Methods for simultaneous amplification of target loci
The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.
1. A method of nested amplification, the method comprising:
(a) performing a first amplification on a plurality of target loci of a nucleic acid sample using a first library of primers comprising a plurality of target-specific primers and optionally a universal primer,
(b) performing a second, nested amplification on the target loci using a second library of primers comprising a plurality of inner target-specific primers and optionally a universal primer to produce amplified products,
wherein, in (a), (b), or both, the length of an annealing step is greater than 3 minutes, and at least two of the primers have a melting temperature lower than an annealing temperature of the annealing step.
2. The method of claim 1 , further comprising (c) sequencing the amplified products by high-throughput sequencing.
3. The method of claim 1 , wherein melting temperature is determined according to Primer3 program.
4. The method of claim 1 , wherein at least two of the primers have a melting temperature that is at least 2° C. lower than the annealing temperature.
5. The method of claim 1 , wherein at least two of the primers have a melting temperature that is at least 3° C. lower than the annealing temperature.
6. The method of claim 1 , wherein the annealing temperature is at least 2° C. greater than the highest melting temperature of the primers.
7. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than the highest melting temperature of the primers.
8. The method of claim 1 , wherein the annealing temperature is at least 2° C. greater than the average melting temperature of the primers.
9. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than the average melting temperature of the primers.
10. The method of claim 1 , wherein the range of melting temperatures of the primers is less than 5° C.
11. The method of claim 1 , wherein the ΔG values for each possible combination of two primers in the library are all equal to or greater than −5 kcal/mol.
12. The method of claim 1 , wherein in (a), (b), or both, at least 10 different target loci are simultaneously amplified.
13. The method of claim 1 , wherein in (a), (b), or both, at least 50 different target loci are simultaneously amplified.
14. The method of claim 1 , wherein in (a), (b), or both, at least 100 different target loci are simultaneously amplified.
15. The method of claim 1 , wherein at least 90% of the amplified products are target amplicons.
16. The method of claim 1 , wherein at least 90% of the target loci are amplified.
17. The method of claim 1 , wherein less than 20% of the amplified products are primer dimers.
18. The method of claim 1 , wherein the concentration of each primer in the library is less than 20 nM.
19. The method of claim 1 , wherein the primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3′ end of the primers.
20. The method of claim 1 , wherein the length of the annealing step of the reaction conditions is 5-60 minutes.
21. The method of claim 1 , wherein the target loci are SNP loci.
22. The method of claim 1 , wherein the nucleic acid sample comprises cell-free DNA, and wherein the cell-free DNA comprises tumor DNA.