Method for fragmenting and ligating adapters onto a nucleic acid and kit for performing the same
Providing herein, among other things, is a method for preparing a nucleic acid for sequencing. In some embodiments, the method comprises a) amplifying a nucleic acid template using a dNTP mix that contains 5-methyl dCTP, thereby producing product nucleic acid molecules that contains methylcytosines; b) digesting the product nucleic acid molecules with a methylation-dependent restriction endonuclease, thereby cleaving the product nucleic acid molecules at sites that are adjacent to at least some of the methylcytosine and producing fragments of the product nucleic acid molecules; and c) ligating double-stranded adaptors onto the ends of the fragments to produce adaptor-ligated products.
1. A method for preparing a nucleic acid for sequencing, comprising:
a) amplifying a nucleic acid template using a dNTP mix that contains 5-methyl dCTP, thereby producing product nucleic acid molecules that contains methylcytosines;
b) digesting the product nucleic acid molecules with a MspJI family restriction endonuclease, thereby cleaving the product nucleic acid molecules at sites that are adjacent to at least some of the methylcytosines and producing fragments of the product nucleic acid molecules, wherein the fragments have overhangs; and
c) directly ligating double-stranded adaptors onto the overhangs of the fragments to produce adaptor-ligated products.
2. The method of claim 1 , further comprising:
d) amplifying the fragments using primers that hybridize to the adaptors to produce amplification products.
3. The method of claim 1 , wherein the amplifying step a) is an isothermal amplification.
4. The method of claim 1 , wherein the amplifying step a) is by PCR.
5. The method of claim 1 , wherein the product nucleic acid molecules of a) are at least 500 bp in length.
6. The method of claim 1 , wherein the nucleic acid template is a microbial genome.
7. The method of claim 6 , wherein the microbial genome is a viral genome or a bacterial genome.
8. The method of claim 1 , wherein the nucleic acid template is a mammalian genome.
9. The method of claim 1 , wherein the amplifying step a) is done using a proof-reading polymerase.
10. The method of claim 1 , wherein the dNTP mix contains both 5-methyl dCTP and dCTP.
11. The method of claim 10 , wherein the molar ratio of 5-methyl dCTP to dCTP in the nucleotide mix is in the range of 0.01 to 99.9.
12. The method of claim 1 , wherein the double-stranded adaptors of step c) comprise a four base 5′ overhang of random sequence.
13. The method of claim 1 , wherein the restriction endonuclease is selected from the group consisting of FspEI, LpnPI, MspJI, and SgeI.
14. The method of claim 1 , wherein the digesting step b) comprises digesting the product nucleic acid molecules with a plurality of different MspJI family restriction endonucleases.
15. The method of claim 1 , wherein the fragments produced in b) are in the range of 150 bp to 1 kb in length.
16. The method of claim 1 , further comprising sequencing the adaptor-ligated products of c) or amplification products thereof.
17. The method of claim 1 , wherein the digesting and ligating occur simultaneously in the same reaction.
18. The method of claim 1 , wherein the amplifying step (a) is done using one or more primers that contain at least one methylcytosine.
19. A kit comprising:
a) a dNTP stock that contains at least 5-methyl dCTP;
b) one or more MspJI family restriction endonucleases; and
c) double-stranded adaptors that comprise a four-base 5′ overhang.
20. The kit of claim 19 , wherein the dNTP stock contains dGTP, dATP, dTTP, dCTP and 5-methyl dCTP.