Compositions and methods for labeling target nucleic acid molecules

1. A method, comprising:

(a) hybridizing a primer to 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, wherein the primer hybridizes to the first adaptor;

(b) extending the 3′ end of the hybridized primer to produce primer extension products;

(c) hybridizing the primer extension products with synthetic oligonucleotides, wherein:

(i) the synthetic oligonucleotides comprise, in 3′ to 5′ order, a non-extendable 3′ end, a hybridization sequence, and a variable barcode sequence; and

(ii) the hybridization sequence hybridizes to a 3′ terminal region of the sequence complementary to the second adapter; and

(d) incubating the hybridized primer extension products of step (b) with a polymerase such that the polymerase extends the 3′ end of the primer extension products to form a further extended primer extension product.

2. The method according to claim 1 , further comprising the step of denaturing the primer extension products from the polynucleotide molecules prior to step (c).

3. The method according to claim 1 , wherein the 5′ terminus of the primer is protected from exonuclease digestion and the method further comprises incubating the primer extension products and the polynucleotide molecules with a 5′ exonuclease to remove a 5′ terminal sequence from the polynucleotide molecules prior to step (c).

4. The method according to claim 3 , wherein the second adaptor contains modified nucleotides that allow only partial digestion by a 5′ exonuclease.

5. The method according to claim 4 , wherein the modified nucleotides are internal phosphorothioate-containing nucleotides.

6. The method according to claim 4 , wherein the 5′ exonuclease removes 10-20 bases from an end of the second adaptor leaving a single strand overhang.

7. The method according to claim 1 , further comprising removing a 5′ terminal sequence from the polynucleotide molecules after step (b) and prior to step (c), leaving a 3′ overhang.

8. The method according to claim 1 , further comprising amplifying the further extended primer extension products produced in step (d).

9. The method of claim 1 , wherein extension of the primer extension products in step (d) adds the complement of the variable barcode sequence onto the primer extension products.