Detection of rare sequence variants, methods and compositions therefor
AssigneeWashington University
InventorsTodd E. Druley, Andrew Young
Life of this patent
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Abstract
The present disclosure encompasses methods of error corrected sequencing (ECS) that enable detection of very rare mutations well below the error rate of convention next generation sequencing (NGS). Further, the methods disclosed herein enable multiplex targeting of genomic DNA.
Claims25
A method of identifying a genetic mutation in a biological sample comprising nucleic acid obtained from a subject, the method comprising:
- a) amplifying one or more regions of interest from the biological sample comprising nucleic acid, wherein a plurality of amplicons for each region of interest are generated;
- b) attaching an adapter and a random component to each amplicon generated in (a) and amplifying;
- c) sequencing the amplicons comprising the random component generated in (b), wherein redundant reads are generated and wherein the redundant reads are grouped by the random component and a consensus sequence is identified;
- and
- d) comparing the consensus sequence to a reference sequence, wherein a consensus sequence that differs from the reference sequence comprises a genetic mutation.
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-13. (canceled)
A method of identifying a genetic mutation in a biological sample comprising nucleic acid obtained from a subject, the method comprising:
- a) hybridizing a primer pool comprising one or more primer pairs specific to one or more regions of interest from the biological sample comprising nucleic acid, extending from an upstream primer of the primer pair to a downstream primer of the primer pair, and ligating the extension product to the downstream primer of the primer pair, wherein products comprising the regions of interest flanked by sequences required for amplification are generated;
- b) attaching an adapter comprising a random component and attaching an adapter comprising an index sequence to the products from (a) and amplifying;
- c) sequencing the products comprising the random component generated in (b), wherein redundant reads are generated and wherein the redundant reads are grouped by the random component and a consensus sequence is identified;
- and
- d) comparing the consensus sequence to a reference sequence, wherein a consensus sequence that differs from the reference sequence comprises a genetic mutation.
-24. (canceled)
A method of detecting minimal residual disease (MRD) in a subject, the method comprising:
- a) hybridizing a primer pool comprising one or more primer pairs specific to one or more regions of interest from a biological sample comprising nucleic acid obtained from the subject, extending from an upstream primer of the primer pair to a downstream primer of the primer pair, and ligating the extension product to the downstream primer of the primer pair, wherein products comprising the regions of interest flanked by sequences required for amplification are generated;
- b) attaching an adapter comprising a random component and attaching an adapter comprising an index sequence to the products from (a) and amplifying;
- c) sequencing the products comprising the random component generated in (b), wherein redundant reads are generated and wherein the redundant reads are grouped by the random component and a consensus sequence is identified;
- and
- d) comparing the consensus sequence to a reference sequence, wherein a consensus sequence that differs from the reference sequence comprises a genetic mutation and is indicative of MRD.
Dependencies are inferred from the claim text.