USE OF ETHYLENE CARBONATE IN NUCLEIC ACID SEQUENCING METHODS

1 . A method of preparing nucleic acid molecules for sequencing, comprising:

contacting double-stranded nucleic acid molecules attached to a surface with ethylene carbonate to generate single-stranded nucleic acid molecules attached to the surface; and,

hybridizing sequencing primers to the single-stranded nucleic acid molecules, thereby generating sequencing hybrids.

2 . The method of claim 1 , wherein the ethylene carbonate has a concentration of between about 10% and about 50% volume/volume.

3 . The method of claim 1 or 2 , wherein the contacting is implemented at a temperature of between about 35° C. and about 50° C.

4 . The method of any one of claims 1-3 , wherein the double-stranded nucleic acid molecules attached to the surface are contacted with the ethylene carbonate for about 5 minutes or more.

5 . The method of any one of claims 1-4 , wherein the sequencing primers comprise a nucleic acid primer.

6 . The method of any one of claims 1-4 , wherein the sequencing primers comprise a peptide nucleic acid (PNA) primer.

7 . The method of claim 6 , wherein the PNA primer has increased hybridization affinity with the single-strand nucleic acid molecules compared to a nucleic acid primer.

8 . The method of any one of claims 1-7 , wherein a concentration of the sequencing primers is selected to favor formation of the sequencing hybrids over re-formation of the double-stranded nucleic acid molecules.

9 . The method of claim 8 , wherein the sequencing primers are in excess concentration compared to the single-stranded nucleic acid molecules.

10 . The method of any one of claims 1-9 , wherein the contacting and hybridizing occur simultaneously.

11 . The method of any one of claims 1-10 , wherein the double-stranded nucleic acid molecules or the single-stranded nucleic acid molecules are derived from a fluidic sample obtained from an individual.

12 . The method of claim 11 , wherein the fluidic sample is a blood sample, a plasma sample, a saliva sample, a urine sample, or a fecal sample.

13 . The method of claim 11 or 12 , wherein the fluidic sample comprises cell-free nucleic acid molecules.

14 . The method of any one of claims 11-13 , wherein the fluidic sample comprises DNA molecules.

15 . The method of any one of claims 11-14 , wherein the fluidic sample comprises cDNA molecules.

16 . The method of any one of claims 1-15 , wherein the double-stranded nucleic acid molecules or the single-stranded nucleic acid molecules comprise a sequencing adaptor sequence, wherein the sequencing adaptor sequence comprises a sequencing primer hybridization sequence.

17 . The method of any one of claims 1-16 , wherein the double-stranded nucleic acid molecules or the single-stranded nucleic acid molecules are amplification products.

18 . The method of any one of claims 1-17 , wherein the double-stranded nucleic acid molecules or the single-stranded nucleic acid molecules are covalently attached to the surface.

19 . The method of any one of claims 1-18 , wherein the double-stranded nucleic acid molecules or the single-stranded nucleic acid molecules are attached to the surface using click chemistry or amine-reactive crosslinker chemistry.

20 . The method of any one of claims 1-19 , wherein the surface is a bead.

21 . The method of claim 20 , wherein the bead is a gel bead.

22 . The method of any one of claims 1-21 , wherein the surface is immobilized to a wafer.

23 . The method of any one of claims 1-22 , further comprising attaching nucleic acid molecules in a sequencing library to the surface prior to the contacting with ethylene carbonate.

24 . The method of claim 23 , further comprising amplifying the nucleic acid molecules in the sequencing library attached to the surface prior to the contacting with ethylene carbonate, thereby generating sequencing colonies comprising the double-stranded nucleic acid molecules attached to the surface.

25 . The method of claim 24 , wherein the nucleic acid molecules are amplified isothermally.

26 . The method of claim 25 , wherein the amplifying occurs between about 30° C. and about 50° C.

27 . The method of any one of claims 24-26 , wherein the amplifying comprises one or more of rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and molten recombinase polymerase amplification (mRPA).

28 . The method of claim 27 , wherein the amplifying comprises use of reagents selected from the group consisting of polymerases, recombinases, single-stranded DNA binding proteins, magnesium acetate, betaine, formamide, tetramethyl ammonium chloride, sodium dodecyl sulfate (SDS), and trimethylamine N-oxide.

29 . The method of any one of claims 24-28 , further comprising removing deoxyuridine primers after amplifying the nucleic acid molecules on the surface.

30 . The method of any one of claims 1-29 , further comprising washing the single-stranded nucleic acid molecules with a wash buffer prior to the hybridizing of sequencing primers to the single-stranded nucleic acid molecules.

31 . The method of any one of claims 1-30 , further comprising washing the sequencing hybrids with a wash buffer.

32 . The method of claim 30 or 31 , wherein the washing is repeated two or more times.

33 . The method of any one of claims 30-32 , wherein the wash buffer comprises tris (hydroxymethyl) aminomethane (tris), ethylenediaminetetraacetic acid (EDTA), triton, or sodium dodecyl sulfate (SDS).

34 . The method of any one of claims 1-33 , further comprising sequencing the single-stranded nucleic acid molecules, thereby generating sequencing data.

35 . The method of claim 34 , wherein the single-stranded nucleic acid molecules are sequenced using a plurality of sequencing flow steps, each sequencing flow step comprising contacting the sequencing hybrids with nucleotides, wherein at least a portion of the nucleotides are labeled, and detecting the presence or absence of an incorporated nucleotide.

36 . The method of claim 35 , wherein the nucleotides in each sequencing flow step comprise nucleotides of a same base type.

37 . The method of claims 35 or 36 , wherein the at least a portion of the nucleotides is less than all of the nucleotides in each sequencing flow step.

38 . The method of any one of claims 35-37 , wherein the nucleotides are non-terminating nucleotides.

39 . The method of any one of claims 35-38 , wherein the sequencing data comprises flow signals at the plurality of sequencing flow steps.

40 . The method of claim 39 , wherein the flow signals are used to determine a base count indicative of a number of bases sequenced at each flow step.

41 . The method of claim 39 or 40 , wherein the flow signals are used to determine a statistical parameter indicative of a likelihood for at least one base count at each flow step, wherein the base count is indicative of a number of bases of a given single-stranded nucleic acid molecule sequenced at a given flow step.

42 . A method of preparing nucleic acid molecules for sequencing, comprising:

providing nucleic acid molecules attached to a surface;

amplifying the nucleic acid molecules on the surface to generate double-stranded nucleic acid molecules;

contacting the double-stranded nucleic acid molecules with ethylene carbonate to generate single-stranded nucleic acid molecules;

washing the single-stranded nucleic acid molecules with a wash buffer; and,

hybridizing sequencing primers to the single-stranded nucleic acid molecules, thereby generating sequencing hybrids.

43 . The method of claim 42 , wherein the amplifying is isothermal.

44 . The method of claim 42 or 43 , wherein the amplifying comprises one or more of rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and molten recombinase polymerase amplification (mRPA).

45 . A method of sequencing nucleic acid molecules, comprising:

providing nucleic acid molecules attached to a surface;

amplifying the nucleic acid molecules on the surface to generate double-stranded nucleic acid molecules;

contacting the double-stranded nucleic acid molecules with ethylene carbonate to generate single-stranded nucleic acid molecules;

washing the single-stranded nucleic acid molecules with a wash buffer;

hybridizing sequencing primers to the single-stranded nucleic acid molecules, thereby generating sequencing hybrids; and,

sequencing the single-stranded nucleic acid molecules using a plurality of sequencing flow steps, each sequencing flow step comprising contacting the sequencing hybrids with non-terminating nucleotides, wherein at least a portion of the non-terminating nucleotides are labeled, and detecting the presence or absence of an incorporated non-terminating nucleotide.

46 . The method of claim 45 , wherein the amplifying is isothermal.

47 . The method of claim 45 or 46 , wherein the amplifying comprises one or more of rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and molten recombinase polymerase amplification (mRPA).

48 . The method of any one of claims 45-47 , further comprising generating sequencing data, wherein the sequencing data comprises flow signals detected at the plurality of sequencing flow steps.

49 . The method of any one of claims 45-48 , wherein the flow signals are used to determine a base count indicative of a number of bases sequenced at each flow step.

50 . The method of claim 48 or 49 , wherein the flow signals are used to determine a statistical parameter indicative of a likelihood for at least one base count at each flow step, wherein the base count is indicative of a number of bases of a given single-stranded nucleic acid molecule sequenced at a given flow step.