METHODS FOR NON-INVASIVE PRENATAL PLOIDY CALLING
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.
1 . A method for amplifying and sequencing DNA, comprising:
ligating adaptors to cell-free DNA isolated from a biological sample, wherein the adaptors each comprises a universal amplification sequence;
performing a first PCR to simultaneously amplify at least 100 target loci using a universal primer and at least 100 target-specific primers in a single reaction volume;
performing a second, nested PCR to simultaneously amplify the at least 100 target loci using a universal primer and at least 100 inner target-specific primers in a single reaction volume;
performing high-throughput sequencing to sequence the amplified DNA comprising the target 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 200 target loci using a universal primer and at least 200 target-specific primers in a single reaction volume.
4 . The method of claim 1 , wherein the first PCR comprises simultaneously amplifying at least 500 target loci using a universal primer and at least 500 target-specific primers in a single reaction volume.
5 . The method of claim 1 , wherein the first PCR comprises simultaneously amplifying at least 1,000 target loci using a universal primer and at least 1,000 target-specific primers in a single reaction volume.
6 . The method of claim 1 , wherein the second PCR comprises simultaneously amplifying at least 200 target loci using a universal primer and at least 200 inner target-specific primers in a single reaction volume.
7 . The method of claim 1 , wherein the second PCR comprises simultaneously amplifying at least 500 target loci using a universal primer and at least 500 inner target-specific primers in a single reaction volume.
8 . The method of claim 1 , wherein the second PCR comprises simultaneously amplifying at least 1,000 target loci using a universal primer and at least 1,000 inner target-specific primers in a single reaction volume.
9 . The method of claim 1 , wherein the inner target-specific primers each comprises a random molecular barcode sequence.
10 . The method of claim 1 , wherein the adaptors each comprises a random molecular barcode sequence.
11 . The method of claim 8 , wherein the amplified DNA comprising the target loci are tagged with up to 1024 different molecular barcode sequences.
12 . The method of claim 8 , wherein the amplified DNA comprising the target loci are tagged with 1024-65536 different molecular barcode sequences.
13 . The method of claim 1 , wherein the concentration of each target-specific primer is less than 20 nM.
14 . The method of claim 1 , wherein the length of the annealing step of the first PCR or the second PCR is greater than 10 minutes.
15 . The method of claim 1 , wherein at least 90% of the amplified DNA map to the target loci.
16 . The method of claim 1 , wherein the target loci are SNP loci.
17 . The method of claim 1 , wherein the cell-free DNA comprises DNA from mixed origin.
18 . The method of claim 17 , wherein the cell-free DNA comprises DNA from a fetus.
19 . The method of claim 17 , wherein the cell-free DNA comprises DNA from a tumor.
20 . The method of claim 17 , wherein the cell-free DNA comprises DNA from a transplant.