Compositions and methods for detecting rare sequence variants

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.

1 .- 22 . (canceled) 
     
     
         23 . 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) circularizing individual 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) to produce amplified polynucleotides; 
 (c) 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; 
 (d) sequencing the sheared 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 sheared polynucleotides. 
 
     
     
         24 . The method of  claim 23 , wherein calling the sequence difference as the sequence variant occurs further when (i) the sequence difference is identified on both strands of a double-stranded input molecule; and/or (ii) the sequence difference occurs in a consensus sequence for a concatemer formed by amplification comprising rolling circle amplification. 
     
     
         25 . The method of  claim 23 , wherein the plurality of polynucleotides is single-stranded. 
     
     
         26 . The method of  claim 23 , wherein circularizing is effected by subjecting the plurality of polynucleotides to a ligation reaction. 
     
     
         27 . The method of  claim 23 , wherein the sequence variant is a single nucleotide polymorphism. 
     
     
         28 . The method of  claim 23 , wherein the reference sequence is a consensus sequence formed by aligning the sequencing reads with one another. 
     
     
         29 . The method of  claim 23 , wherein the reference sequence is a sequencing read. 
     
     
         30 . The method of  claim 23 , wherein circularizing comprises the step of joining an adapter polynucleotide to the 5′ end, the 3′ end, or both the 5′ end and the 3′ end of a polynucleotide in the plurality of polynucleotides. 
     
     
         31 . The method of  claim 23 , wherein amplifying is effected by using a polymerase having strand-displacement activity. 
     
     
         32 . The method of  claim 23 , wherein amplifying comprises subjecting the circular polynucleotides to an amplification reaction mixture comprising random primers. 
     
     
         33 . The method of  claim 23 , wherein amplifying comprises subjecting the circular polynucleotides to an amplification reaction mixture comprising one or more primers, each of which specifically hybridizes to a different target sequence via sequence complementarity. 
     
     
         34 . The method of  claim 23 , wherein the amplified polynucleotides are subjected to the sequencing step without enrichment. 
     
     
         35 . The method of  claim 23 , further comprising enriching one or more target polynucleotides among the amplified polynucleotides by performing an enrichment step prior to sequencing. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 23 , wherein the sample is a sample from a subject. 
     
     
         38 . The method of  claim 37 , wherein the sample is urine, stool, blood, saliva, tissue, or bodily fluid. 
     
     
         39 . The method of  claim 37 , wherein the sample comprises tumor cells. 
     
     
         40 . The method of  claim 37 , wherein the sample is a formalin-fixed paraffin embedded (FFPE) sample. 
     
     
         41 . The method of  claim 37 , further comprising diagnosing, and optionally treating, said subject based on the calling step. 
     
     
         42 . The method of  claim 23 , wherein the sequence variant is a causal genetic variant. 
     
     
         43 . The method of  claim 23 , wherein the sequence variant is associated with a type or stage of cancer. 
     
     
         44 . The method of  claim 23 , wherein the plurality of polynucleotides comprises cell-free polynucleotides. 
     
     
         45 . The method of  claim 44 , wherein the cell-free polynucleotides comprise circulating tumor DNA. 
     
     
         46 .- 103 . (canceled)