Methods of nucleic acid sample preparation
Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids. In some embodiments, methods for preparing nucleic acids for sequence analysis (e.g., using next-generation sequencing) are provided herein.
1. A method of preparing nucleic acids for analysis, the method comprising:
(a) adding one or more nucleotides to a 3′ end of a double-stranded nucleic acid comprising a target nucleotide sequence, wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;
(b) ligating an adapter nucleic acid to at least one strand of the double-stranded nucleic acid to produce a ligation product comprising the one or more capture moiety modified nucleotides; and
(c) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety modified nucleotide.
2. The method of claim 1 , further comprising, after the capturing of (c):
(d) amplifying the ligation product.
3. The method of claim 2 , wherein the amplifying comprises linear amplification or a polymerase chain reaction.
4. The method of claim 3 , wherein the polymerase chain reaction comprises use of a first target-specific primer that specifically anneals to a portion of the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid.
5. The method of claim 4 , further comprising amplifying a product of (d) by a second polymerase chain reaction that comprises (i) use of a second adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid, and (ii) use of a second target-specific primer that specifically anneals to a portion of the target nucleotide sequence and is nested relative to the first target-specific primer.
6. The method of claim 1 , further comprising, after the capturing of (c), determining a sequence of at least a portion of the double-stranded nucleic acid.
7. The method of claim 1 , wherein the double-stranded nucleic acid comprises genomic DNA, fragmented DNA, cell-free DNA (cfDNA) or cDNA.
8. The method of claim 1 , wherein after the adding of (a), the double-stranded nucleic acid comprises a 3′-overhang sequence.
9. The method of claim 8 , wherein the 3′-overhang sequence is complementary to one or more nucleotides at a 3′ end of the adapter nucleic acid.
10. The method of claim 1 , wherein each of the one or more nucleotides is a capture moiety modified nucleotide.
11. The method of claim 1 , wherein only one nucleotide is added to the 3′ end of the double-stranded nucleic acid in step (a), wherein the one nucleotide is a capture moiety modified nucleotide.
12. The method of claim 11 , wherein the one nucleotide comprises adenine.
13. The method of claim 1 , wherein the capture moiety modified nucleotide comprises a biotin capture moiety.
14. The method of claim 1 , wherein the capturing of (c) provides an immobilized ligation product, and the method further comprises washing the immobilized ligation product, releasing the immobilized ligation product, and amplifying at least a portion of the ligation product.
15. The method of claim 1 , wherein the adapter nucleic acid comprises a barcode and a primer binding site.
16. The method of claim 1 , wherein the adapter nucleic acid is double-stranded.
17. A method of preparing nucleic acids for analysis, the method comprising:
(a) adding one or more nucleotides to a 3′ end of each of a plurality of double-stranded nucleic acids comprising a plurality of different target nucleotide sequences, wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;
(b) ligating an adapter nucleic acid to at least one strand of each of the plurality of double-stranded nucleic acids to produce a plurality of ligation products comprising the one or more capture moiety modified nucleotides;
(c) capturing the plurality of ligation products by contacting the plurality of ligation products with a binding partner of the capture moiety modified nucleotide; and
(d) amplifying the plurality of ligation products, thereby providing a plurality of amplicons.
18. The method of claim 17 , wherein the plurality of double-stranded nucleic acids comprises 25 to 1000 different target nucleotide sequences or 1000 to 10,000 different target nucleotide sequences.
19. A method of preparing nucleic acids for analysis, the method comprising:
(a) adding one or more nucleotides to a 3′ end of a double-stranded nucleic acid comprising a target nucleotide sequence, wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;
(b) ligating an adapter nucleic acid to at least one strand of the double-stranded nucleic acid to produce a ligation product comprising the one or more capture moiety modified nucleotides;
(c) capturing the ligation product by contacting the ligation product with a binding partner of the capture moiety modified nucleotide, thereby providing an immobilized ligation product;
(d) optionally washing the immobilized ligation product;
(e) separating the ligation product from the binding partner; and
(f) amplifying the separated ligation product of (e) by a polymerase chain reaction using a first target-specific primer that specifically anneals to the target nucleotide sequence and a first adapter primer that specifically anneals to a complementary sequence of the adapter nucleic acid.
20. The method of claim 19 , wherein the capture moiety modified nucleotide comprises a biotin capture moiety.
21. The method of claim 19 , wherein the binding partner comprises an avidin protein.
22. A method of preparing nucleic acids for analysis, the method comprising:
(a) combining a double-stranded nucleic acid, a modified nucleotide comprising a capture moiety, and an enzyme under conditions in which the enzyme adds the modified nucleotide to a 3′ end of the double-stranded nucleic acid;
(b) combining the double-stranded nucleic acid, an adapter nucleic acid, and a ligase under conditions in which the ligase ligates the adapter nucleic acid to the double-stranded nucleic acid, thereby producing a ligation product comprising the capture moiety; and
(c) combining the ligation product and a binding partner of the capture moiety under conditions in which the binding partner forms a complex with the ligation product.