| CPC C12N 15/1065 (2013.01) [C12Q 1/6869 (2013.01)] | 25 Claims |
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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, and wherein individual primers additionally comprise barcodes, thereby generating barcode-tagged molecules;
(ii) clonally amplifying the barcode-tagged molecules, thereby generating amplified barcode-tagged molecules;
(iii) randomly fragmenting the amplified barcode-tagged molecules using a mechanical method or an enzymatic method, thereby obtaining barcode-containing fragments with barcode-tagged ends and barcode-distal ends;
(iv) circularizing the barcode-containing fragments by intramolecular ligation, thereby juxtaposing the barcode-tagged ends of the barcode-containing fragments with the barcode-distal ends of the barcode-containing fragments and generating a sequencing library of overlapping fragments, wherein the sequencing library is compatible for sequencing on a massively parallel sequencing platform with a maximum read length of around 250 basepairs;
(v) 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
(vi) assembling the demultiplexed reads to obtain extended sequence reads for the template DNA molecules.
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