Next-Generation–Sequencing-Based Noninvasive Prenatal Diagnosis - Next Generation Sequencing Technologies in Medical Genetics - page 46

5.6 Noninvasive Prenatal Diagnosis of Fetal Chromosomal

Aneuploidy by Next-Generation-Sequencing

Chiu et al. applied the same technique as Fan et al., but followed a different strategy
for data analysis (Chiu et al. 2008 ). They used this strategy in order to detect trisomy
21. They tested an algorithm to calculate the percentage of unique sequences for the
chromosome of interest in the test sample and compared it with the reference popula-
tion of that same chromosome. The differences in amounts of chr21 DNA sequences
in maternal plasma contributed by T21 fetuses compared with euploid fetuses were
unambiguously demonstrated due to the ability of NGS to sequence a large number
of molecules. For example, about ten million 36-bp reads were generated for each
plasma sample, which was equivalent to just one-tenth of the human genome.
Furthermore, in this study, only the U0-1-0-0 sequences, representing just 20 % of
all of the reads sequenced from each plasma DNA sample, were used to generate a
quantitative profi le of chromosomal distribution. In contrast to previous methods that
relied on coverage quantifi cation, this method simply sequences a random represen-
tative fraction of the human genome. The relative chromosome size is then deduced
by counting the relative number of sequences aligned to the chromosome. Despite
the randomness of the sequencing, the quantitative estimation of %chr21 sequences
was so precise and robust that the z -scores for chr21 of the T21 pregnancies were
markedly different from the mean of a reference euploid sample set. Similarly, abso-
lute differences in amounts of chrX and chrY DNA sequences in maternal plasma
contributed by male fetuses compared with female fetuses were convincingly
observed. Measurements of the genomic representations for chromosomes 13 and 18
were less precise (Chiu et al. 2010 ). The important advantage of the NGS technique
is that it is gender- and polymorphism-independent, applicable in all pregnancies and
likely to allow analysis of all frequent forms of aneuploidies in the same test.
Currently, the technique is technically demanding, the cost per tested sample is high,
and the throughput per instrument is low (16 samples per week). This prevents its use
as a regular test for all pregnant women. Recently, three large-scale clinical studies
were published showing results that NGS of maternal DNA of pregnant women is a
powerful molecular diagnostic tool for diagnosis of fetal aneuploidies (Chiu et al.
2011 ; Ehrich et al. 2011 ; Palomaki et al. 2011 ). Furthermore, it was shown that NGS
in combination with targeted enrichment of chromosome X is also a suitable approach
for noninvasive detection of fetal-specifi c alleles (Liao et al. 2011 ).

5.7 Noninvasive Prenatal Diagnosis of Fetal Chromosomal

Aneuploidy Using Different Next-Generation-
Sequencing Strategies and Algorithms

Stumm et al. tested whether other sequencing and enrichment platforms could be
used for NIPD of fetal chromosomal aneuploidy (Stumm et al. 2012 ). Specifi cally,
they showed the ability to adopt various NGS technologies and to provide new