Patent ID: 11859171
Assignee: AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
1. A method comprising:
(i) assigning a specific barcode sequence to template DNA molecules in a sample, wherein the assigning comprises a polymerase chain reaction (PCR) amplification reaction using primers comprising universal sequences at the 5′ ends of the primers, wherein for a template DNA molecule of the template DNA molecules, a primer of the primers additionally comprises a barcode, thereby generating barcode-tagged molecules;
(ii) clonally amplifying the barcode-tagged molecules, thereby generating amplified barcode-tagged molecules;
(iii) protecting barcode-proximal ends of the amplified barcode-tagged molecules, thereby generating protected barcode-tagged molecules;
(iv) fragmenting the protected barcode-tagged molecules to obtain barcode-containing fragments, wherein the fragmenting comprises subjecting the protected barcode-tagged molecules to unidirectional deletion from a barcode-distal end of the protected barcode-tagged molecules;
(v) circularizing the barcode-containing fragments by intramolecular ligation, thereby juxtaposing a barcode-tagged end of the barcode-containing fragments with a barcode-distal end of the barcode-containing fragments and generating a sequencing library of overlapping fragments;
(vi) obtaining demultiplexed reads from the sequencing library, wherein demultiplexed reads from the sequencing library comprise sequences of the barcode and the barcode-distal ends of the barcode-containing fragments; and
(vii) assembling the demultiplexed reads to obtain extended sequence reads for the template DNA molecules.