Methods for nested pcr amplification

Abstract

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

What is claimed is: 
     
         1 . A method for nested PCR amplification, comprising:
 ligating at least one adaptor to cell-free DNA isolated from a biological sample or DNA derived therefrom, wherein the adaptor comprise a universal priming site,   performing a first PCR to simultaneously amplify at least 10 target loci in one reaction volume using a first universal primer and at least 10 target-specific primers,   performing a second, nested PCR to simultaneously amplify the at least 10 target loci in one reaction volume using a second universal primer and at least 10 inner target-specific primers to obtain amplified DNA,   wherein primer binding sites of the inner target-specific primers of the second PCR are internal to primer binding sites of the target-specific primers of the first PCR, wherein at least 80% of the amplified DNA maps to the target loci, and wherein the target loci are single nucleotide polymorphism or variant loci.   
     
     
         2 . The method of  claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample. 
     
     
         3 . The method of  claim 1 , wherein the first PCR comprises simultaneously amplifying at least 50 target loci in one reaction volume. 
     
     
         4 . The method of  claim 1 , wherein the second PCR comprises simultaneously amplifying at least 50 target loci in one reaction volume. 
     
     
         5 . The method of  claim 1 , wherein the concentration of each of the target-specific primers in the first and/or second PCR is less than 20 nM. 
     
     
         6 . The method of  claim 1 , wherein the concentration of each of the target-specific primers in the first and/or second PCR is less than 10 nM. 
     
     
         7 . The method of  claim 1 , wherein the length of an annealing step in the first and/or second PCR is at least 3 minutes. 
     
     
         8 . The method of  claim 1 , wherein the length of an annealing step in the first and/or second PCR is at least 5 minutes. 
     
     
         9 . The method of  claim 1 , wherein the adaptor comprises a molecular barcode. 
     
     
         10 . The method of  claim 1 , wherein the amplified DNA are tagged with up to 1024 different molecular barcodes. 
     
     
         11 . The method of  claim 1 , wherein the amplified DNA are tagged with 1024-65536 different molecular barcodes. 
     
     
         12 . The method of  claim 1 , wherein at least 90% of the amplified DNA maps to the target loci. 
     
     
         13 . The method of  claim 1 , wherein at least one of the target specific primers comprises a tail, wherein the tail has no homology to the target loci and comprises a common priming site. 
     
     
         14 . The method of  claim 1 , wherein at least one of the target specific primers comprises a priming site for a subsequent amplification to add barcode sequences for multiplex sequencing. 
     
     
         15 . The method of  claim 1 , wherein the method further comprises barcoding PCR to introduce a sample barcode, and wherein the amplified DNA from multiple samples are pooled together and sequenced in a single sequencing lane. 
     
     
         16 . A method for nested PCR amplification, comprising:
 performing a multiplex targeted pre-amplification on cell-free DNA isolated from a biological sample or DNA derived therefrom, wherein the pre-amplification is a linear amplification,   performing a first PCR to simultaneously amplify at least 10 target loci in one reaction volume using a first universal primer and at least 10 target-specific primers,   performing a second, nested PCR to simultaneously amplify the at least 10 target loci in one reaction volume using a second universal primer and at least 10 inner target-specific primers to obtain amplified DNA,   wherein primer binding sites of the inner target-specific primers of the second PCR are internal to primer binding sites of the target-specific primers of the first PCR, wherein at least 80% of the amplified DNA maps to the target loci, and wherein the target loci are single nucleotide polymorphism or variant loci.   
     
     
         17 . The method of  claim 16 , wherein the biological sample is a blood, plasma, serum, or urine sample. 
     
     
         18 . The method of  claim 16 , wherein the first PCR comprises simultaneously amplifying at least 50 target loci in one reaction volume. 
     
     
         19 . The method of  claim 16 , wherein the second PCR comprises simultaneously amplifying at least 50 target loci in one reaction volume. 
     
     
         20 . The method of  claim 16 , wherein the concentration of each of the target-specific primers in the first and/or second PCR is less than 20 nM. 
     
     
         21 . The method of  claim 16 , wherein the concentration of each of the target-specific primers in the first and/or second PCR is less than 10 nM. 
     
     
         22 . The method of  claim 16 , wherein the length of an annealing step in the first and/or second PCR is at least 3 minutes. 
     
     
         23 . The method of  claim 16 , wherein the length of an annealing step in the first and/or second PCR is at least 5 minutes. 
     
     
         24 . The method of  claim 16 , wherein the amplified DNA are tagged with molecular barcodes. 
     
     
         25 . The method of  claim 16 , wherein the amplified DNA are tagged with up to 1024 molecular barcodes. 
     
     
         26 . The method of  claim 16 , wherein the amplified DNA are tagged with 1024-65536 molecular barcodes. 
     
     
         27 . The method of  claim 16 , wherein at least 90% of the amplified DNA maps to the target loci. 
     
     
         28 . The method of  claim 16 , wherein at least one of the target specific primers comprises a tail, wherein the tail has no homology to the target loci and comprises a common priming site. 
     
     
         29 . The method of  claim 16 , wherein at least one of the target specific primers comprises a priming site for a subsequent amplification to add barcode sequences for multiplex sequencing. 
     
     
         30 . The method of  claim 16 , wherein the method further comprises barcoding PCR to introduce a sample barcode, and wherein the amplified DNA from multiple samples are pooled together and sequenced in a single sequencing lane.