Biotecnología

Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood
H. Christina Fan*, Yair J. Blumenfeld†, Usha Chitkara†, Louanne Hudgins‡, and Stephen R. Quake*§
*Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, 318 Campus Drive, Clark Center, Room E300, Stanford, CA 94305; †Division of Maternal-Fetal Medicine, Department ofObstetrics and Gynecology, Stanford University, 300 Pasteur Drive, Room HH333, Stanford, CA 94305; and ‡Division of Medical Genetics, Department of Pediatrics, Stanford University, 300 Pasteur Drive, Stanford, CA 94305 Communicated by Leonard A. Herzenberg, Stanford University School of Medicine, Stanford, CA, August 22, 2008 (received for review July 13, 2008)

We directly sequenced cell-free DNA withhigh-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphismindependent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using thismethod, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that thisDNA is enriched for sequences from nucleosomes.
fetal DNA next-generation sequencing Down syndrome trisomy noninvasive prenatal diagnosis

some of origin and enumeration of fragments per chromosome. Recent advances in DNA-sequencing technology allow massively parallel sequencing (20), producing tens of millions of short sequence tags in a single run and enabling a deeper sampling than can beachieved by digital PCR. By counting the number of sequence tags mapped to each chromosome, the over- or underrepresentation 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, and with large enough tag counts, it can be applied to arbitrarily small fractions of fetal DNA. Wedemonstrate here the successful use of shotgun sequencing to detect fetal trisomy 21 (Down syndrome), trisomy 18 (Edward syndrome), and trisomy 13 (T13) (Patau syndrome) noninvasively by using cell-free fetal DNA in maternal plasma. This forms the basis of a universal, polymorphism-independent noninvasive diagnostic test for fetal aneuploidy. The sequence data also allowed us to characterize plasma DNAin unprecedented detail, suggesting that it is enriched for nucleosome-bound fragments. Results
Shotgun Sequencing of Cell-Free Plasma DNA. Cell-free plasma DNA

etal aneuploidy and other chromosomal aberrations affect 9 of 1,000 live births (1). The gold standard for diagnosing chromosomal abnormalities is karyotyping of fetal cells obtained via invasive procedures such as chorionic villussampling and amniocentesis. These procedures impose small but potentially significant risks to both the fetus and the mother (2). Noninvasive screening of fetal aneuploidy using maternal serum markers and ultrasound are available but have limited reliability (3–5). There is therefore a desire to develop noninvasive genetic tests for fetal chromosomal abnormalities. Since the discovery of intactfetal cells in maternal blood, there has been intense interest in trying to use them as a diagnostic window into fetal genetics (6–9). Although this has not yet moved into practical application (10), the later discovery that significant amounts of cell-free fetal nucleic acids also exist in maternal circulation has led to the development of new noninvasive prenatal genetic tests for a variety of...