Cell-free fetal DNA is present in the plasma of pregnant women. It consists of short DNA fragments among primarily maternally derived DNA fragments. We sequenced a maternal plasma DNA sample at up to 65-fold genomic coverage. We showed that the entire fetal and maternal genomes were represented in maternal plasma at a constant relative proportion. Plasma DNA molecules showed a predictable fragmentation pattern reminiscent of nuclease-cleaved nucleosomes, with the fetal DNA showing a reduction in a 166-base pair (bp) peak relative to a 143-bp peak, when compared with maternal DNA. We constructed a genome-wide genetic map and determined the mutational status of the fetus from the maternal plasma DNA sequences and from information about the paternal genotype and maternal haplotype. Our study suggests the feasibility of using genome-wide scanning to diagnose fetal genetic disorders prenatally in a noninvasive way.

Chromosomal aneuploidy is the major reason why couples opt for prenatal diagnosis. Current methods for definitive diagnosis rely on invasive procedures, such as chorionic villus sampling and amniocentesis, and are associated with a risk of fetal miscarriage. Fetal DNA has been found in maternal plasma but exists as a minor fraction among a high background of maternal DNA. Hence, quantitative perturbations caused by an aneuploid chromosome in the fetal genome to the overall representation of sequences from that chromosome in maternal plasma would be small. Even with highly precise single molecule counting methods such as digital PCR, a large number of DNA molecules and hence maternal plasma volume would need to be analyzed to achieve the necessary analytical precision. Here we reasoned that instead of using approaches that target specific gene loci, the use of a locus-independent method would greatly increase the number of target molecules from the aneuploid chromosome that could be analyzed within the same fixed volume of plasma. Hence, we used massively parallel genomic sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. Twenty-eight first and second trimester maternal plasma samples were tested. All 14 trisomy 21 fetuses and 14 euploid fetuses were correctly identified. Massively parallel plasma DNA sequencing represents a new approach that is potentially applicable to all pregnancies for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidies.Down syndrome ͉ Solexa sequencing ͉ trisomy 21

Objectives To validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling. Design Diagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples. Setting Prenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands. Participants 753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing. Main outcome measures Proportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection. Results Results were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%. ConclusionMultiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

Trisomy 21 is the most common reason that women opt for prenatal diagnosis. Conventional prenatal diagnostic methods involve the sampling of fetal materials by invasive procedures such as amniocentesis. Screening by ultrasonography and biochemical markers have been used to risk-stratify pregnant women before definitive invasive diagnostic procedures. However, these screening methods generally target epiphenomena, such as nuchal translucency, associated with trisomy 21. It would be ideal if noninvasive genetic methods were available for the direct detection of the core pathology of trisomy 21. Here we outline an approach using digital PCR for the noninvasive detection of fetal trisomy 21 by analysis of fetal nucleic acids in maternal plasma. First, we demonstrate the use of digital PCR to determine the allelic imbalance of a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome 21, in the maternal plasma of women bearing trisomy 21 fetuses. We named this the digital RNA SNP strategy. Second, we developed a nonpolymorphism-based method for the noninvasive prenatal detection of trisomy 21. We named this the digital relative chromosome dosage (RCD) method. Digital RCD involves the direct assessment of whether the total copy number of chromosome 21 in a sample containing fetal DNA is overrepresented with respect to a reference chromosome. Even without elaborate instrumentation, digital RCD allows the detection of trisomy 21 in samples containing 25% fetal DNA. We applied the sequential probability ratio test to interpret the digital PCR data. Computer simulation and empirical validation confirmed the high accuracy of the disease classification algorithm.circulating fetal nucleic acids ͉ noninvasive prenatal diagnosis ͉ sequential probability ratio test ͉ trisomy 21 ͉ RNA SNP