| CPC C12Q 1/6874 (2013.01) [C12N 15/1065 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6855 (2013.01)] | 24 Claims |
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1. A method of reducing base errors in sequencing double-stranded DNA targets, wherein each primary clone of a DNA target is subdivided into subclones along the course of DNA amplifications, wherein base errors are reduced by finding consensus sequence in each subclone and then in each primer clone, the method comprising:
forming primary clones from double-stranded DNA molecules by:
ligating an adapter to both ends of each of a plurality of double-stranded DNA molecules, wherein the adapter comprises a unique molecular identifier (UMI) and a first universal primer binding site for PCR amplification, and the UMI comprises 12 or more bases including degenerate or semi-degenerate base sequences, and
amplifying the adapter-DNA complexes with a first universal primer, resulting in each strand of the DNA molecule producing a clone of itself;
subdividing each primary clone into subclones comprising:
annealing and extending a plurality of target-specific primers to the primary clones, wherein each of the target-specific primer comprises a target-specific region, a UMI and a second universal primer binding site for PCR amplification, resulting in each primary clone being subdivided into multiple subclones defined by the UMIs on the target-specific primers on one side of the resulting molecules, while each primary clone is still identifiable by the UMI from the adapter on the other side of the resulting molecules,
enzymatically removing single-stranded regions from 3′ ends in the primary clones, and
amplifying the resulting products using the first universal primer and a second universal primer;
sequencing the resulting products; and
removing base errors after sequencing, comprising:
sorting sequences into primary clones by UMIs on the adapters on one side of the molecules, then sorting each primary clone into subclones by the UMIs on the target-specific primers on the other side of the molecules, and
deducing consensus sequence from each subclone, then deducing consensus sequence in each primary clone from the consensus sequences obtained from the subclones within each primary clone.
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