Methods of nucleic acid sample preparation for analysis of cell-free DNA
Aspects of the technology disclosed herein relate to methods of preparing and analyzing nucleic acids, e.g, cfDNA. 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, and wherein the double-stranded nucleic acid is obtained from cell-free DNA;
(b) ligating an adapter nucleic acid to the double-stranded nucleic acid to which the capture moiety modified nucleotide has been added to produce a ligation product, wherein a sequence of one or more nucleotides at a 3′ end of the adapter nucleic acid is complementary with the one or more nucleotides added to the 3′ end of the double-stranded nucleic acid in step (a); and
(c) capturing the ligation product by contacting the ligation product with a binding partner of a capture moiety of the capture moiety modified nucleotide.
2. The method of claim 1 further comprising:
(d) amplifying the ligation product by 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.
3. The method of claim 2 further comprising:
(e) amplifying an amplification product of step (d) by polymerase chain reaction using a second adapter primer and a second target-specific primer.
4. The method of claim 3 , wherein the second target-specific primer is nested relative to the first target-specific primer.
5. The method of claim 3 , wherein the second target-specific primer comprises a 5′ tail that does not anneal to the target nucleotide sequence.
6. The method of claim 5 , further comprising adding an additional primer comprising a 3′ portion that is identical to the 5′ tail of the second target-specific primer.
7. The method of claim 1 , wherein step (b) comprises combining the adapter nucleic acid, the double-stranded nucleic acid, and a ligase under conditions in which the ligase ligates the adapter nucleic acid to the double-stranded nucleic acid, wherein the adapter nucleic acid that is combined with the double-stranded nucleic acid comprises a duplex portion and an overhang sequence, wherein the overhang sequence comprises the sequence of one or more nucleotides at the 3′ end of the adapter nucleic acid that is complementary with the one or more nucleotides added to the 3′ end of the double stranded nucleic acid in step (a).
8. The method of claim 1 , wherein step (b) comprises combining the adapter nucleic acid, the double-stranded nucleic acid, and a ligase under conditions in which the ligase ligates the adapter nucleic acid to the double-stranded nucleic acid, wherein the adapter nucleic acid that is combined with the double-stranded nucleic acid is single-stranded.
9. The method of claim 1 , wherein the capture moiety is a biotin moiety, optionally wherein the biotin moiety is covalently linked to the nucleotide via a linker of 5 to 20 atoms in length.
10. The method of claim 1 , wherein the capture moiety modified nucleotide comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, uracil, and cytosine, or a derivative thereof.
11. The method of claim 10 , wherein the capture moiety modified nucleotide comprises an adenine nucleobase or derivative thereof, optionally wherein the capture moiety is covalently linked to the adenine nucleobase or derivative thereof at position 5, 6, 7 or 8, optionally wherein position 7 of the adenine nucleobase is a carbon atom.
12. The method of claim 1 , wherein the binding partner is streptavidin, optionally wherein the streptavidin is attached to a paramagnetic bead.
13. The method of claim 1 , wherein, in step (a), one nucleotide is added to the 3′ end of the double-stranded nucleic acid comprising the target nucleotide sequence.
14. The method of claim 1 further comprising a washing step after step (a) and before step (b).
15. The method of claim 1 further comprising, prior to step (a), 5′ phosphorylating the double-stranded nucleic acid.
16. The method of claim 1 , wherein, in step (b), the double-stranded nucleic acid is ligated to the adapter nucleic acid in the presence of a crowding agent, optionally wherein the crowding agent is polyethylene glycol in an amount representing 5% to 50% of a ligation mixture.
17. The method of claim 1 , wherein the target nucleotide sequence is within a sequence corresponding to a T-cell receptor constant region or to an immunoglobulin heavy or light chain constant region.
18. A method of preparing nucleic acids for analysis, the method comprising:
(a) preparing a cDNA by conducting a randomly-primed first strand synthesis reaction using a cell-free RNA (cfRNA) preparation as a template and a second strand synthesis reaction using a product of the randomly-primed first strand synthesis reaction as a template, wherein the cfRNA preparation comprises a target nucleotide sequence;
(b) end repairing the cDNA to produce a blunt-ended, double-stranded nucleic acid comprising the target nucleotide sequence;
(c) washing the blunt-ended, double-stranded nucleic acid;
(d) adding one or more nucleotides to the 3′ end of the nucleic acid washed in step (c), wherein at least one of the one or more nucleotides is a capture moiety modified nucleotide;
(e) washing the nucleic acid produced in step (d);
(f) ligating an adapter nucleic acid that comprises a ligatable duplex portion and an overhang sequence to the nucleic acid washed in step (e) to produce a ligation product, wherein the overhang sequence is complementary with the one or more nucleotides added in step (d);
(g) amplifying the ligation product by 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;
(h) amplifying an amplification product of step (g) by polymerase chain reaction using a second adapter primer and a second target-specific primer, wherein the second target-specific primer is nested relative to the first target-specific primer; and
(i) washing the amplification product of step (h).
19. The method of claim 18 , wherein the capture moiety modified nucleotide comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, uracil, and cytosine, or a derivative thereof.
20. The method of claim 19 , wherein the capture moiety modified nucleotide comprises an adenine nucleobase or derivative thereof, optionally wherein the capture moiety is covalently linked to the adenine nucleobase or derivative thereof at position 5, 6, 7 or 8, optionally wherein position 7 of the adenine nucleobase is a carbon atom.
21. The method of claim 19 , wherein the capture moiety is a biotin moiety.
22. The method of claim 21 , wherein the biotin moiety is covalently linked to the nucleotide via a linker of 5 to 20 atoms in length.
23. The method of claim 19 , wherein the method further comprises, following step (f) and before step (g), capturing the ligation product using an immobilized binding partner of the capture moiety of the capture moiety modified nucleotide.
24. The method of claim 23 , wherein the binding partner is streptavidin, optionally wherein the streptavidin is attached to a paramagnetic bead.
25. The method of claim 23 , further comprising purifying the captured ligation product.
26. The method of claim 18 , wherein the washing steps are performed using a solid-phase reversible immobilization technique.
27. The method of claim 18 , wherein the second adapter primer is nested relative to the first adapter primer.