Biomedical Engineering Reference
In-Depth Information
aneuploidy detection and specifi cally the %chrN values should be refl ective of the
genomic representation of the maternal and fetal DNA fragments in maternal plasma.
NGS must permit capture and sequencing of the small fetal DNA fraction alongside
the maternal DNA. Moreover, the captured and sequenced pool of plasma DNA must
represent the total DNA pool with similar interchromosomal distribution to that in
the original maternal plasma. In addition, a minimal bias in ability to sequence DNA
fragments originating from each chromosome is assumed. Furthermore, if both the
maternal and the fetal genomes are evenly represented in maternal plasma, the pro-
portional contribution of plasma DNA sequences per chromosome should in turn
bear correlation with the relative size of each chromosome in the human genome.
5.5
Noninvasive Diagnosis of Fetal Aneuploidy
by Shotgun Sequencing DNA
Fan et al. applied NGS on cfDNA from plasma of pregnant women with a gestational
age of 10-35 weeks (Fan et al.
2008
). They directly sequenced cfDNA with a high-
throughput shotgun sequencing technology (Illumina's platform) and obtained fi ve
million sequence tags per patient from plasma of 18 pregnant women. An average
of 154,000, 135,000, and 65,700 sequence tags mapped to chromosomes 13, 18,
and 21 were obtained, respectively. This enabled the measurement of over- and
underrepresentation of chromosomes from an aneuploid fetus. The distribution of
chromosome 21 sequence tag density for all nine T21 pregnancies was clearly sepa-
rated from that of pregnancies bearing disomy 21 fetuses. The coverage of chromo-
some 21 for T21 cases was 4-18 % higher (average 11 %) than that of the disomy
21 cases. Moreover, plasma DNA of pregnant women carrying T18 fetuses (two
cases) and a T13 fetus (one case) were also directly sequenced. Overrepresentation
was observed for chromosomes 18 and 13 in T18 and T13 cases, respectively.
Although there were not enough positive samples to measure a representative dis-
tribution, it was encouraging that all of these three positives were outliers from the
distribution of disomy values. The T18 were large outliers and were clearly statisti-
cally signifi cant, whereas the statistical signifi cance of the single T13 case was
marginal. An advantage of using direct sequencing to measure aneuploidy noninva-
sively is that it is able to make full use of the sample, whereas PCR-based methods
analyze only a few targeted sequences. Fan et al. showed successful development of
a truly universal, polymorphism-independent noninvasive test for fetal aneuploidy.
The sequencing approach is polymorphism-independent and therefore universally
applicable for the noninvasive detection of fetal aneuploidy. Using this method,
authors successfully identifi ed all nine cases of trisomy 21 (Down syndrome), two
cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syn-
drome) 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 the
study of the characteristics of cell-free plasma DNA, and evidence was found that
this DNA is enriched for sequences from nucleosomes.
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