Biomedical Engineering Reference
algorithms for the quantifi cation of fetal aneuploidies. Clearly, NGS was successfully
applied for the detection of fetal trisomy 21. The study focused on sequencing 42
samples using the GAIIx and sequencing of 38 samples using the HiSeq 2000. On the
basis of the algorithm hg18_rm_0mm for z -score calculation described by Chiu et al.
all trisomy 21 samples were identifi ed. Furthermore, the power of quantifi cation
detection of trisomy 21 was improved by establishing new algorithms to calculate the
z -score. The algorithm hg18_par_X_Y_0mm_autosomes based on using only UMR-
0mm of autosomes was the most suitable calculation method for this assay. In addi-
tion, for the fi rst time a combination of the 38 Mb SureSelect Human All Exome
target enrichment system followed by sequencing allowed the identifi cation of fetal
trisomy 13 and fetal trisomy 21. However, further studies are necessary to prove that
it might be an alternative approach to detect all chromosomal aneuploidies.
Single Molecule Sequencing for the Detection
of Trisomy 21
van den Oever et al. described the application of single molecule NGS (with the
Helicos BioSciences platform) to the detection of trisomy 21 (van den Oever et al.
2012 ). NGS has developed substantially over the past 5 years, but most platforms
require the preliminary amplifi cation of genomic DNA sequences which introduces
sequence-specifi c artifacts. Single molecule NGS bypasses this requirement, and
the authors have compared the effi cacy of this approach with that of amplifi cation-
based NGS (Illumina's Genome Analyzer II) by using identical maternal plasma
samples obtained from mothers with (control) euploid and trisomy 21 fetuses. The
Helicos platform uses visual imaging across the fl ow cell for direct DNA measure-
ment by recording the incorporation of fl uorescently labeled nucleotides (Milos
2009 ). This approach largely overcomes the limitations associated with PCR ampli-
fi cation and bias. The sequencing time on the Helicos platform is longer (4 vs. 2
days, respectively), sample preparation is simple, relatively inexpensive, and
requires a low DNA input compared to Illumina.
A successful fetal T21 detection using free DNA from maternal plasma by single
molecule sequencing on the Helicos platform was demonstrated (van den Oever
et al. 2012 ). The greater sensitivity achieved with this platform is clearly advanta-
geous because it permits early diagnosis, which the authors of this study reported
being successful as early as 9 weeks, 3 days gestation. In addition, the Helicos
platform is not biased in GC-rich areas, thereby leading to increased accuracy of
analysis compared to the Illumina platform. This study shows for the fi rst time that
single molecule sequencing can be a reliable and easy-to-use alternative for nonin-
vasive T21 detection in diagnostics. By using single molecule sequencing, previ-
ously described experimental noise associated with PCR amplifi cation, such as GC
bias, can be overcome. The work clearly shows that further refi nement of NGS will
pave the way for routine use of NIPD for aneuploidy.