Non-invasive prenatal diagnosis of fetal genetic condition using cellular dna and cell free dna

Patent metadata

Field	Value
Publication number	US-2016186253-A1
Application number	US-201514802873-A
Country	US
Kind code	A1
Filing date	Jul 17, 2015
Priority date	Jul 18, 2014
Publication date	Jun 30, 2016
Grant date	—

Abstract

Disclosed are methods for determining at least one sequence of interest of a fetus of a pregnant mother. In various embodiments, the method can determine one or more sequences of interest in a test sample that comprises a mixture of fetal cellular DNA and mother-and-fetus cfDNA. In some embodiments, methods are provided for determining whether the fetus has a genetic disease. In some embodiments, methods are provided for determining whether the fetus is homozygous in a disease causing allele when the mother is heterozygous of the same allele. In some embodiments, methods are provided for determining whether the fetus has a copy number variation (CNV) or a non-CNV genetic sequence anomaly.

First claim

What is claimed is:

1 . A method for determining at least one sequence of interest of a fetus of a pregnant mother, the method comprising:
(a) obtaining cellular DNA from the blood of the pregnant mother, wherein the cellular DNA comprises fetal cellular DNA; (b) obtaining mother-and-fetus mixed cfDNA from the blood of the pregnant mother; (c) applying an indicator to at least one of the fetal cellular DNA and the mixed cfDNA, wherein the indicator identifies a source of DNA as being from the fetal cellular DNA or the mixed cfDNA; (d) combining the fetal cellular DNA and the mixed cfDNA to provide a sample of combined cellular DNA and cfDNA; (e) sequencing the sample of combined cellular DNA and cfDNA to provide a plurality of sequence tags; and (f) analyzing the plurality of sequence tags to determine the presence and/or abundance of the at least one sequence of interest in the fetus's DNA, wherein at least a portion of the plurality of sequence tags map to the at least one sequence of interest.

2 . The method of claim 1 , wherein (e) sequencing said sample of combined cellular and cfDNA comprises: sequencing said sample of combined cellular and cfDNA to produce a plurality of sequence reads; and aligning the plurality of sequence reads to a reference sequence to provide the plurality of sequence tags, wherein sources of the plurality of sequence tags are indicated by the indicator identifying the source of DNA.

3 . The method of claim 1 , wherein the fetal cellular DNA is obtained from one or more fetal nucleated red blood cells (NRBCs) in the blood of the pregnant mother.

4 . The method of claim 3 , further comprising separating the fetal NRBCs from maternal erythrocytes in a cellular component of a blood sample of the pregnant mother.

5 . The method of claim 4 , wherein separating the fetal NRBCs from the maternal erythrocytes comprises differentially lysing maternal erythrocytes.

6 . The method of claim 4 , wherein separating the fetal NRBCs from the maternal erythrocytes comprises size-based separation and/or capture-based separation.

7 . The method of claim 6 , wherein the capture-based separation comprises capturing the fetal NRBCs through binding one or more cellular markers expressed by fetal NRBCs.

8 . The method of claim 7 , wherein the one or more cellular markers expressed by fetal NRBCs are selected from the group consisting of CD71, CD36, CD34, antigen-i, galactose, glycophorin-A, fetal haemoglobin, and any combinations thereof.

9 . The method of claim 7 , wherein the one or more cellular markers comprise a surface marker expressed by fetal NRBCs but not, or to a lesser degree, by maternal NRBCs.

10 . The method of claim 7 , wherein the one or more cellular markers comprises a 4B9-antigen and/or a 4B8-antigen.

11 . The method of claim 7 , wherein the capture-based separation comprise binding magnetically responsive particles to fetal NRBCs, wherein the magnetically responsive particles have an affinity to one or more cellular markers expressed by fetal NRBCs.

12 . The method of claim 11 , wherein the capture-based separation is performed by an automated immunomagnetic separation device.

13 . The method of claim 7 , wherein the capture-based separation comprise binding fluorescent labels to fetal NRBCs, wherein the fluorescent labels have an affinity to one or more cellular markers expressed by fetal NRBCs.

14 . The method of claim 1 , further comprising:
obtaining a blood sample from the pregnant mother; separating an erythrocyte fraction and a plasma fraction of the blood sample; obtaining the fetal cellular DNA from the erythrocyte fraction the blood sample; and obtaining the cfDNA from the plasma fraction of the blood sample.

15 . The method of claim 1 , further comprising preparing a first sequencing library of the fetus-only cellular DNA and a second sequencing library of the cfDNA, wherein applying the indicator in (c) comprises incorporating indexes in each of said sequencing libraries, wherein the indexes incorporated in said first library differ from the indexes incorporated in said second library, and the indexes are identifiable from said plurality of sequence tags.

16 . The method of claim 15 , wherein incorporating indexes in each of said sequencing libraries comprises hybridizing and extending adapter oligonucleotides comprising the indexes.

17 . The method of claim 16 , wherein the adapter oligonucleotides comprise locus-specific extension oligonucleotides.

18 . The method of claim 17 , wherein the locus-specific extension oligonucleotides are selective for two or more alleles of a disease related gene.

19 . The method of claim 16 , wherein each of the adapter oligonucleotides comprises an adapter sequence or a portion thereof, wherein the adapter sequence is configured to hybridize to an oligonucleotide attached to a substrate of a flow cell of a sequencing apparatus.

20 . The method of claim 15 , wherein incorporating indexes in each of said sequencing libraries comprises ligating or transposing sequences comprising the indexes to the fetal cellular DNA and the mixed cfDNA.

21 . The method of claim 15 , further comprising incorporating an individual-specific index to the sequencing libraries, wherein the individual-specific index indicates the identity of the pregnant mother, thereby allowing the pregnant mother's DNA to be processed with other individuals' DNA for parallel sequencing.

22 . The method of claim 15 , wherein the sequencing libraries are transposon insertion libraries.

23 . The method of claim 1 , further comprising determining whether the fetus has a genetic disease from the at least one sequence of interest of the fetus.

24 . The method of claim 1 , wherein the at least one sequence of interest comprises a disease associated allele selected from the group consisting: a single nucleotide polymorphism, a tandem repeat, a micro-deletion, an insertion, an indel, and any combinations thereof.

25 . The method of claim 1 , further comprising determining a complete or partial aneuploidy.

26 . A method, implemented at a computer system that includes one or more processors and system memory, for determining a condition of a fetus related to a sequence of interest, the method comprising:
obtaining, by the computer system, sequence reads of fetus-only cellular DNA obtained from a blood sample of the mother carrying the fetus, the cellular DNA having been specifically enriched for a sequence of interest; computing, by the computer system, a count of sequence tags mapping to the sequence of interest for the cellular DNA; obtaining, by the computer system, sequence reads of mother-and-fetus mixed cfDNA obtained from the mother, the cfDNA having been specifically enriched for the sequence of interest; computing, by the computer system, a count of sequence tags mapping to the sequence of interest for the cfDNA; comparing, by the computer system, the sequence tag counts mapping to the sequence of interest between the cellular DNA and the cfDNA; and determining, by the computer system, the condition of the fetus related to the sequence of interest.

27 . The method of 26, wherein the specifically enriched cellular DNA and the specifically enriched cfDNA were combined for amplification and/or sequencing.

28 . A computer program product comprising a non-transitory m

Assignees

• Illumina Inc

Inventors

• Talasaz Amirali Hajhossein
• Cann Gordon M

Classifications

• G16B20/10

  Ploidy or copy number detection · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• G16B40/20

  Supervised data analysis · CPC title

• G16B30/20

  Sequence assembly · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title