Compositions and methods for detecting rare sequence variants
AssigneeAccuragen Holdings Limited
InventorsShengrong Lin, Zhaohui Sun, Grace Qizhi Zhao, Paul Ling-Fung Tang
Life of this patent
Estimating the term…
Abstract
In some aspects, the present disclosure provides methods for identifying sequence variants in a nucleic acid sample. In some embodiments, a method comprises identifying sequence differences between sequencing reads and a reference sequence, and calling a sequence difference that occurs in at least two different circular polynucleotides, such as two circular polynucleotides having different junctions, as the sequence variant. In some aspects, the present disclosure provides compositions and systems useful in the described methods.
Claims26
A method of distinguishing a sequence variant from an error in a nucleic acid sample comprising a plurality of cell-free polynucleotides, each cell-free polynucleotide of the plurality having a 5′ end and a 3′ end, the method comprising:
- (a) circularizing individual cell-free polynucleotides of said plurality to form a plurality of circular polynucleotides, each of which having a junction between the 5′ end and 3′ end;
- (b) amplifying the circular polynucleotides of (a);
- (c) sequencing the amplified polynucleotides to produce a plurality of sequencing reads;
- and
- (d) identifying a sequence difference between sequencing reads and a reference sequence as the sequence variant only when the sequence difference occurs in at least two circular polynucleotides having different junctions and not when the sequence difference only occurs in circular polynucleotides having the same junction thereby distinguishing the sequence variant from the error.
A method of distinguishing a sequence variant from an error in a nucleic acid sample comprising less than 75 ng of cell-free polynucleotides, each polynucleotide having a 5′ end and a 3′ end, the method comprising:
- (a) obtaining a nucleic acid sample comprising less than 75 ng of cell-free polynucleotides;
- (b) circularizing with a ligase individual cell-free polynucleotides in said sample to form a plurality of circular polynucleotides;
- (c) upon separating said ligase from said circular polynucleotides, amplifying the circular polynucleotides to form concatemers;
- (d) sequencing the concatemers to produce a plurality of sequencing reads;
- and
- (e) identifying a sequence difference between the plurality of sequencing reads and a reference sequence as a sequence variant that occurs with a frequency of 1% or lower in said plurality of reads from said nucleic acid sample of less than 75 ng polynucleotides as the sequence variant only when the sequence difference occurs in at least two circular polynucleotides having different junctions and not when the sequence difference only occurs in circular polynucleotides having the same junction thereby distinguishing the sequence variant from the error.
Dependencies are inferred from the claim text.