Non-invasive prenatal testing at early stage of pregnancy
A highly accurate non-invasive prenatal testing methodology that allows accurate fetal rhesus determination and fetal DNA fraction measurement in a single assay is provided. The disclosed testing may be administered as early as at week 10 of pregnancy and can be conveniently combined with prenatal genetic disease testing and detection.
1. A non-invasive method for accurate determination of fetal Rhesus D genotype and measurement of fetal fraction in one assay at an early stage of pregnancy, wherein the method comprises:
(i) collecting a blood sample from a pregnant woman subject;
(ii) extracting cell-free DNA from the blood sample;
(iii) performing multiplex polymerase chain reaction of the cell-free DNA to simultaneously amplify polymorphic allele loci and multiple RhD gene locations;
(iv) sequencing amplified DNA;
(v) choosing polymorphic alleles from the sequenced amplified DNA whose allele fraction is less than a predetermined percentage;
(vi) averaging the allele fraction for a plurality of the chosen polymorphic alleles to determine a fetal fraction;
(vii) quantifying total cell free DNA;
(viii) multiplying fetal fraction and the total cell free DNA to determine an expected number of fetal molecules in the blood sample;
(ix) determining a per sample normalization based on the expected number of fetal molecules in the blood sample;
(x) computationally aligning sequencing reads to a human reference genome using a Burrows-Wheeler algorithm;
(xi) identifying and selecting amplified RhD gene regions to obtain quantitative fetal RhD data;
(xii) determining the fetal Rhesus D genotype by applying the per sample normalization to the quantitative fetal RhD data and comparing the normalized result with a predetermined value for the Rhesus D genotype; and
(xiii) administering anti-D immunoglobulin to the pregnant woman where the pregnant woman has an Rh-D negative blood type and the determined fetal Rhesus D genotype is an Rh-D-positive blood type.
2. The method of claim 1 , wherein the pregnant woman subject is at a gestational stage between 10 and 20 weeks.
3. The method of claim 1 , wherein the pregnant woman subject is at 20 weeks or later stages of gestation.
4. The method of claim 1 , wherein the pregnant woman subject has Rhesus D negative genotype.
5. The method of claim 1 , wherein the pregnant woman subject has Rhesus RhD-CE-D genotype.
6. The method of claim 1 , wherein the pregnant woman subject has Rhesus D pseudogene (RHDO genotype.
7. The method of claim 1 , wherein the method further comprises analyzing the blood sample to determine the fetus' sex.
8. The method of claim 7 , wherein the method further comprises analyzing the blood sample to determine presence or absence of fetal aneuploidy for genetic disease detection.
9. A non-invasive method for accurate determination of fetal Rhesus D genotype and measurement of fetal fraction in one assay at an early stage of pregnancy, wherein the method comprises:
(i) collecting a blood sample from a pregnant woman subject;
(ii) extracting cell-free DNA from the blood sample;
(iii) performing multiplex polymerase chain reaction of the cell-free DNA to simultaneously amplify polymorphic allele loci and multiple RhD gene locations;
(iv) sequencing amplified DNA;
(v) choosing polymorphic alleles from the sequenced amplified DNA whose allele fraction is less than a predetermined percentage;
(vi) averaging the allele fraction for a plurality of the chosen polymorphic alleles to determine a fetal fraction;
(vii) quantifying total cell free DNA;
(viii) multiplying fetal fraction and the total cell free DNA to determine an expected number of fetal molecules in the blood sample;
(ix) determining a per sample normalization based on the expected number of fetal molecules in the blood sample;
(x) identifying and selecting amplified RhD gene regions to obtain quantitative fetal RhD data;
(xi) determining the fetal Rhesus D genotype by applying the per sample normalization to the quantitative fetal RhD data and comparing the normalized result with a predetermined value for the Rhesus D genotype; and
(xii) administering anti-D immunoglobulin to the pregnant woman where the pregnant woman has an Rh-D negative blood type and the determined fetal Rhesus D genotype is an Rh-D-positive blood type.
10. The method of claim 9 , wherein the pregnant woman is at a gestational period from 10 weeks to 20 weeks.
11. The method of claim 9 , wherein the pregnant woman subject is at 20 weeks or later stages of gestation.
12. The method of claim 9 , wherein the pregnant woman subject has Rhesus D negative genotype.
13. The method of claim 9 , wherein the pregnant woman subject has Rhesus RhD-CE-D genotype.
14. The method of claim 9 , wherein the pregnant woman subject has Rhesus D pseudogene (RHDψ) genotype.
15. The method of claim 9 , wherein the method further comprises analyzing the blood sample to determine the fetus' sex.
16. The method of claim 15 , wherein the method further comprises analyzing the blood sample to determine presence or absence of fetal aneuploidy for genetic disease detection.