Patent ID: 11964997
Assignee: REALSEQ BIOSCIENCES, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method for preparing a sequencing library for a plurality of sample polynucleotides in a sample, comprising:
a) ligating a plurality of combo adapters to the plurality of sample polynucleotides to produce a plurality of adapter-polynucleotide ligation products, wherein said plurality of combo adapters comprises a 5′-proximal segment, a 3′-proximal segment, and a template-deficient segment linking the 5′ proximal segment and the 3′ proximal segment that restricts primer extension by a polymerase over the template-deficient segment, and wherein at least one of the 5′ proximal segment or 3′ proximal segment comprises a sequencing adapter, wherein the combo adapter is single-stranded;
b) circularizing said plurality of adapter-polynucleotide ligation products by ligating their 5′ ends to their 3′ ends to produce a plurality of circularized adapter-polynucleotide ligation products;
c) hybridizing a first primer comprising a sequence at least partially complementary to the 5′-proximal segment of said combo adapter, to said circularized adapter-polynucleotide ligation products;
d) extending said first primer with a polymerase to produce a plurality of monomeric nucleic acids, wherein said template-deficient segment allows one cycle of primer extension on said circularized adapter-polynucleotide ligation product, thereby preventing rolling-circle amplification, wherein each of the monomeric nucleic acids is complementary to: at least one sample polynucleotide of the plurality of sample polynucleotides flanked by at least a portion of the sequencing adapter; and
e) amplifying said plurality of monomeric nucleic acids using said first primer and a second primer, wherein the sequence of the second primer is at least partially complementary to the 3′-proximal segment of said combo adapter, to produce amplicon(s) comprising the sequencing library.