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DOI: 10.1002/pd.

4111

REVIEW

Benefits 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

2

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 benefits and limitations and discusses the implications for
future noninvasive prenatal testing. © 2013 John Wiley & Sons, Ltd.

Funding sources: None
Conflicts 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 first 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 ~10–20%
(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 identified 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 first 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 first trimester,4 for noninvasive aneuploidy
Prenatal Diagnosis 2013, 33, 563–568

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 specific 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 significant) 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 identification 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.

Inc. Inc. Using single-molecule-sequencing on. Boon and B. W.23 With sequencing-by-ligation. Ltd. especially when used for NIPT. namely the Illumina Genome Analyzer/Illumina HiSeq 2000 Genome Analyzer.4 h. this bias can be eliminated. and sequenced through sequencing-bysynthesis. one can even choose between a rapid run and a high output run. the use of four have been reported for NIPT. makes this platform very suitable for multiplexing of samples and thus high throughput runs. a better distinction between fetal disomy and trisomy 21 could be achieved than with PCR-based platforms. 33. and subsequently. M. making it possible to produce even faster results. Prenatal Diagnosis 2013. and with the 5500xl SOLiDTM. Inc) and SOLiDTM [Life TechnologiesTM/ Applied BiosystemsR (ABI)] platforms. Both benchtop sequencers have relatively low set-up costs and reduced running times compared with most of the high throughput sequencers. Therefore. Illumina’s MiSeqTM is based on the existing sequencing-by-synthesis chemistry. H. can be introduced by the amplification step. especially for GC-rich regions.) and Ion TorrentTM PGM (Life Technologies). Among the available platforms. benefits. The 5500xl SOLiDTM machine uses two fully configurable flowchips. the amount of data that can be generated has increased to 1.18 and the IonTorrent (Life Technologies) platform.7 billion single-end reads per run. Theoretically. whereas the Ion TorrentTM PGM uses the semiconductor sequencing technology. However. achieved via multiplexing.4 billion single-end reads per run. for most applications. and this difference in length has been used to specifically enrich for fetal DNA. indeed. The attached DNA fragments are extended. depending on the experimental set-up. With the rapid run. Although this platform produces slightly less data per run than the Illumina HiSeqTM platform. this does not seem to be timeefficient and cost-efficient.6. It is debatable whether these machines have the capacity to deliver sufficient data for reliable noninvasive aneuploidy detection. The forthcoming Ion ProtonTM benchtop sequencer may resolve these issues as it can produce up to 10 Gb of data. attached to beads.11–14 the Applied Biosystems (ABI) SOLiD Analyzer. especially for NIPT. However. the performance remains to be tested. These differ from each other in that they use polymerase chain reaction (PCR)-based sequencing-by-synthesis or sequencing-by-ligation. thereby limiting possibilities for high throughput. 563–568 SolidTM [Life TechnologiesTM/Applied BiosystemsR (ABI)] For the library construction of the ABI SOLiDTM system. 300 million single-end reads (~10 Gb) can be produced within 7 h. first. DNA fragments are ligated to adapters and attached to reaction chambers located on a flow cell.17 However. HiseqTM (Illumina. because for NIPT in routine clinical practice. With the arrival of the HiSeqTM 2500. and limitations. as ccffDNA shows a fragmentation pattern of around 146 bp. particularly if samples are multiplexed. Faas 564 ensure the analysis of sufficient chromosome 21 fragments to statistically detect significant differences between normal and trisomic fetuses.15. depending on read length. each with six independent lanes.E. whereas only paying reagent costs for the lanes used rather than the whole flow cell. different NGS platforms are commercially available.4 Gb in 16. In the succeeding text.21.22 Given the fact that DNA in maternal plasma is highly fragmented. is inevitable. each with its own costs. J. a small study reported that when applying this single-molecule-sequencing platform. with the recently marketed upgrade 5500xl W platform offering even more possibilities. NGS PLATFORMS Today. and subsequently clonally amplified by emulsion PCR. and the Ion TorrentTM PGM (Ion 318 chip) 200 bp reads up to 1 Gb (~5 million reads) in 4. and these will therefore not be discussed here.16 the Helicos Heliscope. The run time for the HiSeqTM 2000 varies from 5 to 14 days.) For the Illumina platforms.5 h. for instance.17. including benefits and limitations. the HeliScopeR Single Molecule Sequencer. such as the Roche/454 (454 Life Sciences Branfort USA) and PacBio RS (Pacific Biosciences CA USA) for NIPT. For NIPT. both whole genome and targeted (MPS) approaches for NIPT. DNA fragments are ligated to adapters. . which. © 2013 John Wiley & Sons. this singlemolecule-sequencing platform is not very well suited as multiplexing of samples is cumbersome. very well suited. 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. Runtime on the SOliDTM instrument varies between 5 and 10 days. will be discussed in more detail. an important limitation of the current benchtop machines is the amount of data that can be generated. allowing the user to use between one and six lanes. the Illumina HiSeqTM 2000 can produce up to three billion single-end reads per run. The MiSeqTM Personal Sequencer can produce for a 2  100 bp run up to 3. several benchtop sequencers are available on the market as well that might be suitable for NIPT. the different next-generation sequencing (NGS) platforms and their specifications in general terms. including the MiSeqTM (Illumina. HeliScopeR single molecule sequencer (HelicosTM Biosciences) A weakness of the aforementioned platforms is that through the PCR-based sample preparation experimental bias. The most widely used platforms are the high-throughput HiSeqTM (Illumina.8. amplified by bridge amplification. Others In addition to the high throughput sequencers mentioned previously. which are.7.19 These platforms are all short-read platforms. high throughput analysis.13. with only one chromosome of interest. the SOLiD4 can produce up to 0. it is also flexible and can be used for high throughput runs. Using this technology.20 There is evidence that fetal DNA is shorter than maternal DNA. targeted alternatives to whole genome MPS have been developed. one can envision that this is the reason why no papers have appeared on the use of longer-sequence-read platforms.

theoretically. Assuming a minimal of 5 to 10 million mappable reads per sample.3 million (8-plex) 100 (2-plex) 79. the relatively low number of achievable reads reduces the possibilities for multiplexing and therefore high throughput sequencing. as fewer samples can be multiplexed per run.s. In an attempt to reduce sequencing costs. the mean sequence coverage was 213-fold higher than that of the nonenriched samples. . as assumed by others. and thus. can be performed with high (nearly diagnostic) accuracy (Table 1). In 2011. Indeed.6 99. The application of the Ion TorrentTM PGM for NIPT.3 million uniquely mapped reads are required to ensure sufficient chromosome 21 count. ~6.9%.11 showed that when the mean number of mappable sequenced reads per sample was 2. 100% of the fetal Down syndrome cases could be correctly diagnosed. Ltd.25 Table 1 Overview of large-scale validation studies for NIPT for Down syndrome No. with one machine being equipped with two flow chips. Too many reads. as the statistical significance of any difference in number of reads will drop. In terms of costs and efficiency.7 Chiu 201111 2-plex n = 314 8-plex n = 753 86 Illumina GAIIx WG 2. as reported upon recently by Yuan et al.3 million. might be applicable too if sufficient reads can be obtained.7 2882 (6-plex) 89 Illumina High Seq 2000 WG n. With the ABI 5500xl SOLiDTM. the detection rate of fetal trisomy 21 was only 79. The advantage of lower set-up costs for such platforms therefore needs to be weighed against these disadvantages.5 million reads per sample. noninvasive prenatal testing. insufficient numbers of sequenced reads will reduce the reliability. taking 8 to 10 days. As mentioned earlier.9 (2-plex) 98. Targeted approaches When using nontargeted approaches. 98.19 resulted in a mean of 3. even though there is (are) only one (or several) chromosomes(s) of interest.s. not specified. n. thus when using all 16 lanes of the dual-flow cell system. guidelines are desired.s. and thus the minimal number of reads. Furthermore. Sparks et al. ~70 samples can be pooled on one flowchip.9 (8-plex) Palomaki 201114 4664 (4-plex) 212 Illumina High Seq 2000 WG n. 33.. the number of samples that can be sequenced simultaneously. In 2011. however. or Ion ProtonTM System. resulting in increased costs per sample.1% (Table 1). will increase the costs significantly. T n. 100 100 Ehrich 2011 12 Norton 2012 28 Bianchi 201231 Number of mapped reads per sample Sensitivity (%) Specificity (%) 3228 81 n. 100 100 NIPT. such as the MiSeqTM.3 million. with the Illumina HiSeqTM 2000. less expensive benchtop sequencing platforms. for the regions targeted by enrichment.s. next-generation sequencing. theoretically.5% to 95.s..8 Sparks 201226 298 39 Illumina High Seq 2000 T 204 000/410 000/ 620 000 100 100 Sparks 201224 163 35 Illumina High Seq 2000 T 1 million Ashoor 201227 397 50 n. T n. To address important issues such as the minimal number of reads required for reliable results and quality controls of the test. whereas when the mean number was 0. Ion TorrentTM PGM. sequencing of the complete genome is carried out.24 stated that when using whole genome MPS. several groups therefore have studied the application of targeted approaches.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. The applicability of the Ion TorrentTM PGM therefore needs to be validated in larger studies. of samples T21 samples NGS platform Whole genome (WG)/Targeted (T) approach 449 (4-plex) 39 Illumina GAIIx WG 3–5 million 100 99.3 million (2-plex) 0. This means that much of the information that is obtained remains unused. Prenatal Diagnosis 2013. Chiu et al. a total of ~140 samples can be sequenced in one run. There is still an ongoing debate on how many DNA fragments should be sequenced and quantified for a reliable result (Table 1). Clearly. 100 99. which is on the lower limit of the minimal number of reads necessary for a reliable NIPT result.s.25 used the SureSelect Target Enrichment System (Agilent Technologies) to enrich for exons on chromosome X. Liao et al. 563–568 © 2013 John Wiley & Sons. NGS. particularly for Down syndrome. 192 samples can be sequenced per run in 4 to 5 days (depending on read length).s. This increased the coverage of fetalspecific alleles within the targeted region from 3.1 (8-plex) 97. 12 samples can be multiplexed per lane. is an important issue.

as well as the sample and library preparation costs. Reduced sensitivity and specificity rates for the detection of trisomy 13 Prenatal Diagnosis 2013.E. and subsequent MPS analysis.). the capital costs of the machines. with prices ranging from ~$500 to $1700.85 million mappable reads per sample. will probably be the same for the targeted and whole genome approach.31 can thus be solved. because these approaches have not been used and validated widely. they were able to correctly identify fetal disomy or trisomy for chromosomes 21. they obtained an average of 8. BGI. This means that for medium to high throughput of samples. Furthermore.X and 47. 13. The authors therefore state that the parental support method increases clinical coverage of viable chromosomal abnormalities by approximately twofold. They enriched for 11 000 SNP loci and used a method they called parental support for analyzing the MPS data. They designed DANSR assays against 576 nonpolymorphic loci on each chr18 and chr21.XXY. It is likely that costs will fall © 2013 John Wiley & Sons. 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. fetal trisomy 21 and 18 cases could be identified with high accuracy with ~1 million raw reads per sample. only the region(s) of interest can be studied. they were only able to correctly identify trisomy 21 when the extra chromosome was paternal in origin. NIPT IN CLINICAL PRACTICE On the basis of the evidence of the clinical performance of NIPT tests. All companies offer MPS-based tests using Illumina equipment for the detection of fetal trisomy 13.3 million per chromosome). as described in previous studies. Ariosa Diagnostics. and 13 and also correctly identified fetal 45.24. For the 145 samples that met the quality criteria. some of them whole genome (Verinata Health.28 showed that by selective amplification of specific regions on chromosome 21 and 18. the number of reads obtainable using benchtop platforms will be sufficient. 33.30 described a modified version of this approach. In maternally derived trisomy 21. high-throughput possibilities. Berry Genomics) and others targetedbased (Ariosa Diagnostics. realize that when using a targeted approach. both using whole genome approaches. GENNET) outside these countries. NIPT is commercially offered as a service for so-called ‘high risk’ pregnant women in parts of the USA. however. charges $795 in the USA. Liao et al. whereas others only include the test. NateraTM) and China (BGI. Methods that do not require MPS at all are expected to be cost efficient too. however.. and costs remain to be determined in more detail. Middle East. Sequenom. and the median depth per SNP was 255. and in addition. In an attempt to further refine this method. This suggests that at this stage. One should. Boon and B. such as DANSR. The laboratory test is mostly performed by companies in the USA (Verinata Health Inc. One should. Natera). to simultaneously determine the fetal fraction in the sample. During library preparation for MPS analysis. as more samples can be sequenced simultaneously (theoretically greater than tenfold for the DANSR method). Zimmermann et al. it was less accurate.29 reported on targeted MPS for the detection of fetal trisomy 21 by allelic ratio analysis. Nevertheless.33 However. M. 18. accompanied by a decrease in sequencing costs as well as in data storage. H. Differences in prices are obvious. whereas companies such as Verinata Health and Sequenom. to determine trisomy risk for each pregnant woman.26 Ashoor et al. but the mean sequencing depth of the enriched samples was 225-fold higher than the nonenriched samples (0. J. W. An advantage of this method is that by comparing the relative amounts of alleles at a set of loci.. assays were designed against single nucleotide polymorphism (SNP) containing loci on chromosomes 1 to 12. however. They found that the number of mappable paired-end reads in nonenriched and enriched samples was comparable. prices differ from country to country and some prices include counseling. respectively. Inc. 18. fetal-fraction optimized risk of trisomy evaluation.6. 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. The average depth of read was 344. it is hard to draw any conclusions regarding costeffectiveness of whole genome compared with the targeted approach.g. of which 6. as internal control. Asia.. using their analysis algorithm and the enrichment approach. realize that these are not cost prices but commercial prices.24 Using the combination of DANSR and fetal-fraction optimized risk of trisomy evaluation. which means a five to tenfold decrease in number of reads as compared with the whole genome approach. and these techniques will therefore not be discussed here. and 21. targeted sequencing can be a good alternative for the still rather expensive whole genome approach. currently. Unfortunately. problems with chromosometo-chromosome amplification variation are eliminated. Ltd. as summarized in Table 1. the possibilities for high throughput are still rather limited. charge $1200 and $1700 in the USA. their performance. applying the targeted DANSR approach. and/or 21 and/or sex chromosomal aberrations.15. but at this stage. Ariosa Diagnostics.27 and Norton et al. 18. Furthermore. In 2012. a method referred to as digital analysis of selected regions (DANSR). with the majority offering the test via healthcare providers and companies (e. Although for targeted methods. In the future. Faas 566 Sparks et al.. these targeted approaches might also be used to target specific regions for the detection of submicroscopic imbalances (microdeletions/microduplications). they included an enrichment step to enrich for 2906 SNP loci on chromosomes 7.12 times versus 27 times). higher costs are still associated with the whole genome approach. Two of such approaches that have been described are digital PCR32 and methylated DNA immunoprecipitation in combination with real-time quantitative PCR. Data were analyzed in combination with a novel algorithm. They concluded that either adding more informative SNP loci or increasing the sequencing depth would solve this problem.. Sequenom Inc. Using this method. the fetal fraction in the sample could be determined simultaneously. . Berry Genomics Co. and Europe.47 million mapped to the targeted SNPs (~1. 563–568 and sex chromosomal abnormalities. LifeCodexx AG. the latter. the amount of sequenced reads required to reliably detect fetal trisomy 18 or 21 is significantly less than that required for whole genome MPS approaches.

Non-invasive prenatal diagnosis of fetal aneuploidies using massively parallel sequencing-by-ligation and evidence that cell-free fetal DNA in the maternal plasma originates from cytotrophoblastic cells. several groups already reported the detection of submicroscopic chromosomal aberrations. Lo YM.11:31–46. Tsui NB. Lancet 1997. 563–568 10. Lo YM. Metzker ML. Venter JC. for example. a new era in the development of noninvasive aneuploidy testing was opened by the first successful application of massively parallel sequencing for this purpose. SUMMARY At the moment.105:20458–63. as technological advances are being made rapidly. Sequencing technologies . BMJ 2011. Moreover. and this test can improve health outcomes. Genet Med 2011. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. • Since 2008. WHAT’S ALREADY KNOWN ABOUT THIS TOPIC? • Since the discovery of cell-free fetal DNA in maternal plasma. Verweij EJ et al. Expert Opin Biol Ther 2012. Liu L. 3. REFERENCE 1. Prenatal Diagnosis 2013. Rapid clearance of fetal DNA from maternal plasma. more expensive per sample than targeted approaches. the complete fetal genome was assembled from maternal plasma.342:c7401. sample handling and library preparation are comparable for both approaches. Am J Hum Genet 1999.13:913–20. Li Y.350:485–7. Zwiefelhofer T et al.39–41 Whatever approach will be used in the future.. as fewer samples can be multiplexed. Clin Chem 2012.Whole genome versus targeted approaches for NIPT in the near future and only then a valid comparison can be made. one should bear in mind that. Lau TK et al. 4. Proc Natl Acad Sci U S A 2008. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. 5. Kloza EM.58:699–706. Maternal plasma DNA analysis with massively parallel sequencing by ligation for noninvasive prenatal diagnosis of trisomy 21. 9. Leung TN et al. 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