Diagnosing fetal chromosomal aneuploidy using massively parallel genomic sequencing

Abstract

Embodiments of this invention provide methods, systems, and apparatus for determining whether a fetal chromosomal aneuploidy exists from a biological sample obtained from a pregnant female. Nucleic acid molecules of the biological sample are sequenced, such that a fraction of the genome is sequenced. Respective amounts of a clinically-relevant chromosome and of background chromosomes are determined from results of the sequencing. A parameter derived from these amounts (e.g. a ratio) is compared to one or more cutoff values, thereby determining a classification of whether a fetal chromosomal aneuploidy exists.

1 - 23 . (canceled) 
     
     
         24 . 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.   
     
     
         25 . The method of  claim 24  wherein the sample is a maternal serum or plasma sample, wherein the abnormal distribution of said first chromosome is a fetal aneuploidy, and wherein said second chromosome is a euploid chromosome. 
     
     
         26 . The method of  claim 25  wherein the sequencing comprises massively parallel sequencing of the predefined subsequences. 
     
     
         27 . The method of  claim 26  wherein said massively parallel sequencing comprises attaching DNA fragments to an optically transparent surface, conducing solid phase amplification of the attached DNA fragments to create a high density sequencing flow cell with millions of DNA clusters, and sequencing the DNA clusters by a four-color DNA sequencing-by-synthesis method employing reversible terminators with removable fluorescent dyes. 
     
     
         28 . The method of  claim 25  wherein the fetal aneuploidy is an aneuploidy of a chromosome selected from the group consisting of chromosome 13, chromosome 18 and chromosome 21. 
     
     
         29 . The method of  claim 25  wherein the step of assigning sequence tags to corresponding chromosome portions allows one mismatch. 
     
     
         30 . The method of  claim 25  wherein the length of the sequence tags is from about 25 bp to about 100 bp in length. 
     
     
         31 . The method of  claim 25  wherein the DNA is genomic DNA. 
     
     
         32 . The method of  claim 25  wherein said sequencing comprises selectively sequencing nucleic acid molecules comprising the predefined sequences. 
     
     
         33 . The method of  claim 32  wherein said sequencing comprises the use of a sequencing array. 
     
     
         34 . The method of  claim 33  wherein said selected defined subsequences of the genomic DNA are rendered single-stranded and captured under hybridizing conditions by single-stranded probes physically separated on an array. 
     
     
         35 . The method of  claim 25  further comprising determination of fetal DNA fraction of the DNA obtained from the maternal serum or plasma sample. 
     
     
         36 . The method of  claim 35  wherein the fetal DNA fraction is determined by digital PCR. 
     
     
         37 . 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 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.   
     
     
         38 . The method of  claim 37  wherein the sample is a maternal serum or plasma sample, wherein the abnormal distribution of said first chromosome is a fetal aneuploidy, and wherein said second chromosome is a euploid chromosome.