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 preparing a deoxyribonucleic acid (DNA) fraction from a biological sample useful for analyzing genotypes for DNA from cancer cells in the biological sample, comprising:
(a) extracting cell-free DNA from the biological sample, wherein the extracted cell-free DNA comprises DNA from cancer cells;
(b) producing a fraction of the DNA extracted in (a) by performing targeted multiplex amplification on the cell-free DNA extracted in (a) to amplify at least 50 different target loci in a single reaction volume, followed by a nested PCR amplification of the at least 50 different target loci in a single reaction volume;
(c) analyzing the fraction of the DNA produced in (b) by performing high-throughput sequencing on the DNA to obtain sequence reads and determining the sequence of the target loci based on the sequence reads.
2. The method of claim 1 , wherein the method further comprises enriching the DNA in the sample at a plurality of target loci prior to the high-throughput sequencing.
3. The method of claim 2 , wherein the enrichment comprises:
obtaining a plurality of target-specific forward primers in which each primer targets one of the target loci, wherein the 3′ end of each of the forward primers is designed to hybridize to a region of DNA upstream from the target site of the locus and separated from the target site by 1-60 bases;
obtaining at least one reverse primer;
hybridizing the forward primers and the reverse primer to the DNA; and
amplifying the DNA using the polymerase chain reaction to form amplicons.
4. The method of claim 2 , wherein the enrichment comprises:
obtaining at least one forward primer;
obtaining a plurality of target-specific reverse primers in which each primer targets one of the target loci, wherein the 3′ end of each of the reverse primers is designed to hybridize to a region of DNA upstream from the target site of the locus and separated from the target site by 1-60 bases;
hybridizing the reverse primers and the forward primer to the DNA; and
amplifying the DNA using the polymerase chain reaction to form amplicons.
5. The method of claim 3 , wherein the forward 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.
6. The method of claim 4 , wherein the reverse 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.
7. The method of claim 2 , wherein the enrichment results in an average degree of allelic bias between the sample after enrichment and the sample prior to enrichment of no more than a factor of 1.2.
8. The method of claim 1 , wherein the plurality of target loci are single nucleotide polymorphisms.
9. The method of claim 1 , wherein the biological sample is a blood sample.
10. The method of claim 1 , wherein the nested PCR amplification comprises semi-nested PCR, hemi-nested PCR, one-sided nested PCR, or fully nested PCR.
11. The method of claim 1 , wherein at least 80% of the sequence reads map to the target loci.