Noninvasive diagnosis of fetal aneuoploidy by sequencing
CAFCFirst Claim
1. A method of testing for an abnormal distribution of a chromosome in a sample comprising a mixture of maternal and fetal DNA, comprising the steps of:
- (a) obtaining maternal and fetal DNA from said sample;
(b) sequencing predefined subsequences of the maternal and fetal DNA to obtain a plurality of sequence tags aligning to the predefined subsequences, wherein said sequence tags are of sufficient length to be assigned to a specific predefined subsequence, wherein the predefined subsequences 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;
(c) assigning the plurality of sequence tags to their corresponding predetermined subsequences;
(d) determining a number of sequence tags aligning to the predetermined subsequences of said first chromosome and a number of sequence tags to the predetermined subsequences of the second chromosome; and
(e) comparing the numbers from step (d) to determine the presence or absence of an abnormal distribution of said first chromosome.
2 Assignments
1 Petition
Accused Products
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.
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Citations
15 Claims
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1. A method of testing for an abnormal distribution of a chromosome in a sample comprising a mixture of maternal and fetal DNA, comprising the steps of:
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(a) obtaining maternal and fetal DNA from said sample; (b) sequencing predefined subsequences of the maternal and fetal DNA to obtain a plurality of sequence tags aligning to the predefined subsequences, wherein said sequence tags are of sufficient length to be assigned to a specific predefined subsequence, wherein the predefined subsequences 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; (c) assigning the plurality of sequence tags to their corresponding predetermined subsequences; (d) determining a number of sequence tags aligning to the predetermined subsequences of said first chromosome and a number of sequence tags to the predetermined subsequences of the second chromosome; and (e) comparing the numbers from step (d) to determine the presence or absence of an abnormal distribution of said first chromosome. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of testing for an abnormal distribution of a chromosome in a sample comprising a mixture of maternal and fetal DNA, comprising the steps of:
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(a) obtaining maternal and fetal DNA from said sample; (b) sequencing predefined subsequences of the maternal and fetal DNA to obtain a plurality of sequence tags aligning to the predefined subsequences, wherein said sequence tags are of sufficient length to be assigned to a specific predefined subsequence, wherein the predefined subsequences 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; (c) assigning the plurality of sequence tags to their corresponding predetermined subsequences; (d) determining a relative number of sequence tags aligning to the predetermined subsequences of said first chromosome and to the predetermined subsequences of said second chromosome; (e) determining a weight for correcting for G/C bias and applying the weight to the numbers of sequence tags determined in step (d) to obtain a corrected number of sequence tags assigned to the predefined subsequences of the first chromosome and a corrected number of sequence tags assigned to the predefined subsequences of the second chromosome; and (f) comparing the corrected number of sequence tags aligning to the predetermined subsequences of said first chromosome to the corrected number of sequence tags aligning to the predetermined subsequences of said second chromosome to determine the presence or absence of an abnormal distribution of said first chromosome. - View Dependent Claims (15)
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Specification