COMPOSITIONS AND METHODS FOR DETECTING RARE SEQUENCE VARIANTS

1 . A method of identifying a sequence variant comprising:

(a) circularizing individual polynucleotides of a plurality of polynucleotides to form a plurality of circular polynucleotides, each circular polynucleotide having a junction between a 5′ end and a 3′ end;

(b) amplifying said plurality of circular polynucleotides of (a) to produce a plurality of amplified polynucleotides, each amplified polynucleotide having more than one copy of the circular polynucleotide;

(c) shearing said amplified polynucleotides or a derivative thereof to produce a plurality of sheared polynucleotides, each sheared polynucleotide comprising a 5′ end shear point and a 3′ end shear point;

(d) subjecting said plurality of sheared polynucleotides or a derivative thereof to sequencing to identify a plurality of sequence reads of said plurality of sheared polynucleotides;

(e) comparing said plurality of sequence reads to a reference sequence to obtain a sequence difference;

(f) calling a sequence difference as the sequence variant when the sequence difference occurs in (i) at least two copies on one sheared polynucleotide and (ii) at least two different sheared polynucleotides having different 5′ end shear points and/or 3′ end shear points.

2 . The method of claim 1 , further comprising subsequent to (c), attaching a first adapter to said 5′ end shear point and a second adapter to said 3′ end shear point of each of said plurality of sheared polynucleotides or a derivative thereof to create a plurality of adapter-linked sheared polynucleotides.

3 . The method of claim 2 , further comprising amplifying said plurality of adapter-linked sheared polynucleotides using a first primer that binds to said first adapter and a second primer that binds to said second adapter or amplifying one or more target sequences of said plurality of adapter-linked sheared polynucleotides using a first primer that binds to said first adapter and at least a second primer that binds to said one or more target sequences in said sheared polynucleotide.

4 . (canceled)

5 . The method of claim 1 , further comprising subsequent to (c), enriching a target sequence in said plurality of sheared polynucleotides or a derivative thereof.

6 . The method of claim 5 , wherein enriching comprises contacting said plurality of sheared polynucleotides or a derivative thereof with a capture probe that binds to said target sequence or wherein enriching comprises amplification with at least one primer that binds to said target sequence.

7 . (canceled)

8 . The method of claim 1 , wherein (a) comprises ligating ends of each of said plurality of polynucleotides or a derivative thereof to one another.

9 . The method of claim 1 , wherein (a) comprises coupling an adapter to said 5′ end, said 5′ end, or both said 5′ end and said 3′ end of each of said plurality of polynucleotides or a derivative thereof.

10 . The method of claim 1 , wherein (b) is effected by a polymerase having strand-displacement activity or a polymerase having 5′ to 3′ exonuclease activity.

11 . (canceled)

12 . The method of claim 1 , wherein (b) comprises contacting said plurality of circular polynucleotides with an amplification reaction mixture comprising random primers.

13 . The method of claim 1 , wherein (b) comprises contacting said plurality of circular polynucleotides with an amplification mixture comprising at least one primer that hybridizes to a target sequence of at least one of said plurality of circular polynucleotides.

14 . The method of claim 1 , wherein said polynucleotides are single-stranded.

15 . The method of claim 1 , wherein said polynucleotides are double-stranded.

16 . The method of claim 1 , wherein said polynucleotides are cell-free polynucleotides.

17 . The method of claim 1 , wherein said polynucleotides are deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof.

18 . The method of claim 1 , wherein said polynucleotides are from a tumor.

19 . The method of claim 1 , wherein (d) comprises (i) bringing said plurality of sheared polynucleotides or a derivative thereof in contact with a plurality of nucleotides in the presence of a polymerase to incorporate one or more nucleotides of said plurality of nucleotides into a growing strand complementary to a strand of said sheared polynucleotides or derivative thereof, and (ii) detecting one or more signals indicative of incorporation of said one or more nucleotides into said growing strand.

20 . (canceled)

21 . The method of claim 1 , wherein said sequence variant comprises a single nucleotide variant, a fusion, an insertion, a deletion, or an epigenetic modification.

22 . The method of claim 1 , wherein said sequence variant is indicative of minimum residual disease (MRD).

23 . The method of claim 1 , wherein said polynucleotides are from a bodily fluid comprising urine, saliva, blood, serum, or plasma.

24 . (canceled)

25 . The method of claim 1 , further comprising detecting minimum residual disease (MRD).