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:
isolating cell-free DNA from a biological sample and tagging the isolated cell-free DNA with at least one universal priming sequence to obtain tagged DNA;
performing a universal PCR on the tagged DNA using the universal priming sequence;
performing a first targeted PCR using a first universal primer that binds to all or a part of the universal priming sequence and 10 to 1,000 target-specific primers to amplify 10 to 1,000 target loci together in the same reaction volume;
performing a second targeted, nested PCR using a second universal primer that binds to all or a part of the universal priming sequence and 10 to 1,000 inner target-specific primers to amplify the 10 to 1,000 target loci together in the same reaction volume, wherein at least one of the primers comprises a sequencing tag; and
performing high-throughput sequencing to sequence the amplified DNA comprising the target loci.
2 . The method of claim 1 , wherein the cell-free DNA is isolated from a blood, plasma, serum, or urine sample.
3 . The method of claim 1 , wherein the method further comprises subjecting the isolated cell-free DNA to blunt-ending and dA-tailing, prior to the tagging step.
4 . The method of claim 1 , wherein the second targeted PCR is one-sided nested PCR.
5 . The method of claim 1 , wherein the first targeted PCR comprises amplifying 20 to 500 target loci together in the same reaction volume using the first universal primer and 20 to 500 target specific primers.
6 . The method of claim 5 , wherein the second targeted PCR comprises amplifying 20 to 500 target loci together in the same reaction volume using the second universal primer and 20 to 500 target-specific primers.
7 . The method of claim 1 , wherein the first targeted PCR comprises amplifying 20 to 200 target loci together in the same reaction volume using the first universal primer and 20 to 200 inner target-specific primers.
8 . The method of claim 7 , wherein the second targeted PCR comprises amplifying 20 to 200 target loci together in the same reaction volume using the second universal primer and 20 to 200 inner target-specific primers.
9 . The method of claim 1 , wherein at least one target-specific primer in the first and/or second targeted PCR has a concentration of 1-50 nM.
10 . The method of claim 1 , wherein at least one target-specific primer in the first and/or second targeted PCR has a concentration of 1-20 nM.
11 . The method of claim 1 , wherein the first and/or second targeted PCR comprises an annealing step having a duration of longer than 2 minutes.
12 . The method of claim 1 , wherein the first and/or second targeted PCR comprises an annealing step having a duration of longer than 4 minutes.
13 . The method of claim 1 , wherein at least 90% of the amplified DNA maps to the target loci.
14 . The method of claim 1 , wherein the target loci comprise a single nucleotide polymorphism or variant loci.
15 . The method of claim 1 , wherein the method further comprises, after the universal PCR, dividing the amplified DNA into a plurality of aliquots and performing the first and the second targeted PCRs in each aliquot in parallel, and pooling the plurality of aliquots together prior to the high-throughput sequencing.
16 . A method for amplifying and sequencing DNA, comprising:
ligating at least one adaptor comprising a universal priming sequence to cell-free DNA isolated from a biological sample to obtain adaptor-ligated DNA;
performing a universal PCR on the adaptor-ligated DNA using the universal priming sequence;
performing a first targeted PCR using a first universal primer that binds to all or a part of the universal priming sequence and 10 to 1,000 target-specific primers to amplify 10 to 1,000 target loci together in the same reaction volume;
performing a second targeted, nested PCR using a second universal primer that binds to all or a part of the universal priming sequence and 10 to 1,000 inner target-specific primers to amplify 10 to 1,000 target loci together in the same reaction volume, wherein at least one of the primers comprises a sequencing tag; and
performing high-throughput sequencing to sequence the amplified DNA comprising the target loci.
17 . The method of claim 16 , wherein the cell-free DNA is isolated from a blood, plasma, serum, or urine sample.
18 . The method of claim 16 , wherein the method further comprises subjecting the isolated cell-free DNA to blunt-ending and dA-tailing, prior to the ligating step.
19 . The method of claim 16 , wherein the second targeted PCR is one-sided nested PCR.
20 . The method of claim 16 , wherein the first targeted PCR comprises amplifying 20 to 500 target loci together in the same reaction volume using the first universal primer and 20 to 500 target specific primers.
21 . The method of claim 20 , wherein the second targeted PCR comprises amplifying 20 to 500 target loci together in the same reaction volume using the second universal primer and 20 to 500 target-specific primers.
22 . The method of claim 16 , wherein the first targeted PCR comprises amplifying 20 to 200 target loci together in the same reaction volume using the first universal primer and 20 to 200 inner target-specific primers.
23 . The method of claim 22 , wherein the second targeted PCR comprises amplifying 20 to 200 target loci together in the same reaction volume using the second universal primer and 20 to 200 inner target-specific primers.
24 . The method of claim 16 , wherein the concentration of at least one target-specific primer in the first and/or second targeted PCR is 1-50 nM.
25 . The method of claim 16 , wherein the concentration of at least one target-specific primer in the first and/or second targeted PCR is 1-20 nM.
26 . The method of claim 16 , wherein the annealing step of the first and/or second targeted PCR is longer than 2 minutes.
27 . The method of claim 16 , wherein the annealing step of the first and/or second targeted PCR is longer than 4 minutes.
28 . The method of claim 16 , wherein at least 90% of the amplified DNA maps to the target loci.
29 . The method of claim 16 , wherein the target loci comprise a single nucleotide polymorphism or variant loci.
30 . The method of claim 16 , wherein the method further comprises, after the universal PCR, dividing the amplified DNA into a plurality of aliquots and performing the first and the second targeted PCRs in each aliquot in parallel, and pooling the plurality of aliquots together prior to the high-throughput sequencing.