Noninvasive diagnosis of fetal aneuploidy by sequencing

Patent metadata

Field	Value
Publication number	US-12054777-B2
Application number	US-202016852663-A
Country	US
Kind code	B2
Filing date	Apr 20, 2020
Priority date	Sep 20, 2008
Publication date	Aug 6, 2024
Grant date	Aug 6, 2024

Abstract

Disclosed is a method to achieve digital quantification of DNA (i.e., counting differences between identical sequences) using direct shotgun sequencing followed by mapping to the chromosome of origin and enumeration of fragments per chromosome. The preferred method uses massively parallel sequencing, which can produce tens of millions of short sequence tags in a single run and enabling a sampling that can be statistically evaluated. By counting the number of sequence tags mapped to a predefined window in each chromosome, the over- or under-representation of any chromosome in maternal plasma DNA contributed by an aneuploid fetus can be detected. This method does not require the differentiation of fetal versus maternal DNA. The median count of autosomal values is used as a normalization constant to account for differences in total number of sequence tags is used for comparison between samples and between chromosomes.

First claim

What is claimed is:

1. A method of testing for the presence or absence of a fetal aneuploidy in a maternal plasma sample comprising a mixture of maternal and fetal cell-free DNA, comprising the steps of:
(a) sequencing chromosomal locations known to exist in the maternal and fetal cell-free DNA, to obtain a plurality of millions of sequence tags aligning to the known chromosomal locations, wherein the sequencing identifies molecules containing the chromosomal locations, wherein said sequence tags are of sufficient length to be assigned to a known chromosomal location, wherein the known chromosomal locations are from a plurality of different chromosomes, and wherein said plurality of different chromosomes comprise at least one first chromosome suspected of having an abnormal distribution in said sample and at least one second chromosome presumed to be normally distributed in said sample, wherein the sequencing identifies sequences mapping uniquely or with one mismatch to said at least one first chromosome and said at least one second chromosome, and wherein said at least one first chromosome is chromosome 18, 21, 13, X or Y;
(b) assigning the plurality of millions of sequence tags to their corresponding known chromosomal location;
(c) determining a number of sequence tags aligning to the known chromosomal locations of said first chromosome and a number of sequence tags aligning to the known chromosomal locations of said second chromosome; and
(d) using the numbers from step (c) to determine a differential, between the number of sequence tags aligning to the known chromosomal locations of said first chromosome and the number of sequence tags aligning to the known chromosomal locations of said second chromosome, wherein said differential is statistically significant and determinative of a fetal aneuploidy.

2. The method of claim 1 wherein said sequencing comprises attaching DNA fragments to an optically transparent surface, conducting solid phase amplification of the attached DNA fragments to create a high density sequencing flow cell with millions of DNA clusters, and sequencing the DNA clusters by a four-color DNA sequencing-by-synthesis method employing reversible terminators with removable fluorescent dyes.

3. The method of claim 1 wherein the fetal aneuploidy is an aneuploidy of a chromosome selected from the group consisting of chromosome 13, chromosome 18 and chromosome 21.

4. The method of claim 1 wherein the step of assigning sequence tags to corresponding chromosome portions allows one mismatch.

5. The method of claim 1 wherein the length of the sequence tags is from about 25 bp to about 100 bp in length.

6. The method of claim 1 wherein the identification comprises using capture beads with specific genomic sequences as capture probes.

7. The method of claim 1 wherein the identification is directed to genome sequences mapping uniquely to the at least one first chromosome.

8. The method of claim 1 wherein said sequencing comprises the use of a sequencing array.

9. The method of claim 1 wherein the sample is rendered single stranded and said known chromosomal locations are captured under hybridizing conditions by a number of single-stranded probes which are catalogued by bar coding or which are physically separated on an array.

10. The method of claim 1 further comprising determination of fetal DNA fraction of the DNA obtained from the maternal serum or plasma sample.

11. The method of claim 10 wherein the fetal DNA fraction is determined by digital PCR.

Assignees

• Univ Leland Stanford Junior
• The Board Of Trustees Of The Leland Standford Junior Univ

Inventors

• Fan Hei-Mun Christina
• Quake Stephen R

Classifications

• G01N2800/387

  Down syndrome; Trisomy 18; Trisomy 13 · CPC title

• G01N33/48

  Biological material, e.g. blood, urine (G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence); Haemocytometers (counting blood corpuscules distributed over a surface by scanning the surface G06M11/02) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6883 Primary

  for diseases caused by alterations of genetic material · CPC title