METHODS FOR ASSEMBLING AND READING NUCLEIC ACID SEQUENCES FROM MIXED POPULATIONS
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Abstract
The disclosure relates to methods for obtaining nucleic acid sequence information by constructing a nucleic acid library and reconstructing longer nucleic acid sequences by assembling a series of shorter nucleic acid sequences.
36 Citations
37 Claims
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1-6. -6. (canceled)
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7. A method for obtaining nucleic acid sequence information from a nucleic acid molecule comprising a target nucleotide sequence by assembling a series of nucleic acid sequences into a longer nucleic acid sequence, said method comprising:
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(a) attaching a first adapter comprising an outer polymerase chain reaction (PCR) primer region or nucleic acid amplification region, an inner sequencing primer region, and a central barcode region to each end of a plurality of linear nucleic acid molecules to form barcode-tagged molecules; (b) replicating the barcode-tagged molecules to obtain a library of barcode-tagged molecules; (c) breaking the barcode-tagged molecules, thereby generating linear, barcode-tagged fragments comprising the barcode region at one end and a region of unknown sequence at the other end; (d) circularizing the linear, barcode-tagged fragments comprising the barcode region at one end and a region of unknown sequence from an interior portion of the target nucleotide sequence at the other end, thereby bringing the barcode region into proximity with the region of unknown sequence; (e) fragmenting the circularized, barcode-tagged fragments into linear, barcode-tagged fragments; (f) attaching a second adapter to each end of the linear, barcode-tagged fragments to form double adapter-ligated barcode-tagged nucleic acid fragments; (g) replicating all or part of the double adapter-ligated barcode-tagged nucleic acid fragments; (h) sequencing the double adapter-ligated barcode-tagged nucleic acid fragments; (i) sorting a series of sequenced nucleic acid fragments into independent groups; and (j) assembling each group of reads into a longer nucleic acid sequence. - View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 21, 22, 23, 24, 28, 33, 34, 35, 36)
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17-19. -19. (canceled)
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25-27. -27. (canceled)
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29-32. -32. (canceled)
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37-82. -82. (canceled)
Specification