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.

1 . A method for determining a ploidy status of a chromosome or chromosome segment of a cancer in a host, the method comprising:
 measuring the DNA in a mixed sample that comprises DNA from normal cells from the host and DNA from the cancer at a plurality of at least 10 polymorphic loci on the chromosome or chromosome segment;   determining allele counts at the plurality of polymorphic loci from the DNA measurements made on the sample;   fitting, on a computer, the determined allele counts to expected allele counts at the plurality of polymorphic loci for each of a plurality of ploidy hypotheses each specifying a different possible ploidy state of the chromosome or chromosome segment for the cancer, using a joint distribution model that takes into account the expected linkage between the plurality of polymorphic loci on the chromosome or chromosome segment, to determine a relative probability of each of the ploidy hypotheses; and   outputting the hypothesis with the greatest probability of being true as an indication of the ploidy status of the chromosome or chromosome segment of the cancer.   
     
     
         2 . The method of  claim 1 , wherein the sample is a plasma sample of the host. 
     
     
         3 . The method of  claim 1 , wherein the method further comprises preferentially enriching the DNA in the sample at a plurality of polymorphic loci prior to the measuring step. 
     
     
         4 . The method of  claim 3 , wherein the preferentially enriching the DNA in the sample at the plurality of polymorphic loci comprises:
 obtaining a plurality of pre-circularized probes in which each probe targets one of the polymorphic loci, wherein the 3′ and 5′ end of each probe is designed to hybridize to a region of DNA that is separated from the polymorphic site of the locus by a small number of bases, and wherein the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21 to 25,26 to 30,31 to 60, or a combination thereof;   hybridizing the pre-circularized probes to DNA from the sample;   filling the gap between the hybridized probe ends using DNA polymerase; and   circularizing the pre-circularized probe; and amplifying the circularized probe.   
     
     
         5 . The method of  claim 3 , wherein the preferentially enriching the DNA at the plurality of polymorphic loci comprises:
 obtaining a plurality of ligation-mediated PCR probes in which each PCR probe targets one of the polymorphic loci, wherein the corresponding upstream and downstream PCR probes are designed to hybridize to a region of DNA on one strand of DNA that is separated from the polymorphic site of the locus by a small number of bases, and wherein the small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 21 to 25, 26 to 30, 31 to 60, or a combination thereof;   hybridizing the litigation-mediated PCR probes to the DNA from the sample;   filing the gap between the ligation-mediated PCR probe ends using DNA polymerase;   ligating the ligation-mediated PCR probes; and   amplifying the ligated ligation-mediated PCR probes.   
     
     
         6 . The method of  claim 3 , wherein the preferentially enriching the DNA at the plurality of polymorphic loci comprises:
 obtaining a plurality of hybrid capture probes that target the polymorphic loci;   hybridizing the hybrid capture probes to the DNA in the sample; and   physically removing some or all of the unhybridized DNA from the sample of DNA.   
     
     
         7 . The method of  claim 3 , wherein the preferentially enriching the DNA at the plurality of polymorphic loci comprises:
 obtaining a plurality of inner forward primers in which each primer targets one of the polymorphic loci, wherein the 3′ end of each of the inner forward primers is designed to hybridize to a region of DNA upstream from the polymorphic site of the locus and separated from the polymorphic site by a small number of bases, and wherein the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, and 31 to 60 base pairs;   optionally obtaining a plurality of inner reverse primers in which each primer targets one of the polymorphic loci, wherein the 3′ end of each of the inner reverse primers is designed to hybridize to a region of DNA upstream from the polymorphic site of the locus and separated from the polymorphic site by a small number of bases, and wherein the small number is selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, and 31 to 60 base pairs;   hybridizing the inner primers to the DNA; and   amplifying the DNA using the polymerase chain reaction to form amplicons.   
     
     
         8 . The method of  claim 7 , further comprising:
 obtaining a plurality of outer forward primers in which each primer targets one of the polymorphic loci, wherein each of the outer forward primers is designed to hybridize to the region of DNA upstream from the corresponding inner forward primer;   optionally obtaining a plurality of outer reverse primers in which each primer targets one of the polymorphic loci, wherein each of the outer reverse primers is designed to hybridize to the region of DNA immediately downstream from the corresponding inner reverse primer;   hybridizing the first primers to the DNA; and   amplifying the DNA using the polymerase chain reaction.   
     
     
         9 . The method of  claim 7 , further comprising:
 obtaining a plurality of outer reverse primers in which each primer targets one of the polymorphic loci, wherein each of the outer reverse primers is designed to hybridize to the region of DNA immediately downstream from the corresponding inner reverse primer;   optionally obtaining a plurality of outer forward primers in which each primer targets one of the polymorphic loci, wherein each of the outer forward primers is designed to hybridize to the region of DNA upstream from the corresponding inner forward primer;   hybridizing the first primers to the DNA; and amplifying the DNA using the polymerase chain reaction.   
     
     
         10 . The method of  claim 7 , further comprising:
 appending universal adapters to the DNA in the sample; and   amplifying the DNA in the sample using the polymerase chain reaction.   
     
     
         11 . The method of  claim 7 , wherein the amplifying the DNA is done in one or a plurality of individual reaction volumes, wherein each individual reaction volume contains more than 500 different forward and reverse primer pairs. 
     
     
         12 . The method of  claim 7 , further comprises dividing the first sample into a plurality of portions, and wherein the DNA in each portion is preferentially enriched at a subset of the plurality of polymorphic loci. 
     
     
         13 . The method of  claim 7 , wherein the inner primers are selected by identifying primer pairs likely to form undesired primer duplexes and removing from the plurality of primers at least one of the pair of primers identified as being likely to form undesired primer duplexes. 
     
     
         14 . The method of  claim 1 , further comprising obtaining normal host cell genotypic data at the plurality of polymorphic loci from normal cells from the host. 
     
     
         15 . The method of  claim 14 , wherein the step of fitting the allele counts to expected allele counts at each of the plurality of polymorphic loci for each of the plurality of ploidy hypotheses, uses the normal host cell genotypic data. 
     
     
         16 . The method of  claim 14 , wherein the sample is isolated from host plasma, and wherein the obtaining normal host cell genotypic data is done by estimating the normal cell genotypic data from the DNA measurements made on the sample. 
     
     
         17 . The method of  claim 3 , wherein the preferential enrichment results in an average degree of allelic bias between the sample after preferential enrichment and the sample prior to preferential enrichment of no more than a factor of 1.2. 
     
     
         18 . The method of  claim 1 , wherein the plurality of polymorphic loci are single nucleotide polymorphisms. 
     
     
         19 . The method of  claim 1 , wherein the step of measuring the DNA in the sample is done by sequencing. 
     
     
         20 . The method of  claim 1 , wherein the measurements of the DNA in the sample at the plurality of polymorphic loci are analyzed to estimate the haplotypes present on the chromosome or chromosome segment. 
     
     
         21 . The method of  claim 1 , wherein the step of fitting is performed using the joint distribution model that takes into account the probability of chromosomes crossing over at different locations in the chromosome or chromosome segment to model dependence between blocks of polymorphic alleles on the chromosome or chromosome segment. 
     
     
         22 . The method of  claim 1 , wherein the step of fitting the allele counts to expected allele counts is done using a method that does not require the use of a reference chromosome. 
     
     
         23 . The method of  claim 1 , wherein the step of fitting the allele counts to expected allele counts makes use of an estimated fraction of circulating cancer DNA in the sample. 
     
     
         24 . The method of  claim 1 , wherein the step of fitting the allele counts to expected allele counts further comprises determining relative probabilities for each of the ploidy hypotheses using one or more statistical techniques selected from the group consisting of a read count analysis, comparing heterozygosity rates, a statistic that is only available when normal host cell genetic information is used, the probability of normalized genotype signals for certain normal host cell contexts, a statistic that is calculated using an estimated circulating cancer DNA fraction of the sample, and combinations thereof 
     
     
         25 . The method of  claim 1 , wherein a confidence estimate is calculated for the outputted hypothesis. 
     
     
         26 . The method of  claim 1 , wherein the allele counts measured on the sample comprise quantitative measurements of the number of independent observations of each allele at each polymorphic locus, and wherein the expected allele counts comprise expected quantitative measurements of the number of independent observations of each allele at each polymorphic locus. 
     
     
         27 . The method of  claim 1 , wherein determining the relative probability of each of the ploidy hypotheses does not involve calculating the ratio of the alleles counts measured on the sample at a polymorphic locus. 
     
     
         28 . The method of  claim 1 , further comprising:
 measuring the DNA in a normal sample from the host that comprises DNA from normal cells from the host and not from the cancer, at the plurality of at least 10 polymorphic loci on the chromosome or chromosome segment;   determining, on a computer, normal host cell allele counts at the plurality of polymorphic loci from the DNA measurements made on the normal sample;   phasing the normal host cell allele counts taking into account the expected linkage of the polymorphic loci of the plurality of polymorphic loci, and using the phased normal host cell allele counts to determine the expected allele counts at each of the plurality of polymorphic loci.