DOI: 10.1002/pd.

4111

REVIEW

Benets and limitations of whole genome versus targeted

approaches for noninvasive prenatal testing for fetal aneuploidies
Elles M. J. Boon1* and Brigitte H. W. Faas2
1

Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis, Leiden University Medical Center, Leiden, The Netherlands
Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands
*Correspondence to: Elles M. J. Boon. E-mail E.M.J.Boon@LUMC.nl

ABSTRACT
The goal to noninvasively detect fetal aneuploidies using circulating cell-free fetal DNA in the maternal plasma seems
to be achieved by the use of massively parallel sequencing (MPS). To date, different MPS approaches exist, all aiming
to deliver reliable results in a cost effective manner. The most widely used approach is the whole genome MPS
method, in which sequencing is performed on maternal plasma to determine the presence of a fetal trisomy. To
reduce costs targeted approaches, only analyzing loci from the chromosome(s) of interest has been developed. This
review summarizes the different MPS approaches, their benets and limitations and discusses the implications for
future noninvasive prenatal testing. 2013 John Wiley & Sons, Ltd.

Funding sources: None

Conicts of interest: None declared

INTRODUCTION
Assessing the genetic condition of the fetus using fetal DNA
obtained via noninvasive procedures [noninvasive prenatal
testing (NIPT)] has been a dream for many years. In 1997, the
group of Dennis Lo for the rst time reported the presence of
circulating cell-free fetal DNA (ccffDNA) in the maternal
plasma.1 This ccffDNA was shown to be rapidly cleared from
the maternal circulation after delivery,2 and even though it
only represents a minor fraction of the total circulating free
DNA in maternal plasma, its proportion is still ~1020%
(varying between women and depending on gestational age),
whereas the remainder is of maternal origin.3,4
Since its discovery, many characteristics of ccffDNA have
been identied and much progress has been made in the
development of noninvasive prenatal tests using this material.
However, for fetal aneuploidy detection, noninvasive testing is
rather complex, as the chromosome or region of interest is also
carried by the mother, and there is only a quantitative difference
between the maternal and fetal contribution. The rst promising
report on noninvasive aneuploidy detection was from Lo et al. in
2007,5 when using fetal mRNA in the maternal plasma.
Subsequently, in 2008, two research groups independently
opened up a new era with the successful use of massively parallel
sequencing (MPS) for NIPT.6,7

MASSIVELY PARALLEL SEQUENCING FOR NONINVASIVE

ANEUPLOIDY DETECTION
Because the proportion of fetal DNA in the maternal plasma
is only ~10% in the rst trimester,4 for noninvasive aneuploidy
Prenatal Diagnosis 2013, 33, 563568

detection using ccffDNA in maternal plasma, it is essential to

have a method that can accurately determine a very small increase
in the total amount of a specic chromosome and/or to have a
method to enrich for fetal DNA. With MPS, millions to billions of
short DNA molecules can be analyzed simultaneously in a single
sequencing run,8,9 revealing both the identity and quantity of
DNA fragments. Granted it was possible to detect small changes
in copy number variations reliably with MPS, it was a pivotal step
to examine whether this technique could be used to detect fetal
aneuploidies using (nonenriched) ccffDNA. Indeed, the initial
proof-of-concept studies of Fan et al. and Chiu et al.6,7 described
the successful application of MPS for this purpose. The DNA in
maternal plasma was sequenced, and after mapping the
obtained reads to the human reference genome, the (relative)
numbers of fragments per chromosome were counted. If the
fetus carries a trisomy, (statistically signicant) more fragments
of the trisomic chromosome are expected to be present when
compared with a diploid fetus. Indeed, the initial studies of Fan
et al. and Chiu et al. showed that the MPS method allowed for
correct identication of fetal trisomies 21, 18, and 13, without
false negative or positive results. As a consequence of these
promising results, this method has been widely validated as the
base for aneuploidy detection.
However, MPS is not selective in the chromosomal origin
of the sequenced DNA fragments, and in a normal disomic
individual, chromosome 21, for example, represents less
than 1.5% of the sequenced fragments. It is therefore
necessary to sequence many million DNA fragments,
originating from the complete (nonenriched) genome, to
2013 John Wiley & Sons, Ltd.

E. M. J. Boon and B. H. W. Faas

564

ensure the analysis of sufcient chromosome 21 fragments

to statistically detect signicant differences between
normal and trisomic fetuses. Theoretically, with only one
chromosome of interest, this does not seem to be timeefcient and cost-efcient. Therefore, targeted alternatives
to whole genome MPS have been developed. In the
succeeding text, rst, the different next-generation
sequencing (NGS) platforms and their specications in
general terms, and subsequently, both whole genome and
targeted (MPS) approaches for NIPT, including benets
and limitations, will be discussed in more detail.

NGS PLATFORMS
Today, different NGS platforms are commercially available,
each with its own costs, benets, and limitations, especially
when used for NIPT. The costs of the sequencing platforms
have recently been reviewed10 and will not be discussed
further here as these are often dependent on local
negotiations. Among the available platforms, the use of four
have been reported for NIPT, namely the Illumina Genome
Analyzer/Illumina HiSeq 2000 Genome Analyzer,6,7,1114 the
Applied Biosystems (ABI) SOLiD Analyzer,15,16 the Helicos
Heliscope,17,18 and the IonTorrent (Life Technologies)
platform.19 These platforms are all short-read platforms,
which are, especially for NIPT, very well suited, as ccffDNA
shows a fragmentation pattern of around 146 bp.20 There
is evidence that fetal DNA is shorter than maternal DNA,
and this difference in length has been used to specically
enrich for fetal DNA.13,21,22 Given the fact that DNA in
maternal plasma is highly fragmented, one can envision
that this is the reason why no papers have appeared on
the use of longer-sequence-read platforms, such as the
Roche/454 (454 Life Sciences Branfort USA) and PacBio RS
(Pacic Biosciences CA USA) for NIPT, and these will
therefore not be discussed here.
The most widely used platforms are the high-throughput
HiSeqTM (Illumina, Inc) and SOLiDTM [Life TechnologiesTM/
Applied BiosystemsR (ABI)] platforms. These differ from each
other in that they use polymerase chain reaction (PCR)-based
sequencing-by-synthesis or sequencing-by-ligation.

HiseqTM (Illumina, Inc.)

For the Illumina platforms, DNA fragments are ligated to
adapters and attached to reaction chambers located on a ow
cell. The attached DNA fragments are extended, amplied by
bridge amplication, and sequenced through sequencing-bysynthesis. Using this technology, the Illumina HiSeqTM 2000
can produce up to three billion single-end reads per run, which,
for most applications, makes this platform very suitable for
multiplexing of samples and thus high throughput runs. The
run time for the HiSeqTM 2000 varies from 5 to 14 days,
depending on read length. With the arrival of the HiSeqTM
2500, one can even choose between a rapid run and a high
output run, making it possible to produce even faster results.
With the rapid run, 300 million single-end reads (~10 Gb) can
be produced within 7 h.
Prenatal Diagnosis 2013, 33, 563568

SolidTM [Life TechnologiesTM/Applied BiosystemsR (ABI)]

For the library construction of the ABI SOLiDTM system,
DNA fragments are ligated to adapters, attached to beads,
and subsequently clonally amplied by emulsion PCR.8,23
With sequencing-by-ligation, the SOLiD4 can produce up
to 0.7 billion single-end reads per run, and with the
5500xl SOLiDTM, the amount of data that can be generated
has increased to 1.4 billion single-end reads per run.
Runtime on the SOliDTM instrument varies between 5
and 10 days, depending on the experimental set-up. The
5500xl SOLiDTM machine uses two fully congurable
owchips, each with six independent lanes, allowing the
user to use between one and six lanes, whereas only
paying reagent costs for the lanes used rather than the
whole ow cell. Although this platform produces slightly
less data per run than the Illumina HiSeqTM platform, it
is also exible and can be used for high throughput runs,
with the recently marketed upgrade 5500xl W platform
offering even more possibilities.

HeliScopeR single molecule sequencer (HelicosTM Biosciences)

A weakness of the aforementioned platforms is that through
the PCR-based sample preparation experimental bias,
especially for GC-rich regions, can be introduced by the
amplication step. Using single-molecule-sequencing on, for
instance, the HeliScopeR Single Molecule Sequencer, this bias
can be eliminated. For NIPT, a small study reported that when
applying this single-molecule-sequencing platform, indeed, a
better distinction between fetal disomy and trisomy 21 could
be achieved than with PCR-based platforms.17 However,
because for NIPT in routine clinical practice, high throughput
analysis, achieved via multiplexing, is inevitable; this singlemolecule-sequencing platform is not very well suited as
multiplexing of samples is cumbersome.

Others
In addition to the high throughput sequencers mentioned
previously, several benchtop sequencers are available on
the market as well that might be suitable for NIPT, including
the MiSeqTM (Illumina, Inc.) and Ion TorrentTM PGM
(Life Technologies). Illuminas MiSeqTM is based on the existing
sequencing-by-synthesis chemistry, whereas the Ion TorrentTM
PGM uses the semiconductor sequencing technology. Both
benchtop sequencers have relatively low set-up costs and
reduced running times compared with most of the high
throughput sequencers. However, an important limitation
of the current benchtop machines is the amount of data that
can be generated. The MiSeqTM Personal Sequencer can produce
for a 2  100 bp run up to 3.4 Gb in 16.5 h, and the Ion TorrentTM
PGM (Ion 318 chip) 200 bp reads up to 1 Gb (~5 million reads) in
4.4 h. It is debatable whether these machines have the capacity
to deliver sufcient data for reliable noninvasive aneuploidy
detection, particularly if samples are multiplexed, thereby
limiting possibilities for high throughput. The forthcoming Ion
ProtonTM benchtop sequencer may resolve these issues as it
can produce up to 10 Gb of data. However, the performance
remains to be tested.
2013 John Wiley & Sons, Ltd.

Whole genome versus targeted approaches for NIPT

565

WHOLE GENOME VERSUS TARGETED APPROACHES FOR

ANEUPLOIDY NIPT
Whole genome approach
To date, a number of large-scale clinical validation studies
have shown that whole genome MPS of ccffDNA in maternal
plasma for fetal aneuploidy detection, particularly for Down
syndrome, can be performed with high (nearly diagnostic)
accuracy (Table 1). In terms of costs and efciency, the
number of samples that can be sequenced simultaneously,
and thus the minimal number of reads, is an important issue.
Clearly, insufcient numbers of sequenced reads will reduce
the reliability, as the statistical signicance of any difference
in number of reads will drop. Too many reads, however, will
increase the costs signicantly, as fewer samples can be
multiplexed per run. There is still an ongoing debate on how
many DNA fragments should be sequenced and quantied
for a reliable result (Table 1). In 2011, Chiu et al.11 showed that
when the mean number of mappable sequenced reads per
sample was 2.3 million, 100% of the fetal Down syndrome
cases could be correctly diagnosed, whereas when the mean
number was 0.3 million, the detection rate of fetal trisomy 21
was only 79.1% (Table 1). Sparks et al.24 stated that when using
whole genome MPS, ~6.3 million uniquely mapped reads are
required to ensure sufcient chromosome 21 count.
Assuming a minimal of 5 to 10 million mappable reads per
sample, with the Illumina HiSeqTM 2000, theoretically, 12
samples can be multiplexed per lane, thus when using all 16
lanes of the dual-ow cell system, 192 samples can be
sequenced per run in 4 to 5 days (depending on read length).
With the ABI 5500xl SOLiDTM, theoretically, ~70 samples can
be pooled on one owchip, and thus, with one machine being
equipped with two ow chips, a total of ~140 samples can be

sequenced in one run, taking 8 to 10 days. As mentioned

earlier, less expensive benchtop sequencing platforms, such
as the MiSeqTM, Ion TorrentTM PGM, or Ion ProtonTM System,
might be applicable too if sufcient reads can be obtained. The
application of the Ion TorrentTM PGM for NIPT, as reported
upon recently by Yuan et al.,19 resulted in a mean of 3.5 million
reads per sample, which is on the lower limit of the minimal
number of reads necessary for a reliable NIPT result, as
assumed by others. The applicability of the Ion TorrentTM
PGM therefore needs to be validated in larger studies.
Furthermore, the relatively low number of achievable reads
reduces the possibilities for multiplexing and therefore high
throughput sequencing, resulting in increased costs per
sample. The advantage of lower set-up costs for such platforms
therefore needs to be weighed against these disadvantages.
To address important issues such as the minimal number of
reads required for reliable results and quality controls of the
test, guidelines are desired.

Targeted approaches
When using nontargeted approaches, sequencing of the
complete genome is carried out, even though there is (are) only
one (or several) chromosomes(s) of interest. This means that
much of the information that is obtained remains unused. In
an attempt to reduce sequencing costs, several groups therefore
have studied the application of targeted approaches.
In 2011, Liao et al.25 used the SureSelect Target Enrichment
System (Agilent Technologies) to enrich for exons on chromosome
X. Indeed, for the regions targeted by enrichment, the mean
sequence coverage was 213-fold higher than that of the
nonenriched samples. This increased the coverage of fetalspecic alleles within the targeted region from 3.5% to 95.9%.25

Table 1 Overview of large-scale validation studies for NIPT for Down syndrome

No. of samples

T21 samples

NGS platform

Whole genome
(WG)/Targeted
(T) approach

449
(4-plex)

39

Illumina GAIIx

WG

35 million

100

99.7

Chiu 201111

2-plex
n = 314
8-plex
n = 753

86

Illumina GAIIx

WG

2.3 million
(2-plex)
0.3 million
(8-plex)

100 (2-plex)
79.1 (8-plex)

97.9 (2-plex)
98.9 (8-plex)

Palomaki 201114

4664
(4-plex)

212

Illumina High
Seq 2000

WG

n.s.

98.6

99.8

Sparks 201226

298

39

Illumina High
Seq 2000

204 000/410 000/

620 000

100

100

Sparks 201224

163

35

Illumina High
Seq 2000

1 million

Ashoor 201227

397

50

n.s.

n.s.

100

100

Ehrich 2011

12

Norton 2012

28

Bianchi 201231

Number of mapped
reads per sample

Sensitivity (%)

Specicity (%)

3228

81

n.s.

n.s.

100

99.7

2882
(6-plex)

89

Illumina High
Seq 2000

WG

n.s.

100

100

NIPT, noninvasive prenatal testing; NGS, next-generation sequencing; n.s., not specied.

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2013 John Wiley & Sons, Ltd.

E. M. J. Boon and B. H. W. Faas

566

Sparks et al.,24,26 Ashoor et al.,27 and Norton et al.28 showed

that by selective amplication of specic regions on chromosome
21 and 18, and subsequent MPS analysis, a method referred to as
digital analysis of selected regions (DANSR), the amount of
sequenced reads required to reliably detect fetal trisomy 18 or
21 is signicantly less than that required for whole genome
MPS approaches. They designed DANSR assays against 576
nonpolymorphic loci on each chr18 and chr21, and in addition,
to simultaneously determine the fetal fraction in the sample,
assays were designed against single nucleotide polymorphism
(SNP) containing loci on chromosomes 1 to 12. Data were
analyzed in combination with a novel algorithm, fetal-fraction
optimized risk of trisomy evaluation, to determine trisomy risk
for each pregnant woman.24 Using the combination of DANSR
and fetal-fraction optimized risk of trisomy evaluation, fetal
trisomy 21 and 18 cases could be identied with high accuracy
with ~1 million raw reads per sample, which means a ve to
tenfold decrease in number of reads as compared with the
whole genome approach, accompanied by a decrease in
sequencing costs as well as in data storage. Furthermore, as
internal control, the fetal fraction in the sample could be
determined simultaneously.
In 2012, Liao et al.29 reported on targeted MPS for the
detection of fetal trisomy 21 by allelic ratio analysis. During
library preparation for MPS analysis, they included an
enrichment step to enrich for 2906 SNP loci on chromosomes
7, 13, 18, and 21. They subsequently analyzed plasma DNA
libraries with and without target enrichment by MPS and
compared the data with the maternal and fetal SNPs known
from SNP array analysis. They found that the number of
mappable paired-end reads in nonenriched and enriched
samples was comparable, but the mean sequencing depth of
the enriched samples was 225-fold higher than the nonenriched
samples (0.12 times versus 27 times). Unfortunately, using their
analysis algorithm and the enrichment approach, they were only
able to correctly identify trisomy 21 when the extra chromosome
was paternal in origin. In maternally derived trisomy 21, it was
less accurate.
They concluded that either adding more informative SNP
loci or increasing the sequencing depth would solve this problem,
the latter, however, not being very realistic as this would mean the
number of reads would need to be increased to more than the
number necessary in the whole genome approach.
In an attempt to further rene this method, Zimmermann
et al.30 described a modied version of this approach. They
enriched for 11 000 SNP loci and used a method they called
parental support for analyzing the MPS data. For the 145 samples
that met the quality criteria, they obtained an average of 8.85
million mappable reads per sample, of which 6.47 million
mapped to the targeted SNPs (~1.3 million per chromosome).
The average depth of read was 344, and the median depth per
SNP was 255. Using this method, they were able to correctly
identify fetal disomy or trisomy for chromosomes 21, 18, and
13 and also correctly identied fetal 45,X and 47,XXY. An
advantage of this method is that by comparing the relative
amounts of alleles at a set of loci, problems with chromosometo-chromosome amplication variation are eliminated. Reduced
sensitivity and specicity rates for the detection of trisomy 13
Prenatal Diagnosis 2013, 33, 563568

and sex chromosomal abnormalities, as described in previous

studies,6,15,31 can thus be solved. The authors therefore state that
the parental support method increases clinical coverage of viable
chromosomal abnormalities by approximately twofold. In the
future, these targeted approaches might also be used to target
specic regions for the detection of submicroscopic imbalances
(microdeletions/microduplications).
Although for targeted methods, such as DANSR, the number
of reads obtainable using benchtop platforms will be sufcient,
the possibilities for high throughput are still rather limited.
This means that for medium to high throughput of samples,
the capital costs of the machines, as well as the sample and
library preparation costs, will probably be the same for the
targeted and whole genome approach. Nevertheless, targeted
sequencing can be a good alternative for the still rather
expensive whole genome approach, as more samples can be
sequenced simultaneously (theoretically greater than tenfold
for the DANSR method). One should, however, realize that
when using a targeted approach, only the region(s) of interest
can be studied.
Methods that do not require MPS at all are expected to be cost
efcient too. Two of such approaches that have been described
are digital PCR32 and methylated DNA immunoprecipitation in
combination with real-time quantitative PCR.33 However, because
these approaches have not been used and validated widely, their
performance, high-throughput possibilities, and costs remain to
be determined in more detail, and these techniques will therefore
not be discussed here.

NIPT IN CLINICAL PRACTICE

On the basis of the evidence of the clinical performance of
NIPT tests, as summarized in Table 1, currently, NIPT is
commercially offered as a service for so-called high risk
pregnant women in parts of the USA, Asia, Middle East,
and Europe. The laboratory test is mostly performed by
companies in the USA (Verinata Health Inc., Sequenom
Inc., Ariosa Diagnostics, Inc., NateraTM) and China (BGI,
Berry Genomics Co.), with the majority offering the test
via healthcare providers and companies (e.g. LifeCodexx
AG, GENNET) outside these countries. All companies offer
MPS-based tests using Illumina equipment for the detection
of fetal trisomy 13, 18, and/or 21 and/or sex chromosomal
aberrations, some of them whole genome (Verinata Health,
Sequenom, BGI, Berry Genomics) and others targetedbased (Ariosa Diagnostics, Natera), with prices ranging from
~$500 to $1700. Differences in prices are obvious, but at this
stage, it is hard to draw any conclusions regarding costeffectiveness of whole genome compared with the targeted
approach. Ariosa Diagnostics, applying the targeted DANSR
approach, charges $795 in the USA, whereas companies
such as Verinata Health and Sequenom, both using whole
genome approaches, charge $1200 and $1700 in the USA,
respectively. This suggests that at this stage, higher costs
are still associated with the whole genome approach. One
should, however, realize that these are not cost prices but
commercial prices. Furthermore, prices differ from country
to country and some prices include counseling, whereas
others only include the test. It is likely that costs will fall
2013 John Wiley & Sons, Ltd.

Whole genome versus targeted approaches for NIPT

in the near future and only then a valid comparison can be

made. Furthermore, even though not discussed here, one
should bear in mind that issues of patents may also
inuence prices. Moreover, the recent acquisition of
Verinata Health by Illumina might be a potential point of
concern for NIPT providers, as this might affect pricing or
availability of the Illumina machines for NIPT, but so far,
this is only speculative. Noteworthy is that some insurance
companies (in the USA) already decided to cover (part of)
the costs, resulting in differential pricing: Sequenom, for
example, charges $1700 to uninsured women and $235 to
insured women. Future analysis on clinical utility and
economic costs, as already described by Chitty et al.,34
and Song et al.,35 need to determine at what price NIPT
would be cost efcient in the different healthcare systems
throughout the world. It is expected that NIPT will be cost
efcient in the long term because methods keep improving,
costs of sequencing are dropping, and this test can improve
health outcomes.

SUMMARY
At the moment, whole genome MPS approaches for the
detection of fetal aneuploidies using ccffDNA in maternal
plasma are more extensively validated than targeted
approaches with regard to sensitivity and specicity. Cost
wise, whole genome approaches are, if used to study only
one specic chromosome, more expensive per sample
than targeted approaches, as fewer samples can be
multiplexed. However, one should bear in mind that, with
the exception of multiplexing, sample handling and library
preparation are comparable for both approaches, limiting
cost reduction in case of the targeted approach. The
ongoing and rapid reduction in the costs associated with
sequencing and downstream data handling should be taken

567

into account too. Moreover, as technological advances are

being made rapidly, it is questionable whether, in the long
term, MPS NIPT will only be applied for the detection of
full-blown aneuploidies of specic chromosomes. Indeed,
several groups already reported the detection of
submicroscopic chromosomal aberrations,31,3638 and in
2010, the complete fetal genome was assembled from
maternal plasma.20 Recently, three studies followed up on
these ndings and performed whole genome, exome, and
targeted MPS for the detection of paternally inherited or
de novo arisen fetal mutations using ccffDNA from maternal
plasma.3941 Whatever approach will be used in the future,
both benets and limitations will go along with associated
challenges for diagnostic services and healthcare providers.

WHATS ALREADY KNOWN ABOUT THIS TOPIC?

Since the discovery of cell-free fetal DNA in maternal plasma, large
progress has been made in the development of noninvasive
prenatal tests.
The rst applications in noninvasive prenatal diagnosis were single
polymerase chain reaction-based.
Since 2008, a new era in the development of noninvasive
aneuploidy testing was opened by the rst successful application
of massively parallel sequencing for this purpose.

WHAT DOES THIS STUDY ADD?

For fetal aneuploidy testing, whole genome massively parallel
sequencing is still rather expensive and to reduce costs, targeted
sequencing approaches are being developed.
This review highlights benets and limitations of both whole genome
and targeted approaches for noninvasive prenatal testing for fetal
aneuploidy detection for now and the near future as a shift in the
most cost effective approach is anticipated in the near future.

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