Compositions and Methods for Labeling Target Nucleic Acid Molecules

Abstract

Provided herein are methods and compositions for labeling target nucleic acid molecules with molecular barcodes.

What is claimed is: 
     
         1 . A method, comprising:
 (a) hybridizing a library of polynucleotide molecules comprising, in 3′ to 5′ order, a first adapter, a sequence derived from genomic DNA, cDNA or RNA, and a second adapter with a sample index primer, the sample index primer comprising, in 5′ to 3′ order, a sample-specific barcode sequence and a first hybridization sequence, wherein the first hybridization sequence hybridizes to a region of the first adapter;   (b) incubating the hybridized polynucleotide molecules formed in step (a) with a polymerase such that the polymerase extends the 3′ end of the sample index primer to form primer extension products comprising, in 5′ to 3′ order, a sample-specific barcode sequence, a sequence complementary to at least a portion of the first adapter, a sequence complementary to the genomic DNA, cDNA or RNA derived sequence, and a sequence complementary to the second adapter;   (c) hybridizing the primer extension products formed in step (b) with synthetic oligonucleotides comprising, in 3′ to 5′ order, a non-extendable 3′ end, a second hybridization sequence, and a variable barcode sequence, wherein the second hybridization sequence hybridizes to a 3′ terminal region of the sequence complementary to the second adapter;   (d) incubating the hybridized primer extension products of step (c) with a polymerase such the polymerase extends the 3′ end of the primer extension products to form a further extended primer extension product; and   (e) optionally amplifying the further extended primer extension products formed in (d)   
     
     
         2 . The method according to  claim 1 , further comprising the step of denaturing the primer extension products from the polynucleotide molecules between steps (b) and (c). 
     
     
         3 . The method according to  claim 1 , further comprising removing a 5′ terminal sequence from the polynucleotide molecules of the library after step (b) and prior to step (c), leaving a 3′ overhang. 
     
     
         4 . The method of  claim 3 , wherein the 5′ terminus of the sample index primer is protected from exonuclease digestion and the 5′ terminal sequence of the polynucleotides molecules of the library is removed by an exonuclease. 
     
     
         5 . The method of  claim 3 , wherein the second adapter comprises one or more deoxyuridines, and the 5′ terminal sequence of the polynucleotides molecules of the library is removed using uracil DNA glycosylase (UDG) and DNA endonuclease VIII. 
     
     
         6 . The method according to  claim 1 , wherein step (d) further comprises amplifying by PCR the further extended primer extension product. 
     
     
         7 . The method according to  claim 6 , further comprising the step of performing a sequencing process on the amplification products. 
     
     
         8 . The method, comprising performing the method of  claim 1  to label a library of polynucleotide molecules with unique variable sequences; amplifying the library of polynucleotide molecules; and sequencing the unique variable sequence. 
     
     
         9 .- 35 . (canceled)