Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation

Abstract

The present invention provides methods, compositions and kits for targeted nucleic acid sequence enrichment in a nucleic acid sample and for high efficiency nucleic acid library generation for next generation sequencing (NGS). Specifically, the methods, compositions and kits provided herein are useful for the production and capture of amplification-ready, target-specific and strand-specific regions of interest from nucleic acid samples containing complex DNA.

1 .- 50 . (canceled) 
     
     
         51 . A method for enriching a target nucleic acid, the method comprising:
 annealing an oligonucleotide to a sequence of interest in a single-stranded nucleic acid, wherein the single-stranded nucleic acid comprises a first adapter sequence at a 5′ end and the oligonucleotide comprises a 5′ tail portion that is non-complementary to the sequence of interest and comprises a second adapter sequence;   extending the oligonucleotide with a polymerase, to yield an extension product comprising the first adapter sequence at a first end and the second adapter sequence at a second end; and   amplifying the extension product using a first primer that is complementary to a sequence in the first adapter sequence and a second primer that is complementary to a sequence in the second adapter sequence.   
     
     
         52 . The method of  claim 51 , further comprising sequencing the amplified extension products on a massively parallel sequencing platform. 
     
     
         53 . The method of  claim 51 , wherein the first adapter sequence and/or the second adapter sequence comprise(s) a barcode sequence. 
     
     
         54 . The method of  claim 53 , wherein the first adapter sequence comprises a barcode sequence and a universal priming site 5′ of the barcode. 
     
     
         55 . The method of  claim 54 , wherein the first primer is a universal primer. 
     
     
         56 . The method of  claim 55 , wherein the second adapter sequence comprises a barcode sequence that is different from the barcode sequence in the first adapter sequence. 
     
     
         57 . The method of  claim 56 , wherein the amplified products comprise a 3′ end with a sequence complementary to a sequence on a surface. 
     
     
         58 . The method of  claim 57 , further comprising annealing the amplified products to the surface using the 3′ end with the sequence complementary to the sequence on the surface. 
     
     
         59 . The method of  claim 58 , wherein the surface is a surface of a flow cell. 
     
     
         60 . The method of  claim 59 , wherein the sequence of interest comprises cDNA. 
     
     
         61 . A method for enriching a target nucleic acid, the method comprising:
 annealing an oligonucleotide to a sequence of interest in a single-stranded nucleic acid, wherein the single-stranded nucleic acid comprises a first adapter sequence at a 5′ end and a second adapter sequence at a 3′ end and the oligonucleotide comprises a 5′ tail portion that is non-complementary to the sequence of interest and comprises a third adapter sequence;   extending the oligonucleotide with a polymerase, thereby generating an extension product comprising the first adapter sequence at a first end and the third adapter sequence at a second end; and   amplifying the extension product using a first primer that is complementary to a sequence in the first adapter and a second primer that is complementary to a sequence in the third adapter sequence.   
     
     
         62 . The method of  claim 61 , further comprising sequencing the amplified extension products on a massively parallel sequencing platform. 
     
     
         63 . The method of  claim 61 , wherein the first adapter sequence and/or the third adapter sequence comprise(s) a barcode sequence. 
     
     
         64 . The method of  claim 63 , wherein the first adapter sequence comprises a barcode sequence and a universal priming site 5′ of the barcode. 
     
     
         65 . The method of  claim 64 , wherein the first primer is a universal primer. 
     
     
         66 . The method of  claim 65 , wherein the third adapter sequence comprises a barcode sequence that is different from the barcode sequence in the first adapter sequence. 
     
     
         67 . The method of  claim 66 , wherein the amplified products comprise a 3′ end with a sequence complementary to a sequence on a surface. 
     
     
         68 . The method of  claim 67 , further comprising annealing the amplified products to the surface using the 3′ end with the sequence complementary to the sequence on the surface. 
     
     
         69 . The method of  claim 68 , wherein the surface is a surface of a flow cell. 
     
     
         70 . The method of  claim 69 , wherein the sequence of interest comprises cDNA.