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 . (canceled)
2 . A method comprising:
tagging cell-free DNA extracted from a biological sample from a human subject with molecular barcodes to generate tagged DNA;
performing targeted enrichment on the tagged DNA, wherein the targeted enrichment comprises (i) contacting tagged DNA with a library comprising oligonucleotides that specifically hybridize to at least 800 target loci, wherein a target locus comprises a variant position, and (ii) amplifying tagged DNA that was contacted in step (i) in the same reaction volume to generate enriched DNA;
determining the sequence of at least some of the enriched DNA or DNA derived therefrom by performing high-throughput sequencing to generate sequence reads; and
analyzing the sequence reads.
3 . The method of claim 2 , wherein the analyzing comprises detecting one or more variants from the sequence reads.
4 . The method of claim 2 , wherein the biological sample comprises blood or plasma.
5 . The method of claim 2 , wherein the biological sample comprises urine or saliva.
6 . The method of claim 2 , wherein the cell-free DNA comprises cell-free DNA derived from both normal and cancer cells of the human subject.
7 . The method of claim 2 , wherein the library comprises oligonucleotides that specifically hybridize to at least 1,200 different target loci comprising single-nucleotide-variant positions on one or more chromosomes.
8 . The method of claim 2 , wherein the library comprises oligonucleotides that specifically hybridize to 1,000 to 20,000 different target loci comprising single-nucleotide-variant positions on one or more chromosomes.
9 . The method of claim 2 , wherein the library comprises oligonucleotides that specifically hybridize to 1,000 to 10,000 different target loci comprising single-nucleotide-variant positions on one or more chromosomes.
10 . The method of claim 2 , wherein the concentration of each target-specific oligonucleotide in the library of target-specific oligonucleotides is 5 nM or less.
11 . The method of claim 2 , wherein the high-throughput sequencing is sequencing-by-synthesis.
12 . The method of claim 2 , wherein at least 90% of the sequence reads comprises the variants at loci specifically hybridized by the oligonucleotides.
13 . The method of claim 2 , wherein at least 95% of the sequence reads comprises the variants at loci specifically hybridized by the oligonucleotides.
14 . The method of claim 2 , wherein the tagged DNA are tagged with up to 1024 different molecular barcodes.
15 . The method of claim 2 , wherein the tagged DNA are tagged with 1024 to 65536 different molecular barcodes.