Diagnosis of fetal abnormalities using polymorphisms including short tandem repeats
The present invention provides systems, apparatuses, and methods to detect the presence of fetal cells when mixed with a population of maternal cells in a sample and to test fetal abnormalities, i.e. aneuploidy. In addition, the present invention provides methods to determine when there are insufficient fetal cells for a determination and report a non-informative case. The present invention involves quantifying regions of genomic DNA from a mixed sample. More particularly the invention involves quantifying DNA polymorphisms from the mixed sample.
1. A method of analyzing a fetal cell in a mixed sample obtained from a pregnant human female, the method comprising:
(a) obtaining the mixed sample comprising fetal and maternal cells;
(b) enriching the mixed sample for fetal cells to produce an enriched sample comprising fetal cells and maternal cells, wherein the enrichment increases the ratio of fetal cells to maternal cells to about 1/10,000 to about 1/10;
(c) binning fetal cells and maternal cells from the enriched sample by serial dilution, wherein the binning results in at least one bin containing an individual fetal cell from the enriched sample;
(d) identifying bins that contain at least one fetal cell using one or more fetal cell biomarkers;
(e) lysing fetal cells in the identified bins;
(f) amplifying genomes of the lysed fetal cells in the identified bins to produce amplified nucleic acids; and
(g) analyzing the amplified nucleic acids in bins that contain at least one fetal cell for aneuploidy using ultra-deep sequencing.
2. The method of claim 1 , wherein the analyzing comprises analyzing for fetal aneuploidy, wherein the fetal aneuploidy comprises monosomy, trisomy, tetrasomy, or pentasomy of one or more chromosomes.
3. The method of claim 2 , wherein the fetal aneuploidy is a fetal aneuploidy of a chromosome selected from the group consisting of chromosome 13, chromosome 18, chromosome 21, chromosome X, and chromosome Y.
4. The method of claim 2 , wherein the fetal aneuploidy comprises trisomy or monosomy.
5. The method of claim 4 , wherein the fetal aneuploidy comprises trisomy, and wherein the trisomy comprises trisomy 13, trisomy 18, or trisomy 21.
6. The method of claim 4 , wherein the fetal aneuploidy comprises monosomy X and the chromosome suspected of being aneuploid comprises chromosome X.
7. The method of claim 1 , wherein the fetal aneuploidy comprises XXX, XXY, or XYY.
8. The method of claim 1 , wherein the ultra-deep sequencing produces partial genome sequences for analysis.
9. The method of claim 1 , wherein the ultra-deep sequencing produces complete genome sequences for analysis.
10. The method of claim 1 , wherein amplifying the genomes of the lysed fetal cells comprises amplifying whole genomes of the lysed fetal cells.
11. The method of claim 1 , wherein the binning comprises use of a nanofluidic system.
12. The method of claim 11 , wherein the nanofluidic system separates samples into droplets.
13. The method of claim 1 , further comprising positive selection for fetal cells prior to binning.
14. The method of claim 1 , further comprising negative selection for non-target cells prior to binning.
15. The method of claim 1 , wherein the enrichment increases the ratio of fetal cells to maternal cells to about 1/100 to about 1/10.
16. The method of claim 1 , wherein the enrichment increases the ratio of fetal cells to maternal cells to about 1/50 to about 1/10.
17. The method of claim 1 , wherein the enrichment increases the ratio of fetal cells to maternal cells to about 1/10.
18. The method of claim 1 , wherein the mixed sample is obtained from whole blood, bone marrow suspension, or milk.
19. The method of claim 1 , wherein the enriching comprises contacting the mixed sample with particles coupled to antibodies that selectively bind to fetal cells.
20. The method of claim 19 , wherein the particles are magnetic particles.