Highly multiplex pcr methods and compositions

Abstract

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.

What is claimed is: 
     
         1 . A library of test primers that simultaneously amplify at least 1,000 different target loci, wherein (i) less than 30% of the amplified products are test primer dimers, (ii) at least 80% of the amplified products are target amplicons, or (iii) at least 80% of the targeted loci are amplified. 
     
     
         2 . A method of amplifying target loci in a nucleic acid sample, the method comprising:
 (a) contacting the nucleic acid sample with the primer library of  claim 1  to produce a reaction mixture; and   (b) subjecting the reaction mixture to primer extension reaction conditions to produce amplified products comprising target amplicons.   
     
     
         3 . The method of  claim 2 , wherein at least 5,000 different target loci are amplified. 
     
     
         4 . The method of  claim 3 , wherein at least 10,000 different target loci are amplified. 
     
     
         5 . The method of  claim 4 , wherein at least 20,000 different target loci are amplified. 
     
     
         6 . The method of  claim 2 , wherein at least 95% of the amplified products are target amplicons. 
     
     
         7 . The method of  claim 6 , wherein at least 99% of the amplified products are target amplicons. 
     
     
         8 . The method of  claim 7 , wherein at least 95% of the targeted loci are amplified. 
     
     
         9 . The method of  claim 8 , wherein at least 99% of the targeted loci are amplified. 
     
     
         10 . The method of  claim 2 , wherein less than 10% of the amplified products are test primer dimers. 
     
     
         11 . The method of  claim 10 , wherein less than 1% of the amplified products are test primer dimers. 
     
     
         12 . The method of  claim 2 , 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. 
     
     
         13 . The method of  claim 12 , wherein the selection comprises
 (i) calculating on a computer an undesirability score for most or all 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;   (ii) removing the candidate primer with the highest undesirability score from the library of candidate primers;   (iii) if the candidate primer removed in step (ii) is a member of a primer pair, then removing the other member of the primer pair from the library of candidate primers; and   (iv) optionally repeating steps (ii) and (iii).   
     
     
         14 . The method of  claim 12 , wherein the selection comprises
 (i) calculating on a computer an undesirability score for most or all 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;   (ii) 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;   (iii) if the candidate primer removed in step (ii) is a member of a primer pair, then removing the other member of the primer pair from the library of candidate primers; and   (iv) optionally repeating steps (ii) and (iii).   
     
     
         15 . The method of  claim 2 , wherein the concentration of each test primer is less than 10 nM. 
     
     
         16 . The method of  claim 2 , wherein the target loci comprise single nucleotide polymorphisms. 
     
     
         17 . A method of selecting test primers from a library of candidate primers, the method comprising:
 (a) calculating on a computer an undesirability score for most or all 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 the candidate primer with the highest undesirability score from the library of candidate primers;   (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.   
     
     
         18 . A method of selecting test primers from a library of candidate primers, the method comprising:
 (a) calculating on a computer an undesirability score for most or all 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.   
     
     
         19 . The method of  claim 18 , comprising 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. 
     
     
         20 . The method of  claim 18 , comprising 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. 
     
     
         21 . The method of  claim 18 , wherein a candidate primer is selected out of a group of two or more candidate primers with equal undesirability scores for removal from the library of candidate primers based on one or more other parameters. 
     
     
         22 . The method of  claim 18 , wherein the undesirability scores are based at least in part on one or more parameters selected from the group consisting of heterozygosity rate of the target locus, disease prevalence associated with a polymorphism at the target locus, disease penetrance associated with a polymorphism at the target locus, specificity of the candidate primer for the target locus, size of the candidate primer, melting temperature of the target amplicon, GC content of the target amplicon, amplification efficiency of the target amplicon, and size of the target amplicon. 
     
     
         23 . The method of  claim 18 , 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.