# US 10,119,167 B2 · Non-invasive prenatal diagnosis of fetal genetic condition using cellular DNA and cell free DNA

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- Number: US 10,119,167 B2
- Document ID: US10119167B2
- Status: Granted
- Kind: B2
- Application number: 14/802,936
- Filed: 2015-07-17
- Published: 2016-01-21
- Granted: 2018-11-06
- Earliest claimed: 2014-07-18
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Anupama Srinivasan; Darya I. Chudova; Richard P. Rava
- Web page: https://patentgrep.com/patent/US10119167B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6869: Methods for sequencing
- G16B 20/10: Ploidy or copy number detection
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- G16B 30/20: Sequence assembly
- G16B 40/20: Supervised data analysis
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## 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 maternal 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.

## Claims

### Claim 1

A method for determining at least one sequence of interest of a fetus of a pregnant mother, the method comprising:
(a) obtaining mother-only cellular DNA of the pregnant mother, wherein the mother-only cellular DNA has a sequence that maps to the at least one sequence of interest;
(b) obtaining mother-and-fetus mixed cfDNA from the pregnant mother, wherein the mother-and-fetus mixed cfDNA has a sequence that maps to the at least one sequence of interest;
(c) applying a first index sequence to the mother-only cellular DNA and a second index sequence to the cfDNA, wherein
the first index sequence and the second index sequence are different,
the first index sequence specifies a source of DNA as being from the mother-only cellular DNA from the pregnant mother, and
the second index sequence specifies a source of DNA as being from the cfDNA from the pregnant mother;
(d) combining the mother-only cellular DNA and the cfDNA to provide a sample of combined cellular DNA and cfDNA;
(e) sequencing said sample of combined cellular and cfDNA to provide a plurality of sequence reads and mapping the plurality of sequence reads to a reference sequence; and
(f) analyzing the mapped sequence reads 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 mapped sequence reads maps to the at least one sequence of interest.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
obtaining a blood sample from the pregnant mother;
separating the buffy coat and the plasma of the blood sample;
obtaining the mother-only cellular DNA from the buffy coat of the blood sample; and
obtaining the cfDNA from the plasma of the blood sample.

### Claim 3 (depends on 1)

The method of claim 1, further comprising preparing a first sequencing library of the mother-only cellular DNA and a second sequencing library of the cfDNA, wherein (c) comprises incorporating the first and second index sequences in said first and second sequencing libraries respectively, wherein the first and second index sequences are identifiable from said mapped sequence reads.

### Claim 4 (depends on 3)

The method of claim 3, wherein incorporating the first and second index sequences in said first and second sequencing libraries comprises hybridizing and extending adapter oligonucleotides comprising the index sequences.

### Claim 5 (depends on 4)

The method of claim 4, wherein the adapter oligonucleotides comprise locus-specific extension oligonucleotides.

### Claim 6 (depends on 5)

The method of claim 5, wherein the locus-specific extension oligonucleotides are selective for two or more alleles of a disease related gene.

### Claim 7 (depends on 4)

The method of claim 4, 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.

### Claim 8 (depends on 3)

The method of claim 3, wherein incorporating index sequences in said first and second sequencing libraries comprises ligating or transposing the index sequences to the mother-only cellular DNA and the cfDNA.

### Claim 9 (depends on 3)

The method of claim 3, further comprising incorporating an individual-specific index to the sequencing libraries, wherein the individual-specific index indicates an identity of the pregnant mother, thereby allowing the pregnant mother's DNA to be processed with other individuals' DNA for parallel sequencing.

### Claim 10 (depends on 3)

The method of claim 3, wherein the sequencing libraries are transposon insertion libraries.

### Claim 11 (depends on 1)

The method of claim 1, wherein a source of the mapped sequence reads is indicated by the first or second index sequence.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 12)

The method of claim 12, wherein determining whether the fetus has the genetic disease comprises determining whether the fetus is homozygous in a disease causing allele within the sequence of interest when the mother is heterozygous of the same allele.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 14)

The method of claim 14, further comprising determining if the fetus is homozygous or heterozygous for the disease associated allele.

### Claim 16 (depends on 1)

The method of claim 1, wherein the method further comprises enriching the sequence of interest from the mother-only cellular DNA and the cfDNA.

### Claim 17 (depends on 1)

The method of claim 1, wherein the sample of combined cellular DNA and cfDNA provided in (d) comprises about the same quantity and/or about the same concentration of cellular DNA and cfDNA.

### Claim 18 (depends on 1)

The method of claim 1, wherein the at least one sequence of interest comprises a chromosome or a chromosome segment.

### Claim 19 (depends on 1)

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

### Claim 20 (depends on 1)

The method of claim 1, wherein the method further comprises obtaining sequence tags for a plurality of training sequences, wherein zygosity cases of the training sequences are known and the training sequences are different from the sequence of interest.

### Claim 21 (depends on 20)

The method of claim 20, wherein the analyzing of (f) uses information derived from the plurality of training sequences.

### Claim 22 (depends on 20)

The method of claim 20, wherein the plurality of training sequences comprises more than about 10 sequences.

### Claim 23 (depends on 20)

The method of claim 20, wherein the plurality of training sequences comprises more than about 100 sequences.

### Claim 24 (depends on 20)

The method of claim 20, further comprising obtaining distribution statistics of coverage quantities of the sequence tags for the training sequences.

### Claim 25 (depends on 24)

The method of claim 24, further comprising using the distribution statistics to determine the zygosity of the sequence of interest for the fetus.

### Claim 26 (depends on 1)

The method of claim 1, further comprising identifying the sequence of interest in the cfDNA and the cellular DNA, and identifying at least one training sequence in the cfDNA and the cellular DNA.

### Claim 27 (depends on 26)

The method of claim 26, further comprising identifying the sequence of interest in the cfDNA and the cellular DNA, and identifying a plurality of training sequences in the cfDNA and the cellular DNA.

### Claim 28 (depends on 27)

The method of claim 27, further comprising determining a zygosity case of the sequence of interest for the fetus.

### Claim 29 (depends on 26)

The method of claim 26, further comprising comparing relative coverage quantities of cellular DNA versus cfDNA mapping to the sequence of interest and training sequences.

### Claim 30 (depends on 29)

The method of claim 29, further comprising comparing the relative amounts of DNA mapping to two or more alleles.

### Claim 31 (depends on 1)

The method of claim 1, further comprising:
obtaining a test-difference-value based on a difference between a coverage quantity of test sequence tags derived from the cellular DNA and a coverage quantity of test sequence tags derived from the cfDNA, wherein the test sequence tags map to the sequence of interest; and
obtaining a plurality of training-difference-values based on a difference between a coverage quantity of training sequence tags derived from the cellular DNA and a coverage quantity of training sequence tags derived from the cfDNA, wherein the training sequence tags map to a plurality of training sequences.

### Claim 32 (depends on 31)

The method of claim 31, wherein the test-difference-value is also based on a difference of sequence tag coverage quantities between two alleles of the sequence of interest, and wherein the training-difference-values are also based on differences of sequence tag coverage quantities between two alleles of the training sequences.

### Claim 33 (depends on 31)

The method of claim 31, further comprising obtaining distribution statistics for the plurality of training-difference-values.

### Claim 34 (depends on 33)

The method of claim 33, wherein the distribution statistics comprises values of central tendency and spread.

### Claim 35 (depends on 34)

The method of claim 34, wherein the central tendency and spread are mean and standard deviation, respectively.

### Claim 36 (depends on 33)

The method of claim 33, further comprising determining a zygosity of the sequence of interest for the fetus using the test-difference-value and the distribution statistics for the plurality of training-difference-values.

### Claim 37 (depends on 36)

The method of claim 36, further comprising obtaining a fetal fraction that indicates a fraction of fetal DNA in the cfDNA using the determined zygosity of the sequence of interest for the fetus.

### Claim 38 (depends on 1)

The method of claim 1, wherein the reference sequence comprises a consensus sequence or a combination sequence obtained from a plurality of individuals.

### Claim 39 (depends on 1)

The method of claim 1, wherein the reference sequence is obtained from a standard reference genome.

### Claim 40 (depends on 1)

The method of claim 1, wherein the reference sequence is a previously known reference sequence.

### Claim 41 (depends on 1)

The method of claim 1, wherein the previously known reference sequence comprises: a NCB136/hg18 sequence, a GRCh37/hg19 sequence, or a sequence derived from multiple individuals.