Compositions and methods for detecting rare sequence variants
First Claim
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1. A method of identifying a sequence variant in a nucleic acid sample comprising a plurality of polynucleotides, each polynucleotide of the plurality having a 5′
- end and a 3′
end, the method comprising;
(a) in a reaction mixture, circularizing individual polynucleotides of said plurality to form a plurality of circular polynucleotides using a ligase, each of which having a junction between the 5′
end and 3′
end;
(b) adding a heat labile protease to said reaction mixture to degrade the ligase enzyme;
(c) amplifying the circular polynucleotides after step (b) without isolating the circular polynucleotides from said reaction mixture between steps (a) and (c) to produce amplified polynucleotides;
(d) sequencing the polynucleotides to produce a plurality of sequencing reads;
(e) identifying sequence differences between sequencing reads and a reference sequence; and
(f) calling a sequence difference as the sequence variant when the sequence difference occurs in at least two different amplified polynucleotides,wherein the amplifying yields a higher level of recovery of the plurality of polynucleotides as compared to performing a rolling circle amplification comprising steps (a) and (c) alone,wherein the higher level of recovery is evidenced by an increased library complexity, andwherein the increased library complexity is represented by an increased number of unique sequences amplified from said plurality of polynucleotides.
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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, or two different sheared polynucleotides as the sequence variant. In some aspects, the present disclosure provides compositions and systems useful in the described method.
142 Citations
23 Claims
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1. A method of identifying a sequence variant in a nucleic acid sample comprising a plurality of polynucleotides, each polynucleotide of the plurality having a 5′
- end and a 3′
end, the method comprising;(a) in a reaction mixture, circularizing individual polynucleotides of said plurality to form a plurality of circular polynucleotides using a ligase, each of which having a junction between the 5′
end and 3′
end;(b) adding a heat labile protease to said reaction mixture to degrade the ligase enzyme; (c) amplifying the circular polynucleotides after step (b) without isolating the circular polynucleotides from said reaction mixture between steps (a) and (c) to produce amplified polynucleotides; (d) sequencing the polynucleotides to produce a plurality of sequencing reads; (e) identifying sequence differences between sequencing reads and a reference sequence; and (f) calling a sequence difference as the sequence variant when the sequence difference occurs in at least two different amplified polynucleotides, wherein the amplifying yields a higher level of recovery of the plurality of polynucleotides as compared to performing a rolling circle amplification comprising steps (a) and (c) alone, wherein the higher level of recovery is evidenced by an increased library complexity, and wherein the increased library complexity is represented by an increased number of unique sequences amplified from said plurality of polynucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
- end and a 3′
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22. A method of identifying a sequence variant in a nucleic acid sample comprising a plurality of polynucleotides, each polynucleotide of the plurality having a 5′
- end and a 3′
end, the method comprising;(a) in a reaction mixture, circularizing individual polynucleotides of said plurality to form a plurality of circular polynucleotides using a ligase, each of which having a junction between the 5′
end and 3′
end;(b) adding a heat labile protease to said reaction mixture to degrade the ligase enzyme; (c) amplifying the circular polynucleotides after step (b) without isolating the circular polynucleotides from said reaction mixture between steps (a) and (c) to produce amplified polynucleotides; (d) shearing the amplified polynucleotides to produce sheared polynucleotides, each sheared polynucleotide comprising one or more shear points at a 5′
end and/or a 3′
end;(e) sequencing the sheared polynucleotides to produce a plurality of sequencing reads; (f) identifying sequence differences between sequencing reads and a reference sequence; and (g) calling a sequence difference as the sequence variant when the sequence difference occurs in at least two different sheared polynucleotides, wherein the amplifying yields a higher level of recovery of the plurality of polynucleotides as compared to performing a rolling circle amplification comprising steps (a) and (c) alone, wherein the higher level of recovery is evidenced by an increased library complexity, and wherein the increased library complexity is represented by an increased number of unique sequences amplified from said plurality of polynucleotides. - View Dependent Claims (23)
- end and a 3′
Specification
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Current AssigneeAccuragen, Inc.
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Original AssigneeAccuragen, Inc.
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InventorsWeng, Li, Sun, Zhaohui, Lin, Shengrong
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Primary Examiner(s)Flinders, Jeremy C
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Application NumberUS16/172,310Publication NumberTime in Patent Office641 DaysField of SearchUS Class CurrentCPC Class CodesC12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6806 Preparing nucleic acids for...C12Q 1/6816 characterised by the detect...C12Q 1/6827 for detection of mutation o...C12Q 1/6858 Allele-specific amplificationC12Q 1/6869 Methods for sequencingC12Q 2521/501 LigaseC12Q 2531/125 Rolling circleC12Q 2535/122 Massive parallel sequencing
