Compositions and Methods for Labeling Target Nucleic Acid Molecules

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)