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 nucleic acid sample comprising target loci with a library of test primers comprising at least 1,000 different primer pairs to produce a reaction mixture in one reaction volume; wherein each primer pair includes a forward test primer and a reverse test primer that hybridize to the same target locus, and wherein the primers do not include molecular inversion probes (MIPs);
(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons; wherein the concentration of each test primer is less than 10 nM; wherein the length of the annealing step of the reaction conditions is greater than 10 minutes; wherein at least 1,000 different target loci are simultaneously amplified; and wherein (i) less than 20% of the amplified products are test primer dimers, (ii) at least 80% of the amplified products are target amplicons, and (iii) at least 80% of the target loci are amplified; and
(c) sequencing the amplified products.
2. The method of claim 1 , wherein at least 5,000 different target loci are amplified.
3. The method of claim 1 , wherein the length of the target amplicons is less than 100 nucleotides.
4. The method of claim 1 , wherein the test primers are selected from a library of candidate primers based at least in part on the ability of the candidate primers to form primer dimers.
5. The method of claim 1 , wherein the nucleic acid sample is isolated from a blood, plasma, or serum sample.
6. The method of claim 5 , wherein the nucleic acid sample comprises DNA from mixed origin.
7. The method of claim 6 , wherein the nucleic acid sample comprises DNA from a fetus.
8. The method of claim 6 , wherein the nucleic acid sample comprises DNA from a transplant.
9. The method of claim 1 , wherein the target loci are present in the human genome, or wherein the target loci comprise human single nucleotide polymorphisms.
10. The method of claim 1 , wherein the test primers are selected from a library of candidate primers, by a method comprising:
(a) calculating on a computer an undesirability score for at least 90% of the possible combinations of two candidate primers from the library, wherein each undesirability score is based at least in part on the likelihood of dimer formation between the two candidate primers;
(b) removing from the library of candidate primers the candidate primer that is part of the greatest number of combinations of two candidate primers with an undesirability score above a first minimum threshold;
(c) if the candidate primer removed in step (b) is a member of a primer pair, then removing the other member of the primer pair from the library of candidate primers; and
(d) optionally repeating steps (b) and (c), thereby selecting a library of test primers.
11. The method of claim 10 , comprising either
(i) further reducing the number of candidate primers remaining in the library by decreasing the first minimum threshold used in step (b) to a lower second minimum threshold and repeating steps (b) and (c) until the undesirability scores for the candidate primer combinations remaining in the library are all equal to or below the second minimum threshold, or until the number of candidate primers remaining in the library is reduced to a desired number; or
(ii) increasing the number of candidate primers remaining in the library by increasing the first minimum threshold used in step (b) to a higher second minimum threshold and repeating steps (b) and (c) until the undesirability scores for the candidate primer combinations remaining in the library are all equal to or below the second minimum threshold, or until the number of candidate primers remaining in the library is reduced to a desired number.
12. The method of claim 10 , further comprising, prior to step (b), removing a primer pair from the library that produces a target amplicon that overlaps with a target amplicon produced by another primer pair.
13. The method of claim 10 , wherein the undesirability scores are based at least in part on one or more parameters selected from the group consisting of heterozygosity rate of a target locus, disease prevalence associated with a polymorphism or mutation at a target locus, disease penetrance associated with a polymorphism or mutation at a target locus, specificity of the candidate primer for a target locus, size of the candidate primer, melting temperature of a target amplicon, guanine-cytosine (GC) content of a target amplicon, amplification efficiency of a target amplicon, and size of a target amplicon.
14. The method of claim 10 , comprising selecting a library of at least 1,000 different test primers and using at least 1,000 of the selected test primers to simultaneously amplify at least 1,000 different target loci.
15. The method of claim 1 , wherein the melting temperatures of the primers of the primer pairs is 55 to 65° C.
16. The method of claim 1 , wherein the annealing temperature comprises a temperature that is raised.
17. The method of claim 1 , wherein the melting temperatures of the primers of the primer pairs is 57 to 60.5° C.
18. The method of claim 1 , wherein the PCR is nested PCR.
19. The method of claim 1 , wherein range of the length of the target amplicons is between 50 and 100 nucleotides.