Patent ID: 11946104
Assignee: BILLIONTOONE, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C  G | IPC C

Claim 0:
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.