Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood

Abstract

We directly sequenced cell-free DNA with high-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 polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, 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 this DNA is enriched for sequences from nucleosomes.

Conflict of interest statement

Conflict of interest statement: S.R.Q. is a founder, shareholder, and consultant of Fluidigm Corporation. S.R.Q. and H.C.F. have applied for a patent relating to the method described in this study. Other authors declare no conflict of interest.

Figures

Fig. 1.

Fetal aneuploidy is detectable by the overrepresentation of the affected chromosome in maternal blood. (A) Sequence tag density relative to the corresponding value of genomic DNA control; chromosomes are ordered by increasing GC content. (B) Chromosome 21 sequence tag density relative to the median chromosome 21 sequence tag density of the normal cases. Note that the values of three disomy 21 cases overlap at 1.0. The dashed line represents the upper boundary of the 99% confidence interval constructed from all disomy 21 samples. Number of disomy 21 samples = 9. Number of trisomy 21 samples = 9.

Fig. 2.

Fetal DNA fraction and gestational age. The fraction of fetal DNA in maternal plasma correlates with gestational age. Fetal DNA fraction was estimated in three different ways: (i) from the additional amount of chromosomes 13, 18, and 21 sequences for T13, T18, and T21 cases, respectively; (ii) from the depletion in amount of chromosome X sequences for male cases; (iii) from the amount of chromosome Y sequences present for male cases. The horizontal dashed line represents the estimated minimum fetal DNA fraction required for the detection of aneuploidy. For each sample, the values of fetal DNA fraction calculated from the data of different chromosomes were averaged. There is a statistically significant correlation between the average fetal DNA fraction and gestational age (P = 0.0051). The dashed line represents the simple linear regression line between the average fetal DNA fraction and gestational age. The R2 value represents the square of the correlation coefficient.

Fig. 3.

Size distribution of maternal and fetal DNA in maternal plasma. A histogram showing the size distribution of total and chromosome Y-specific fragments obtained from 454 sequencing of maternal plasma DNA from a normal male pregnancy is presented. The distribution is normalized to sum to 1. The numbers of total reads and reads mapped to the Y chromosome are 144,992 and 178, respectively. (Inset) Cumulative fetal DNA fraction as a function of sequenced fragment size. The error bars correspond to the standard error of the fraction estimated, assuming that the error of the counts of sequenced fragments follow Poisson statistics.

Fig. 4.

Distribution of sequence tags around transcription start sites (TSS) of ReSeq genes on all autosomes and chromosome X from plasma DNA sample of a normal male pregnancy (Upper) and randomly sheared genomic DNA control (Lower). The number of tags within each 5-bp window was counted within ±1,000-bp region around each TSS, taking into account the strand to which each sequence tag mapped. The counts from all transcription start sites for each 5-bp window were summed and normalized to the median count among the 400 windows. A moving average was used to smooth the data. A peak in the sense strand represents the beginning of a nucleosome, whereas a peak in the antisense strand represents the end of a nucleosome. In the plasma DNA sample shown here, five well positioned nucleosomes are observed downstream of transcription start sites and are represented as gray ovals. The number within each oval represents the distance in base pairs between adjacent peaks in the sense and antisense strands, corresponding to the size of the inferred nucleosome. No obvious pattern is observed for the genomic DNA control.