Rapid aneuploidy detection

Abstract

Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.

We claim: 
     
         1 . A method for detecting aneuploidy, the method comprising the steps of:
 exposing a sample comprising genomic nucleic acid to a plurality of identical primer pairs that anneal to a genomic region present in a chromosome that is expected to be aneuploid and in one or more chromosomes expected not to be aneuploid in the sample;   amplifying the nucleic acid using the primer pairs in order to produce a plurality of amplicons that contain a sufficient amount of unique sequence to allow a plurality of the amplicons to be distinguished from one another;   aligning the amplicons to one or more reference sequences in order to produce a distribution of the amplicons in the genome; and   identifying aneuploidy based upon an imbalance in the distribution.   
     
     
         2 . The method of  claim 1  wherein the chromosome that is expected to be aneuploid is selected from the group consisting of chromosome 18, chromosome 21, chromosome 13, an X chromosome, and a Y chromosome. 
     
     
         3 . The method of  claim 1  wherein the sufficient amount of unique sequence is from about 3 nucleotides to about 100 nucleotides. 
     
     
         4 . The method of  claim 1  wherein the sample is maternal plasma or serum and the chromosome that is expected to be aneuploid is a fetal chromosome. 
     
     
         5 . The method of  claim 1  wherein the aligning step is performed in silico using a reference set of chromosomes expected not to be aneuploid in the sample. 
     
     
         6 . The method of  claim 1  wherein the imbalance is a statistically significant difference between an amount of amplicon produced in the amplifying step from the genomic region present in a chromosome that is expected to be aneuploid and an amount in the reference sequences. 
     
     
         7 . The method of  claim 6  wherein the amounts are normalized. 
     
     
         8 . The method of  claim 1  wherein unique sequence tags are incorporated at a 5′ end of the primers. 
     
     
         9 . The method of  claim 8  further comprising counting the amplicons based upon the unique sequence tags in order to produce a quantitative measure of amplified chromosomal regions. 
     
     
         10 . The method of  claim 1  wherein the chromosome expected to be aneuploid is a fetal chromosome and the chromosomes expected not to be aneuploid are maternal chromosomes. 
     
     
         11 . The method of  claim 1  wherein the sample is derived from an embryo. 
     
     
         12 . The method of  claim 1  where each chromosome is treated as the expected aneuploid chromosome and the reference sequence is external to the sample. 
     
     
         13 . The method of  claim 1  wherein the aligning step comprises determining relative frequency at which the amplicons map to the chromosomes. 
     
     
         14 . The method of  claim 13  further comprising the step of identifying an imbalance in the relative frequency. 
     
     
         15 . The method of  claim 1  wherein the chromosomes expected to be aneuploid and the chromosomes expected not to be aneuploid are obtained from different samples. 
     
     
         16 . The method of  claim 1  further comprising the step of comparing the distribution to a distribution with known euploid content. 
     
     
         17 . The method of  claim 1  wherein the imbalance is a statistically significant difference between an amount of amplicon amplified from the genomic region present in the chromosome expected to be aneuploid and an amount of amplicon amplified from the genomic region present in the one or more chromosomes expected not to be aneuploid in the sample. 
     
     
         18 . The method of  claim 1  wherein the reference sequences are amplicons that align to the genomic region present in one or more chromosomes expected not to be aneuploid in said sample.