# US 10,319,463 B2 · Combined size- and count-based analysis of maternal plasma for detection of fetal subchromosomal aberrations

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- Number: US 10,319,463 B2
- Document ID: US10319463B2
- Status: Granted
- Kind: B2
- Application number: 14/994,023
- Filed: 2016-01-12
- Published: 2016-07-28
- Granted: 2019-06-11
- Earliest claimed: 2015-01-23
- Assignees: The Chinese University of Hong Kong
- Applicants: The Chinese University of Hong Kong
- Inventors: Yuk-Ming Dennis Lo; Rossa Wai Kwun Chiu; Kwan Chee Chan; Peiyong Jiang; Cheuk Yin Jandy Yu
- Web page: https://patentgrep.com/patent/US10319463B2

## CPC

- G16B 20/10: Ploidy or copy number detection
- C12Q 1/6883: for diseases caused by alterations of genetic material
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- 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

## Abstract

An aberration in a fetal genome can be identified by analyzing a sample of fetal and maternal DNA. Classifications of whether an aberration (amplification or deletion) exists in a subchromosomal region are determined using count-based and size-based methods. The count classification and the size classification can be used in combination to determine whether only the fetus or only the mother, or both, have the aberration in the subchromosomal region, thereby avoiding false positives when the mother has the aberration and the fetus does not.

## Claims

### Claim 1

A method of identifying a subchromosomal aberration in a fetal genome of a fetus by analyzing a biological sample from a female subject pregnant with the fetus, the biological sample including cell-free DNA molecules from the female subject and the fetus, the method comprising:
for each of a plurality of DNA molecules in the biological sample:
measuring a size of the DNA molecule;
identifying a location of the DNA molecule in a reference genome, thereby obtaining locations of the plurality of DNA molecules;
detecting, by a computer system, an aberration in the biological sample of a first subchromosomal region by:
determining a first amount of the plurality of DNA molecules located in the first subchromosomal region using the locations of the plurality of DNA molecules;
determining a second amount of the plurality of DNA molecules located in a second region using the locations of the plurality of DNA molecules;
computing a count parameter from the first amount and the second amount; and
comparing the count parameter to one or more count threshold to determine a count classification of a type of aberration existing in the biological sample for the first subchromosomal region;
determining, by the computer system, a size classification for the first subchromosomal region by:
calculating a first statistical value of sizes of the plurality of DNA molecules located in the first subchromosomal region;
calculating a reference statistical value of sizes of DNA molecules located in a reference region;
determining a separation value between the first statistical value and the reference statistical value; and
comparing the separation value to one or more size thresholds to obtain the size classification; and
determining whether the fetus has the aberration in the first subchromosomal region based on the size classification and the count classification.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
identifying the first subchromosomal region by:
determining a respective bin count parameter for each of a plurality of non-overlapping bins in the reference genome, each respective bin count parameter determined from a respective amount of DNA molecules location in a respective bin; and
performing a segmentation process that merges consecutive bins into a merged segment based on the consecutive bins having a same count classification, wherein the first subchromosomal region corresponds to a first merged segment.

### Claim 3 (depends on 2)

The method of claim 2, wherein the segmentation process includes at least one of: a binary circular segmentation and a Hidden Markov model.

### Claim 4 (depends on 1)

The method of claim 1, further comprising:
detecting a plurality of aberrations in the biological sample for a plurality of other subchromosomal regions by comparing other count parameters to the one or more count thresholds to determine other count classifications; and
determining whether the fetus has the plurality of aberrations in the plurality of other subchromosomal regions based on the other count classifications and other size classifications for the plurality of other subchromosomal regions.

### Claim 5 (depends on 1)

The method of claim 1, wherein the count classification is over-represented or under-represented.

### Claim 6 (depends on 1)

The method of claim 1, wherein the aberration is a deletion or a duplication.

### Claim 7 (depends on 1)

The method of claim 1, wherein the size classification is one of longer, shorter, or equal.

### Claim 8 (depends on 1)

The method of claim 1, wherein the fetus is determined to have an amplification in the first subchromosomal region when:
the count classification indicates the amplification and the size classification indicates the aberration does not exist in the first subchromosomal region, or
the count classification indicates the amplification and the size classification indicates the amplification.

### Claim 9 (depends on 8)

The method of claim 8, further comprising:
determining the female subject to also have the amplification in the first subchromosomal region when the count classification indicates the amplification and the size classification indicates the aberration does not exist in the first subchromosomal region.

### Claim 10 (depends on 8)

The method of claim 8, further comprising:
determining the female subject not to have the amplification in the first subchromosomal region when the count classification indicates the amplification and the size classification indicates the amplification.

### Claim 11 (depends on 1)

The method of claim 1, wherein the fetus is determined to have a deletion in the first subchromosomal region when:
the count classification indicates the deletion and the size classification indicates the aberration does not exist in the first subchromosomal region, or
the count classification indicates the deletion and the size classification indicates the deletion.

### Claim 12 (depends on 11)

The method of claim 11, further comprising:
determining the female subject to also have the deletion in the first subchromosomal region when the count classification indicates the deletion and the size classification indicates the aberration does not exist in the first subchromosomal region.

### Claim 13 (depends on 11)

The method of claim 11, further comprising:
determining the female subject not to have the deletion in the first subchromosomal region when the count classification indicates the deletion and the size classification indicates the deletion.

### Claim 14 (depends on 1)

The method of claim 1, wherein the fetus is determined not to have the aberration when:
the count classification indicates an amplification and the size classification indicates a deletion, or
the count classification indicates the deletion and the size classification indicates the amplification.

### Claim 15 (depends on 14)

The method of claim 14, further comprising:
determining the female subject to have the amplification when the count classification indicates the amplification and the size classification indicates the deletion.

### Claim 16 (depends on 14)

The method of claim 14, further comprising:
determining the female subject to have the deletion when the count classification indicates the deletion and the size classification indicates the amplification.

### Claim 17 (depends on 1)

The method of claim 1, further comprising:
determining a first bin count parameter for a first bin of the first subchromosomal region, the first subchromosomal region including one or more bins, the first bin count parameter determined from a third amount of DNA molecules located in a third region and a fourth amount of DNA molecules located in the first bin;
computing a mean of control bin count parameters for the first bin using control samples;
computing a first score for the first bin by subtracting the mean of control bin count parameters from the first bin count parameter; and
determining whether the first score is greater than a cutoff value to identify whether the female subject has the aberration for the first subchromosomal region.

### Claim 18 (depends on 17)

The method of claim 17, further comprising:
measuring a fetal DNA concentration in the biological sample;
computing a difference between the first score and the fetal DNA concentration, and wherein the determining whether the first score is greater than the cutoff value includes determining whether the difference is greater than a high threshold value.

### Claim 19 (depends on 18)

The method of claim 18, further comprising:
comparing the difference to a low threshold; and
determining that only the fetus has the aberration for the first subchromosomal region when the difference is below a low threshold value.

### Claim 20 (depends on 17)

The method of claim 17, wherein subtracting the mean of control bin count parameters from the first bin count parameter provides a result, and wherein computing the first score further includes dividing the result by the mean of control bin count parameters.

### Claim 21 (depends on 17)

The method of claim 17, wherein subtracting the mean of control bin count parameters from the first bin count parameter provides a result, and wherein computing the first score further includes dividing the result by a standard deviation of the control bin count parameters.

### Claim 22 (depends on 17)

The method of claim 17, wherein the first subchromosomal region includes only one bin, and wherein the fourth amount is of DNA molecules in the first subchromosomal region.

### Claim 23 (depends on 17)

The method of claim 17, wherein the first subchromosomal region includes a plurality of bins, the method further comprising:
computing a respective score for each of the plurality of bins, wherein the first score is determined using a sum of the respective scores.

### Claim 24 (depends on 23)

The method of claim 23, wherein the first score is the sum divided by a square root of a number of bins used to determine the sum.

### Claim 25 (depends on 17)

The method of claim 17, further comprising:
determining no aberration to exist in the first subchromosomal region when the first score is less than the cutoff value and when:
the count classification indicates an amplification and the size classification indicates the aberration does not exist in the first subchromosomal region, or
the count classification indicates a deletion and the size classification indicates the aberration does not exist in the first subchromosomal region.

### Claim 26

A computer product comprising a non-transitory computer readable medium storing a plurality of instructions for controlling a computer system to perform:
for each of a plurality of DNA molecules in a biological sample from a female subject pregnant with a fetus, the biological sample including cell-free DNA molecules from the female subject and the fetus:
measuring a size of the DNA molecule;
identifying a location of the DNA molecule in a reference genome, thereby obtaining locations of the plurality of DNA molecules;
detecting an aberration in the biological sample of a first subchromosomal region by:
determining a first amount of the plurality of DNA molecules located in the first subchromosomal region using the locations of the plurality of DNA molecules;
determining a second amount of the plurality of DNA molecules located in a second region using the locations of the plurality of DNA molecules;
computing a count parameter from the first amount and the second amount; and
comparing the count parameter to one or more count threshold to determine a count classification of a type of aberration existing in the biological sample for the first subchromosomal region;
determining a size classification for the first subchromosomal region by:
calculating a first statistical value of sizes of the plurality of DNA molecules located in the first subchromosomal region;
calculating a reference statistical value of sizes of DNA molecules located in a reference region;
determining a separation value between the first statistical value and the reference statistical value; and
comparing the separation value to one or more size thresholds to obtain the size classification; and
determining whether the fetus has the aberration in the first subchromosomal region based on the size classification and the count classification.

### Claim 27 (depends on 1)

The method of claim 1, further comprising:
collecting the biological sample from the female subject.

### Claim 28 (depends on 26)

The computer product of claim 26, wherein the plurality of instructions control the computer system to further perform:
identifying the first subchromosomal region by:
determining a respective bin count parameter for each of a plurality of non-overlapping bins in the reference genome, each respective bin count parameter determined from a respective amount of DNA molecules location in a respective bin; and
performing a segmentation process that merges consecutive bins into a merged segment based on the consecutive bins having a same count classification, wherein the first subchromosomal region corresponds to a first merged segment.

### Claim 29 (depends on 28)

The computer product of claim 28, wherein the segmentation process includes at least one of: a binary circular segmentation and a Hidden Markov model.

### Claim 30 (depends on 26)

The computer product of claim 26, wherein the plurality of instructions control the computer system to further perform:
detecting a plurality of aberrations in the biological sample for a plurality of other subchromosomal regions by comparing other count parameters to the one or more count thresholds to determine other count classifications; and
determining whether the fetus has the plurality of aberrations in the plurality of other subchromosomal regions based on the other count classifications and other size classifications for the plurality of other subchromosomal regions.

### Claim 31 (depends on 26)

The computer product of claim 26, wherein the count classification is over-represented or under-represented.

### Claim 32 (depends on 26)

The computer product of claim 26, wherein the aberration is a deletion or a duplication.

### Claim 33 (depends on 26)

The computer product of claim 26, wherein the size classification is one of longer, shorter, or equal.

### Claim 34 (depends on 26)

The computer product of claim 26, wherein the fetus is determined to have an amplification in the first subchromosomal region when:
the count classification indicates the amplification and the size classification indicates the aberration does not exist in the first subchromosomal region, or
the count classification indicates the amplification and the size classification indicates the amplification.

### Claim 35 (depends on 34)

The computer product of claim 34, wherein the plurality of instructions control the computer system to further perform:
determining the female subject to also have the amplification in the first subchromosomal region when the count classification indicates the amplification and the size classification indicates the aberration does not exist in the first subchromosomal region.

### Claim 36 (depends on 35)

The computer product of claim 35, wherein the plurality of instructions control the computer system to further perform:
determining the female subject not to have the amplification in the first subchromosomal region when the count classification indicates the amplification and the size classification indicates the amplification.

### Claim 37 (depends on 26)

The computer product of claim 26, wherein the fetus is determined to have a deletion in the first subchromosomal region when:
the count classification indicates the deletion and the size classification indicates the aberration does not exist in the first subchromosomal region, or
the count classification indicates the deletion and the size classification indicates the deletion.

### Claim 38 (depends on 37)

The computer product of claim 37, wherein the plurality of instructions control the computer system to further perform:
determining the female subject to also have the deletion in the first subchromosomal region when the count classification indicates the deletion and the size classification indicates the aberration does not exist in the first subchromosomal region.

### Claim 39 (depends on 37)

The computer product of claim 37, wherein the plurality of instructions control the computer system to further perform:
determining the female subject not to have the deletion in the first subchromosomal region when the count classification indicates the deletion and the size classification indicates the deletion.

### Claim 40 (depends on 26)

The computer product of claim 26, wherein the fetus is determined not to have the aberration when:
the count classification indicates an amplification and the size classification indicates a deletion, or
the count classification indicates the deletion and the size classification indicates the amplification.

### Claim 41 (depends on 40)

The computer product of claim 40, wherein the plurality of instructions control the computer system to further perform:
determining the female subject to have the amplification when the count classification indicates the amplification and the size classification indicates the deletion.

### Claim 42 (depends on 40)

The computer product of claim 40, wherein the plurality of instructions control the computer system to further perform:
determining the female subject to have the deletion when the count classification indicates the deletion and the size classification indicates the amplification.

### Claim 43 (depends on 26)

The computer product of claim 26, wherein the plurality of instructions control the computer system to further perform:
determining a first bin count parameter for a first bin of the first subchromosomal region, the first subchromosomal region including one or more bins, the first bin count parameter determined from a third amount of DNA molecules located in a third region and a fourth amount of DNA molecules located in the first bin;
computing a mean of control bin count parameters for the first bin using control samples;
computing a first score for the first bin by subtracting the mean of control bin count parameters from the first bin count parameter; and
determining whether the first score is greater than a cutoff value to identify whether the female subject has the aberration for the first subchromosomal region.

### Claim 44 (depends on 43)

The computer product of claim 43, wherein the plurality of instructions control the computer system to further perform:
measuring a fetal DNA concentration in the biological sample;
computing a difference between the first score and the fetal DNA concentration, and wherein the determining whether the first score is greater than the cutoff value includes determining whether the difference is greater than a high threshold value.

### Claim 45 (depends on 44)

The computer product of claim 44, wherein the plurality of instructions control the computer system to further perform:
comparing the difference to a low threshold; and
determining that only the fetus has the aberration for the first subchromosomal region when the difference is below a low threshold value.

### Claim 46 (depends on 43)

The computer product of claim 43, wherein subtracting the mean of control bin count parameters from the first bin count parameter provides a result, and wherein computing the first score further includes dividing the result by the mean of control bin count parameters.

### Claim 47 (depends on 43)

The computer product of claim 43, wherein subtracting the mean of control bin count parameters from the first bin count parameter provides a result, and wherein computing the first score further includes dividing the result by a standard deviation of the control bin count parameters.

### Claim 48 (depends on 43)

The computer product of claim 43, wherein the first subchromosomal region includes only one bin, and wherein the fourth amount is of DNA molecules in the first subchromosomal region.

### Claim 49 (depends on 43)

The computer product of claim 43, wherein the first subchromosomal region includes a plurality of bins, wherein the plurality of instructions control the computer system to further perform:
computing a respective score for each of the plurality of bins, wherein the first score is determined using a sum of the respective scores.

### Claim 50 (depends on 49)

The computer product of claim 49, wherein the first score is the sum divided by a square root of a number of bins used to determine the sum.

### Claim 51 (depends on 43)

The computer product of claim 43, wherein the plurality of instructions control the computer system to further perform:
determining no aberration to exist in the first subchromosomal region when the first score is less than the cutoff value and when:
the count classification indicates an amplification and the size classification indicates the aberration does not exist in the first subchromosomal region, or
the count classification indicates a deletion and the size classification indicates the aberration does not exist in the first subchromosomal region.