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 biological sample obtained from a single individual for use in a genetic testing assay, the method comprising:
identifying at least 10 polymorphic loci for targeting DNA of the single individual in the biological sample;
isolating DNA fragments from the biologic sample, wherein the isolated DNA fragments comprise DNA from the at least 10 targeted polymorphic loci;
tagging the isolated DNA fragments with a first known tail sequence and a molecular barcode such that most or all of the tagged DNA fragments comprising a same targeted locus have a unique molecular barcode;
amplifying at least some of the tagged DNA fragments comprising DNA from the at least 10 targeted polymorphic loci using a one-sided nested PCR reaction such that the amplified tagged DNA fragments comprise the first known tail sequence, a molecular barcode, and a target sequence; and
sequencing at least some of the amplified DNA fragments by clonal sequencing to generate allelic data for the at least 10 targeted loci.
2. The method of claim 1 , wherein the allelic data is measured by measuring the number of amplified DNA fragments comprising the same molecular barcode, the same targeted locus, and the same target sequence.
3. The method of claim 1 , wherein the clonal sequencing is performed using high-throughput sequencing.
4. The method of claim 1 , wherein a sequence of sufficient length is obtained for at least some of sequenced amplified DNA fragments such that the sequence spans a molecule barcode as well a sufficient number of unique bases corresponding to a target sequence to allow unambiguous identification of one of the at least 10 targeted polymorphic loci.
5. The method of claim 1 , wherein the biological sample is derived from a blood sample.
6. The method of claim 1 , wherein the biological sample is derived from a plasma sample.
7. The method of claim 1 , wherein the DNA fragments are circulating DNA, and wherein the individual is suspected of having cancer.
8. The method of claim 1 , wherein the known tail sequence comprises a known forward or reverse sequence to facilitate sequencing on a sequencing platform.
9. The method of claim 1 , wherein the molecular barcodes are randomly generated.
10. The method of claim 1 , wherein the molecular barcodes are used to quantitate the amplified DNA fragments after sequencing.
11. The method of claim 1 , wherein the one-sided nested PCR reaction is performed such that the amplified tagged DNA fragments further comprise a second known tail sequence.
12. The method of claim 11 , wherein the first and second known tail sequences are different.
13. The method of claim 12 , further comprising performing universal amplification on the amplified tagged DNA fragments using a forward primer and a reverse primer, wherein the forward primer binds a first universal priming sequence in the first known tail sequence and the reverse primer binds a second universal priming sequence in the second known tail sequence.
14. The method of claim 13 , wherein the reverse primer comprises a third known tail sequence comprising a sequence to facilitate sequencing on a sequencing platform.
15. The method of claim 1 , wherein the one-sided nested PCR reaction comprises first and second PCR amplification rounds, wherein the second amplification round is performed using a second primer configured to bind to a first primer used in the first amplification round.
16. The method of claim 15 , wherein the one-sided nested PCR reaction comprises multiplexing more than 50 primers in a single pool.
17. The method of claim 1 , wherein the one-sided nested PCR reaction comprises using a target-specific primer on one side of a tagged fragment and a universal tag-specific primer on another side of the tagged fragment.
18. A method for preparing a biological sample obtained from a single individual for use in a genetic testing assay, the method comprising:
identifying a plurality of polymorphic loci for targeting in the biological sample;
isolating DNA fragments from the biologic sample, wherein the isolated DNA fragments are between 100 bp and 220 bp and wherein at least some of the isolated DNA fragments are target DNA molecules, and wherein the target DNA molecules are DNA fragments comprising one or more of the plurality of targeted polymorphic loci;
tagging at least some of the isolated DNA fragments with a known tail sequence and a molecular barcode such that most or all of the target DNA molecules in the sample have a unique molecular barcode;
amplifying at least some of the tagged DNA fragments in a one-sided nested PCR reaction such that at least some of the amplified tagged DNA fragments are target DNA molecules comprising the known tail sequence and a molecular barcode; and
sequencing at least some of the amplified tagged DNA fragments by clonal sequencing to generate allelic data for at least some of the target DNA molecules.
19. The method of claim 18 , further comprising measuring the number of amplified DNA fragments comprising the same molecular barcode, the same targeted locus, and the same target sequence, thereby determining the number of target DNA molecules in the sample.
20. The method of claim 18 , wherein the allelic data is measured by measuring the number of amplified DNA fragments comprising the same molecular barcode, the same targeted locus, and the same target sequence.
21. The method of claim 18 , wherein the clonal sequencing is performed using high-throughput sequencing.
22. The method of claim 18 , wherein a sequence of sufficient length is obtained for at least some of sequenced amplified DNA fragments such that the sequence spans a molecule barcode as well a sufficient number of unique bases corresponding to a target sequence to allow unambiguous identification of one of the plurality of targeted loci.
23. The method of claim 18 , wherein the biological sample is derived from a blood sample.
24. The method of claim 18 , wherein the biological sample is derived from a plasma sample.
25. The method of claim 18 , wherein the DNA fragments are circulating DNA, and wherein the individual is suspected of having cancer.
26. The method of claim 18 , wherein the known tail sequence comprises a known forward or reverse sequence to facilitate sequencing on a sequencing platform.
27. The method of claim 18 , wherein the one-sided nested PCR reaction is performed such that the amplified tagged DNA fragments further comprise a second known tail sequence.
28. The method of claim 27 , wherein the first and second known tail sequences are different.
29. The method of claim 27 , further comprising performing universal amplification on the amplified tagged DNA fragments using a forward primer and a reverse primer, wherein the forward primer binds a first universal priming sequence in the first known tail sequence and the reverse primer binds a second universal priming sequence in the second known tail sequence.
30. The method of claim 29 , wherein the reverse primer comprises a third known tail sequence comprising a sequence to facilitate sequencing on a sequencing platform.
31. The method of claim 18 , wherein the plurality of targeted loci comprises more than 100 loci.