METHODS FOR DUPLEX SEQUENCING

1 . A method of processing a nucleic acid comprising:

(a) providing a reaction mixture comprising: (i) a plurality of double stranded nucleic acid molecules,

(ii) a plurality of oligonucleotides, wherein at least one of said plurality of oligonucleotides comprises a 5′ end and a 3 ′ end that are capable of base pairing with each other to yield a stem loop structure in said reaction mixture;

(b) subjecting said reaction mixture to a ligation reaction to ligate (i) a first oligonucleotide of said plurality of oligonucleotides to a 5′ end of a double stranded nucleic acid molecule of said plurality of double stranded nucleic acid molecules and (ii) a second oligonucleotide of said plurality of oligonucleotides to a 3′ end of said double stranded nucleic acid molecule of said plurality of double stranded nucleic acid molecules, thereby creating at least one nucleic acid molecule without an exposed 5′ or 3′ end;

(c) subjecting said nucleic acid molecule or derivative thereof to amplification with a primer to create a linear product comprising one or more copies of said nucleic acid molecule; and

(d) sequencing said linear product or a derivative thereof to obtain a sequence of a first strand and a second strand of said double stranded nucleic acid molecule.

2 . The method of claim 1 , wherein said first oligonucleotide or said second oligonucleotide comprises a methylated nucleotide.

3 . The method of claim 1 or claim 2 , wherein said double stranded nucleic acid molecule comprises a methylated nucleotide.

4 . The method of claim 1 or claim 2 , wherein said first oligonucleotide or said second oligonucleotide comprises one or more methylated cytosines.

5 . The method of claim 2 or claim 3 , wherein said methylated nucleotide comprises methylated cytosine or methylated adenine.

6 . The method of any one of claims 1 to 5 , wherein said polymerase is a strand displacing polymerase.

7 . The method of any one of claims 1 to 6 , wherein subsequent to (b), modifying at least one nucleotide of said nucleic acid molecule to create a mismatched base pair within said nucleic acid molecule to yield a modified nucleic acid molecule.

8 . The method of claim 7 , wherein said modifying comprises a chemical or enzymatic modification that converts a methylated cytosine to a uracil or an unmethylated cytosine to a uracil.

9 . The method of any one of claims 1 to 8 , wherein said amplification product is a concatemer.

10 . The method of any one of claims 1 to 9 , further comprising processing said sequence to identify a sequence variant when a sequence difference is present in said sequence of said first strand and said sequence of said second strand.

11 . The method of claim 10 , wherein said sequence variant comprises a single nucleotide variant, a fusion, an insertion, or a deletion.

12 . The method of any one of claims 1 to 10 , further comprising processing said sequence to identify an epigenetic modification when an epigenetic difference is present in said sequence of said first strand and confirmed by said sequence of said second strand.

13 . The method of any one of claims 1 to 12 , wherein said primer is a random primer.

14 . The method of any one of claims 1 to 12 , wherein said primer is a target specific primer.

15 . The method of any one of claims 1 to 14 , wherein said primer binds to a sequence of said first oligonucleotide or said second oligonucleotide.

16 . The method of any one of claims 1 to 14 , wherein said primer binds to a sequence of said first strand or said second strand of said double stranded nucleic acid molecule.

17 . The method of any one of claims 1 to 16 , wherein said plurality of double stranded nucleic acid molecules comprise double stranded deoxyribonucleic acid, double stranded ribonucleic acid, or a combination thereof.

18 . The method of any one of claims 1 to 17 , wherein said plurality of double stranded nucleic acid molecules is derived from a cell-free nucleic acid sample.

19 . The method of any one of claims 1 to 18 , wherein sequencing 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 . The method of any one of claims 1 to 19 , wherein said sequencing comprises sequencing by ligation.

21 . The method of any one of claims 1 to 20 , wherein said plurality of double stranded nucleic acid molecules is derived from a bodily fluid.

22 . The method of claim 21 , wherein said bodily fluid comprises urine, saliva, blood, serum, sweat, or plasma.

23 . The method of any one of claims 1 to 22 , wherein said plurality of double stranded nucleic acids is derived from a biological sample of a subject, which biological sample comprises blood, saliva, serum, plasma, urine, menstrual fluid, sweat, skin, bone marrow, lung tissue, liver tissue, pancreatic tissue, stomach tissue, intestinal tissue, kidney tissue, bladder tissue, brain tissue, spinal cord tissue, tongue tissue, larynx tissue, esophageal tissue, spleen tissue, thymus tissue, thyroid tissue, breast tissue, prostate tissue, testicular tissue, or a tumor thereof.

24 . The method of any one of claims 1 to 23 , wherein said plurality of double stranded nucleic acid molecules is derived from a tumor sample of a subject.