 . The method of  claim 1 , further comprising depleting non-desired RNA from the RNA sample. 
     
     
         25 . The method of  claim 24 , wherein the non-desired RNA comprises ribosomal RNA (rRNA). 
     
     
         26 . The method of  claim 1 , wherein the RNA in the RNA sample comprises poly(A)+ RNA. 
     
     
         27 . The method of  claim 13 , wherein the massively parallel sequencing comprises bridge amplification. 
     
     
         28 . The method of  claim 13 , wherein the massively parallel sequencing comprises use of labeled reversible terminators.