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 amplifying target loci in a nucleic acid sample, the method comprising:
(a) contacting a sample comprising target human loci with a library of at least 50 non-immobilized, non-identical primers that simultaneously hybridize to at least 50 non-identical target human loci to produce a reaction mixture; wherein the primers do not include molecular inversion probes (MIPs);
(b) subjecting the reaction mixture to primer extension reaction conditions to produce amplified products comprising target amplicons; wherein the annealing temperature for the reaction conditions is greater than a melting temperature of the at least 50 non-identical primers; wherein the length of the annealing step of the reaction conditions is greater than 5 minutes; and wherein at least 50 non-identical target human loci are simultaneously amplified; and
(c) sequencing the amplified products.
2. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than the melting temperature of the at least 50 non-identical primers.
3. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than the highest melting temperature of the at least 50 non-identical primers.
4. The method of claim 3 , wherein the annealing temperature is at least 8° C. greater than the highest melting temperature of the at least 50 non-identical primers.
5. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than the average melting temperature of the at least 50 non-identical primers.
6. The method of claim 5 , wherein the annealing temperature is at least 8° C. greater than the average melting temperature of the at least 50 non-identical primers.
7. The method of claim 1 , wherein the range of melting temperature of the at least 50 non-identical primers is between 1 to 5° C., inclusive.
8. The method of claim 1 , wherein the range of melting temperatures of the at least 50 non-identical primers is less than 5° C.
9. 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.
10. The method of claim 1 , further comprising performing universal amplification on nucleic acids in the sample prior to step (a).
11. The method of claim 1 , wherein at least 50 of the primers have at least 90% identity to the corresponding region of SEQ ID Nos. 1-44,610.
12. The method of claim 1 , wherein the library comprises at least 500 non-immobilized, non-identical primers that simultaneously hybridize to the at least 500 non-identical target human loci, wherein the annealing temperature for the reaction conditions is greater than a melting temperature of the at least 500 non-identical primers, and wherein at least 500 non-identical target human loci are simultaneously amplified.
13. The method of claim 1 , wherein at least 90% of the amplified products are target amplicons.
14. The method of claim 1 , wherein at least 90% of the target human loci are amplified.
15. The method of claim 1 , wherein less than 20% of the amplified products are primer dimers.
16. The method of claim 1 , wherein the concentration of each primer in the library is less than 20 nM.
17. The method of claim 1 , wherein the at least 50 non-identical primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3′ end of the primers.
18. The method of claim 1 , wherein the at least 50 non-identical primers comprise a 5′ region that is not specific for a target human locus followed by a region that is specific for a target human locus, an internal region that is not specific for the target human locus and forms a loop structure, and a 3′ region that is specific for the target human locus.
19. The method of claim 1 , wherein the sample comprises maternal DNA from the pregnant mother of a fetus and fetal DNA, and wherein the method further comprises determining the presence or absence of a fetal chromosome abnormality from the sequencing data.
20. The method of claim 1 , wherein the sample comprises DNA from a single cell.
21. The method of claim 1 , wherein the annealing temperature is 1 to 15° C. greater than the highest melting temperature of the at least 50 non-identical primers.
22. The method of claim 1 , wherein the annealing temperature is 1 to 10° C. greater than the highest melting temperature of the at least 50 non-identical primers.
23. The method of claim 1 , wherein the library comprises at least 1000 non-immobilized, non-identical primer pairs that simultaneously hybridize to at least 1000 non-identical target human loci, wherein the annealing temperature for the reaction conditions is greater than a melting temperature of the at least 1000 non-identical primers, and wherein the at least 1000 non-identical target human loci are simultaneously amplified.
24. The method of claim 1 , wherein the length of the annealing step of the reaction conditions is between 5 and 60 minutes.