Noninvasive prenatal diagnostic methods

Patent metadata

Field	Value
Publication number	US-12416047-B2
Application number	US-201715587811-A
Country	US
Kind code	B2
Filing date	May 5, 2017
Priority date	May 6, 2016
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

Abstract

Prenatal genetic testing allows early detection of genetic disease in a fetus. Described herein are methods of detecting the presence or absence of a genetic variant in a region of interest in the genome of a fetus in a pregnant woman. The methods are noninvasive, and can use cell-free DNA (cfDNA) present in the plasma of the pregnant woman. A DNA library is constructed from the cfDNA, and DNA molecules comprising the region of interest or portions thereof are enriched and analyzed, for example by sequencing. The methods described herein can also rely on constructing a maternal haplotype to provide even higher resolution fetal genetic variant determination.

First claim

The invention claimed is:

1. A method of detecting the presence or absence of a genetic variant in a region of interest in the genome of a fetus in a pregnant woman, comprising:
(a) extracting circulating cell-free DNA from a plasma sample of the pregnant woman;
(b) extracting maternal genomic DNA (gDNA) from a sample of the pregnant woman;
(c) preparing a first DNA library from said cell-free DNA, the first DNA library comprising a first population of DNA molecules comprising covalently linked molecular barcodes and that is enriched for a region of interest in said cell-free DNA, wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(d) preparing a second DNA library from said gDNA, the second DNA library comprising a second population of DNA molecules comprising first and second covalently linked molecular barcodes and that is enriched for a region of interest in said gDNA, wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(e) sequencing the region of interest in the first population of DNA molecules from said first DNA library to produce a plurality of cell-free DNA sequencing reads;
(f) sequencing a region of interest in the second population of DNA molecules from said second DNA library that corresponds to the region of interest in the cell-free DNA to produce a plurality of gDNA sequencing reads by performing long read-length sequencing technologies, wherein said long read-length sequencing technologies produces sequence reads greater than 500 bases, and constructing a maternal haplotype by phasing genetic variants present only in the gDNA sequencing reads; and
(g) detecting the presence or absence of a fetal genetic variant based on the cell-free DNA sequencing reads and the maternal haplotype,
wherein the fetal genetic variant is a single-nucleotide variant, a multi-nucleotide variant, an indel variant of 100 bp or less, or a copy number loss variant within a region of interest of 50,000 bp or fewer.

2. A method of detecting the presence or absence of a genetic variant in a region of interest in the genome of a fetus in a pregnant woman, comprising:
(a) extracting circulating cell-free DNA from a plasma sample of the pregnant woman;
(b) extracting maternal genomic DNA (gDNA) from a sample of the pregnant woman;
(c) preparing a first DNA library from said cell-free DNA, wherein the first DNA library comprises a first plurality of DNA molecules comprising molecular barcodes, wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(d) preparing a second DNA library from said gDNA, wherein the second DNA library comprises a second plurality of DNA molecules comprising a first and a second set of molecular barcodes, wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(e) sequencing the region of interest in the first plurality of DNA molecules to produce a plurality of cell-free DNA sequencing reads; and
(f) sequencing a region of interest in the second plurality of DNA molecules that corresponds to the region of interest in the first plurality of DNA molecules to produce a plurality of gDNA sequencing reads by performing long read-length sequencing technologies, wherein said long read-length sequencing technologies produces sequence reads greater than 500 bases, and constructing a maternal haplotype by phasing genetic variants present only in the gDNA sequencing reads; and
(g) detecting the presence or absence of a fetal genetic variant based on the cell-free DNA sequencing reads and the maternal haplotype,
wherein the fetal genetic variant is a single-nucleotide variant, a multi-nucleotide variant, an indel variant of 100 bp or less, or a copy number loss variant within a region of interest of 50,000 bp or fewer.

3. A method of detecting the presence or absence of a genetic variant in a region of interest in the genome of a fetus in a pregnant woman, comprising:
(a) obtaining a first population of DNA molecules from a first DNA library comprising a first plurality of DNA molecules comprising molecular barcodes, wherein the first DNA library is prepared from circulating cell-free DNA extracted from a plasma sample of the pregnant woman, wherein the first population of DNA molecules is enriched for a region of interest in said cell-free DNA, wherein the molecular barcodes are incorporated into the first plurality of DNA molecules prior to enrichment, and wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(b) obtaining a second population of DNA molecules from a second DNA library comprising a second plurality of DNA molecules comprising a first and a second set of molecular barcodes, wherein the second DNA library is prepared from gDNA extracted from a sample of the pregnant woman, wherein the second population of DNA molecules is enriched for a region of interest in said gDNA, wherein the first and second sets of molecular barcodes are incorporated into the second plurality of DNA molecules prior to enrichment, and wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(c) sequencing the region of interest in the first population of DNA molecules to produce a plurality of cell-free DNA sequencing reads; and
(d) sequencing a region of interest in the second population of DNA molecules that corresponds to the region of interest in the cell-free DNA to produce a plurality of maternal sequencing reads by performing long read-length sequencing technologies, wherein said long read-length sequencing technologies produces sequence reads greater than 500 bases, and constructing a maternal haplotype by phasing genetic variants present only in the maternal sequencing reads; and
(e) detecting the presence or absence of a fetal genetic variant based on the cell-free DNA sequencing reads and the maternal haplotype,
wherein the fetal genetic variant is a single-nucleotide variant, a multi-nucleotide variant, an indel variant of 100 bp or less, or a copy number loss variant within a region of interest of 50,000 bp or fewer.

4. A method of detecting the presence or absence of a genetic variant in a region of interest in the genome of a fetus in a pregnant woman, comprising:
(a) extracting circulating cell-free DNA from a plasma sample of the pregnant woman;
(b) extracting gDNA from a sample of the pregnant woman;
(c) preparing a first DNA library from said cell-free DNA, wherein the first DNA library comprises a first plurality of DNA molecules comprising covalently linked molecular barcodes, wherein a portion of the first plurality of DNA molecules is enriched for a region of interest in said cell-free DNA, wherein the molecular barcodes are incorporated into the first plurality of DNA molecules prior to enrichment, and wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(d) preparing a second DNA library from said gDNA, wherein the second DNA library comprises a second plurality of DNA molecules comprising a first and a second set of covalently linked molecular barcodes, wherein a portion of the second plurality of DNA molecules is enriched for a region of interest that corresponds to the region of interest in said cell-free DNA, wherein the first and second sets of molecular barcodes are incorporated into the second plurality of DNA molecules prior to enrichment, and wherein the sequence of each molecular barcode differs from the sequence of every other molecular barcode;
(e) sequencing the region of interest in the portion of the first DNA library to produce a plurality of cell-free DNA sequencing reads; and
(f) sequencing the region of interest in the sec

Assignees

• Myriad Womens Health Inc

Inventors

• Haque Imran Saeedul
• Maguire Jared Robert
• Chu Clement
• Evans Eric Andrew

Classifications

• C12Q2600/172

  Haplotypes · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6881

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• C12Q1/6883

  for diseases caused by alterations of genetic material · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title