Noninvasive Diagnosis of Fetal Aneuploidy by Sequencing

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.

1 . A method for determining a fetal abnormality, the method comprising:
 obtaining a maternal sample;   sequencing at least a portion of nucleic acids in the sample;   comparing obtained sequence information to a reference sequence;   identifying fetal nucleic acid, if present, in the sample;   optionally, if fetal nucleic acid is present, determining whether the fetus has an abnormality.   
     
     
         2 . The method of  claim 1 , wherein said reference sequence is selected from a maternal reference sequence, a fetal reference sequence, or a consensus human genomic sequence. 
     
     
         3 . The method of  claim 2 , wherein said maternal reference sequence is selected from a sequence obtained from a buccal sample, a saliva sample, a urine sample, a breast nipple aspirate sample, a sputum sample, a tear sample, and an amniotic fluid sample. 
     
     
         4 . The method of  claim 1 , wherein the sequencing reaction is a single molecule sequencing reaction. 
     
     
         5 . The method of  claim 4 , wherein the single molecule sequencing reaction comprises sequencing by synthesis and/or sequencing by nanopore detection. 
     
     
         6 . The method according to  claim 1 , wherein the maternal sample is a tissue or body fluid. 
     
     
         7 . The method according to  claim 6 , wherein the body fluid is maternal blood, blood plasma, or serum. 
     
     
         8 . The method according to  claim 1 , wherein the fetal nucleic acid is cell free circulating fetal nucleic acid. 
     
     
         9 . The method according to  claim 1 , wherein prior to the sequencing step, the method further comprises enriching for fetal nucleic acid in the sample. 
     
     
         10 . The method of  claim 1 , wherein said identifying step comprises a technique selected from sparse allele calling, targeted gene sequencing, identification of Y chromosomal material, enumeration, copy number analysis, and breakpoint analysis. 
     
     
         11 . A method for determining whether a fetus has an abnormality, the method comprising:
 obtaining a maternal sample comprising both maternal and fetal nucleic acids;   attaching a plurality of unique tags to nucleic acids in the sample, wherein each type of tag is associated with a different genomic region;   performing a sequencing reaction on the tagged nucleic acids to obtain tagged sequences;   and determining whether the fetus has an abnormality by quantifying the tagged sequences.   
     
     
         12 . The method according to  claim 11 , wherein the different genomic region is at least a portion of a chromosome. 
     
     
         13 . A method determining the presence of fetal nucleic acid, the method comprising:
 obtaining a maternal sample; sequencing nucleic acids in the sample, wherein said sequencing has an associated error rate;   determining whether fetal nucleic acid is present in the sample based at least in part on a quantitative measure of nucleic acid identified as fetal by said sequencing wherein the quantitative measure has a confidence level determined at least in part by the error rate.   
     
     
         14 . A method for determining whether a fetus has an abnormality, the method comprising:
 obtaining a maternal sample suspected to contain maternal and fetal nucleic acids;   sequencing nucleic acids in the sample;   comparing obtained sequence information to a reference sequence;   identifying regions of sequence match or mismatch between the obtained sequence information and the reference sequence;   confirming the presence of fetal nucleic acid in the sample;   and optionally determining whether the fetus has an abnormality based upon the results of the identifying step.   
     
     
         15 . A method for determining whether fetal nucleic acid is present in a maternal sample, the method comprising:
 obtaining a maternal sample suspected to include fetal nucleic acids;   and performing a sequencing reaction on the sample that is capable of detecting presence of at least a portion of a Y chromosome in the sample if such portion is present;   and determining that fetal nucleic acid is present in the sample.   
     
     
         16 . The method according to  claim 15 , wherein the sequencing reaction is a single molecule sequencing reaction. 
     
     
         17 . The method according to  claim 15 , wherein the maternal sample is a tissue or body fluid. 
     
     
         18 . The method according to  claim 17 , wherein the body fluid is maternal blood, blood plasma, or serum. 
     
     
         19 . The method according to  claim 15 , further comprising: performing a quantitative analysis on the obtained sequences to detect presence of fetal nucleic acid if the Y chromosome is not detected in the sample. 
     
     
         20 . A method for determining proper function of an assay used for detection of an abnormality in a fetus, the method comprising:
 obtaining a maternal sample suspected to include fetal nucleic acids;   determining whether at least a portion of a Y chromosome is present in the sample;   and optionally performing a quantitative analysis on the obtained sequences to detect presence of nucleic acid from a normal female fetus if the Y chromosome is not detected in the sample,   thereby determining that that the assay is functioning properly.   
     
     
         21 . A method for determining whether a fetus has an abnormality, the method comprising:
 obtaining a maternal sample comprising both maternal and fetal nucleic acids;   performing a sequencing reaction on the sample to obtain sequence information on nucleic acids in the sample;   comparing the obtained sequence information to sequence information from a reference genome, thereby determining whether the fetus has an abnormality;   detecting presence of at least a portion of a Y chromosome in the sample;   and distinguishing false negatives from true negatives if the Y chromosome is not detected in the sample.   
     
     
         22 . The method according to  claim 21 , wherein distinguishing comprises: performing a quantitative analysis selected from the group consisting of copy number analysis; sparse allele calling; targeted resequencing; and inversion analysis. 
     
     
         23 . A method for determining whether a fetus has an abnormality, the method comprising:
 obtaining a maternal sample comprising both maternal and fetal nucleic acids;   performing a sequencing reaction on the sample to obtain sequence information on nucleic acids in the sample;   comparing the obtained sequence information to sequence information from a reference genome,   thereby determining whether the fetus has an abnormality;   and distinguishing false negatives from true negatives.   
     
     
         24 . The method according to  claim 23 , wherein distinguishing comprises:
 assaying the sample for presence of at least a portion of a Y chromosome;   and optionally performing a quantitative analysis on the obtained sequences to detect presence of nucleic acid from a normal female fetus if the Y chromosome is not detected in the sample.   
     
     
         25 . The method according to  claim 24 , wherein the quantitative analysis is accomplished by a technique selected from the group consisting of: copy number analysis; sparse allele calling; targeted resequencing; and breakpoint analysis. 
     
     
         26 . The method according to  claim 23 , wherein the distinguishing comprises performing a quantitative analysis on the obtained sequences to detect presence of nucleic acid from a normal fetus. 
     
     
         27 . The method according to  claim 26 , wherein the quantitative analysis is accomplished by a technique selected from the group consisting of copy number analysis; sparse allele calling; targeted resequencing; and breakpoint analysis. 
     
     
         28 . The method according to  claim 23 , wherein prior to the performing step, the method further comprises enriching for the fetal nucleic acids in the sample. 
     
     
         29 . The method according to  claim 23 , wherein the sequencing reaction is a single molecule sequencing by synthesis reaction. 
     
     
         30 . The method according to  claim 23 , wherein the maternal sample is a tissue or body fluid. 
     
     
         31 . The method according to  claim 30 , wherein the body fluid is maternal blood, blood plasma, or serum. 
     
     
         32 . The method according to  claim 23 , wherein the abnormality results from a chromosomal aberration. 
     
     
         33 . The method according to  claim 23 , wherein the abnormality results from fetal aneuploidy. 
     
     
         34 . The method according to  claim 23 , wherein the abnormality is selected from the group consisting of Down syndrome (trisomy of chromosome 21), Edward syndrome (trisomy of chromosome 18), and Patau syndrome (trisomy of chromosome 13). 
     
     
         35 . A method for determining whether a fetus has an abnormality, the method comprising:
 obtaining a maternal sample comprising both maternal and fetal nucleic acids;   performing a sequencing reaction on the sample to obtain sequence information on nucleic acids in the sample;   comparing the obtained sequence information to sequence information from a reference genome,   thereby determining whether the fetus has an abnormality;   detecting presence of at least a portion of a Y chromosome in the sample;   and distinguishing false negatives from true negatives if the Y chromosome is not detected in the sample.   
     
     
         36 . The method according to  claim 35 , wherein prior to the performing step, the method further comprises enriching for the fetal nucleic acids in the sample. 
     
     
         37 . The method according to  claim 35 , wherein distinguishing comprises: performing a quantitative analysis selected from the group consisting of: copy number analysis; sparse allele calling; targeted resequencing; and breakpoint analysis. 
     
     
         38 . The method according to  claim 35 , wherein the comparing step comprises comparison of measured depth of sequencing coverage in a region of interest to expected depth of coverage in a diploid sample with the same total coverage. 
     
     
         39 . The method according to  claim 38 , wherein the region of interest is an entire chromosome. 
     
     
         40 . The method according to  claim 38 , wherein the region of interest is a part of a chromosome. 
     
     
         41 . The method according to  claim 38 , wherein where the expected depth of coverage is determined by average coverage from a control population. 
     
     
         42 . The method according to  claim 38 , wherein the expected depth of coverage is determined by comparison to majority of regions of the same sample. 
     
     
         43 . The method according to  claim 38 , wherein the expected depth of coverage is determined by a model of the observed distribution of coverage values in the sample which partitions the space of coverage depth into ranges corresponding to different copy numbers. 
     
     
         44 . A method for determining whether a fetus has an abnormality, the method comprising:
 obtaining a maternal sample comprising both maternal and fetal nucleic acids;   attaching unique tags to nucleic acids in the sample, wherein each tag is associated with a different chromosome;   performing a sequencing reaction on the tagged nucleic acids to obtain tagged sequences;   and optionally determining whether the fetus has an abnormality by quantifying the tagged sequences.   
     
     
         45 . The method according to  claim 44 , wherein the tags comprise unique nucleic acid sequences. 
     
     
         46 . A method for determining whether fetal nucleic acid is present in a maternal sample, the method comprising:
 obtaining a maternal sample suspected to include fetal nucleic acids; selecting at least two unique k-mers for detection in the sample;   and determining whether fetal nucleic acid is present in the maternal sample based on the ratio of the unique k-mers.   
     
     
         47 . The method according to  claim 46 , further comprising diagnosing an abnormality in the fetus based on analysis of the detected sequence. 
     
     
         48 . The method according to  claim 46 , wherein the unique sequences comprise one or more single nucleotide polymorphisms. 
     
     
         49 . The method according to  claim 46 , further comprising the step of determining an amount of each of said unique k-mers. 
     
     
         50 . The method according to  claim 46 , wherein said determining step comprises sequencing at least a portion of said nucleic acid. 
     
     
         51 . The method according to  claim 46 , wherein the maternal sample is a tissue or body fluid. 
     
     
         52 . The method according to  claim 46 , wherein the body fluid is maternal blood, blood plasma, or serum. 
     
     
         53 . A method for identifying a fetal abnormality, the method comprising the steps of:
 sequencing nucleic acid from a maternal sample;   distinguishing fetal nucleic acid from maternal nucleic acid;   confirming presence or absence of said fetal nucleic acid;   and optionally identifying an abnormality based upon a sequence variant in said fetal nucleic acid.   
     
     
         54 . The method of  claim 53 , wherein said confirming step comprises identifying false negative results in said distinguishing step. 
     
     
         55 . The method of  claim 53 , wherein said confirming step comprises identifying false positive results in said distinguishing step. 
     
     
         56 . The method of  claim 53 , wherein said confirming step comprises identification of nucleic acid associated with a Y chromosome. 
     
     
         57 . The method of  claim 53 , wherein said distinguishing step comprises comparing obtained sequence to one or more reference sequence. 
     
     
         58 . The method of  claim 57 , wherein said reference sequence is selected from a human genome consensus sequence, a maternal reference sequence, a paternal reference sequence, and a fetal reference sequence. 
     
     
         59 . A method for analyzing nucleic acids in a sample, the method comprising:
 sequencing a sample to obtain nucleic acid sequence information;   determining an amount of GC bias in the sequence information;   correcting the sequence information to account for the GC bias;   and analyzing the corrected information.   
     
     
         60 . The method according to  claim 59 , wherein determining comprises:
 partitioning the sequence information into bins;   and measuring a correlation between a number of chromosome counts in each bin and its GC content,   wherein a statistically significant negative or positive correlation indicates existence of GC bias.   
     
     
         61 . The method according to  claim 59 , wherein correcting comprises: selecting a subset of bins within a given range such that average GC content per chromosome is equalized. 
     
     
         62 . The method according to  claim 59 , wherein correcting comprises:
 modeling a correlation between GC content and chromosome counts across a set of bins;   and adjusting the effect of the GC bias by subtracting the GC-dependent component from the chromosome count in each bin based upon the modeling.   
     
     
         63 . The method according to  claim 59 , wherein correcting comprises:
 obtaining an average sequence coverage per bin over a number of controls;   and dividing the obtained coverage in the sample by the mean of the controls.   
     
     
         64 . The method according to  claim 59 , wherein the sample is a tissue or body fluid. 
     
     
         65 . The method according to  claim 64 , wherein the sample is suspected to contain fetal nucleic acid. 
     
     
         66 . The method according to  claim 65 , wherein the body fluid is maternal blood, blood plasma, or serum. 
     
     
         67 . The method according to  claim 59 , wherein sequencing is single molecule sequencing and/or sequencing by nanopore detection. 
     
     
         68 . The method according to  claim 67 , wherein single molecule sequencing is sequencing by synthesis and/or sequencing by nanopore detection. 
     
     
         69 . A method for identifying a fetal abnormality, the method comprising:
 obtaining a maternal sample;   sequencing at least a portion of nucleic acids in the sample to obtain sequence information;   determining an amount of GC bias in the sequence information;   correcting the sequence information to account for the GC bias;   comparing corrected sequence information to a reference sequence;   identifying fetal nucleic acid, if present, in the sample; optionally, if fetal nucleic acid is present,   determining whether the fetus has an abnormality.   
     
     
         70 . The method according to  claim 69 , wherein the reference sequence is selected from a maternal reference sequence, a fetal reference sequence, or a consensus human genomic sequence. 
     
     
         71 . The method according to  claim 69 , wherein said maternal reference sequence is selected from a sequence obtained from a buccal sample, a saliva sample, a urine sample, a breast nipple aspirate sample, a sputum sample, a tear sample, and an amniotic fluid sample. 
     
     
         72 . The method according to  claim 69 , wherein sequencing is single molecule sequencing. 
     
     
         73 . The method according to  claim 72 , wherein single molecule sequencing comprises sequencing by synthesis and/or sequencing by nanopore detection. 
     
     
         74 . The method according to  claim 69 , wherein the maternal sample is a tissue or body fluid. 
     
     
         75 . The method according to  claim 74 , wherein the body fluid is maternal blood, blood plasma, or serum. 
     
     
         76 . The method according to  claim 69 , wherein the fetal nucleic acid is cell free circulating fetal nucleic acid. 
     
     
         77 . The method according to  claim 69 , wherein prior to the sequencing step, the method further comprises enriching for fetal nucleic acid in the sample. 
     
     
         78 . The method according to  claim 69 , wherein the identifying step comprises a technique selected from sparse allele calling, targeted gene sequencing, identification of Y chromosomal material, enumeration, copy number analysis, and inversion analysis. 
     
     
         79 . The method according to  claim 69 , wherein determining comprises:
 partitioning the sequence information into bins;   and measuring a correlation between a number of chromosome counts in each bin and its GC content,   wherein a statistically significant negative or positive correlation indicates existence of GC bias.   
     
     
         80 . The method according to  claim 69 , wherein correcting comprises: selecting a subset of bins within a given range such that average GC content per chromosome is equalized. 
     
     
         81 . The method according to  claim 69 , wherein correcting comprises:
 modeling a correlation between GC content and chromosome counts across a set of bins;   and adjusting the effect of the GC bias by subtracting the GC-dependent component from the chromosome count in each bin based upon the modeling.   
     
     
         82 . The method according to  claim 69 , wherein correcting comprises:
 obtaining an average sequence coverage per bin over a number of controls   and dividing the obtained coverage in the sample by the mean of the controls.   
     
     
         83 . The method according to  claim 69 , wherein the determining comprises: comparing measured depth of coverage in chromosome regions to a normal control that was processed with the sample. 
     
     
         84 . The method according to  claim 59 , wherein determining comprises:
 distributing the sequence information into bins;   and measuring a correlation between a number of chromosome counts in each bin and its GC content,   wherein a statistically significant negative or positive correlation indicates existence of GC bias.   
     
     
         85 . The method according to  claim 59 , wherein correcting comprises:
 correlating GC content and chromosome counts across a set of bins;   and adjusting the effect of the GC bias by subtracting the GC-dependent component from the chromosome count in each bin based upon the modeling.   
     
     
         86 . The method according to  claim 59 , wherein the sample is a maternal sample. 
     
     
         87 . The method according to  claim 86 , wherein the sample is suspected to contain fetal nucleic acid. 
     
     
         88 . The method according to  claim 86 , wherein the maternal sample is maternal blood or blood plasma. 
     
     
         89 . The method according to  claim 59 , wherein sequencing is single molecule sequencing. 
     
     
         90 . The method according to  claim 67 , wherein single molecule sequencing is sequencing by synthesis. 
     
     
         91 . The method according to  claim 69 , wherein the body fluid is maternal blood or plasma. 
     
     
         92 . The method according to  claim 69 , wherein the identifying step comprises a technique selected from targeted gene sequencing, identification of Y chromosomal material, enumeration, and copy number analysis. 
     
     
         93 . The method according to  claim 69 , wherein determining comprises:
 distributing the sequence information into bins;   and measuring a correlation between a number of chromosome counts in each bin and its GC content,   wherein a statistically significant negative or positive correlation indicates existence of GC bias.