ISSN 0003-2654

PAPER Yong Guo, Lingxiang Zhu et al. A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies

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Volume 144 Number 7 7 April 2019 Pages 2197–2444

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PAPER

Cite this: Analyst, 2019, 144, 2239

Received 20th October 2018,Accepted 28th December 2018

DOI: 10.1039/c8an02018c

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A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies†

Chianru Tan, ‡a Xihua Chen,‡b Fang Wang,a Dong Wang,c Zongfu Cao, b

Xiurui Zhu,a Chao Lu,b Wenjun Yang,c Na Gao,c Huafang Gao,b Yong Guo *a andLingxiang Zhu *b

Higher multiplexing in droplet digital PCR (ddPCR) can simplify the detection process of ddPCR-based

non-invasive prenatal testing (NIPT) and improve its reliability, making it a practical approach in clinical

practice. However, a high level of multiplex ddPCR-based NIPT has rarely been reported. In this study, we

developed a multiplex ddPCR assay using universal locked nucleic acid (LNA) probes to reliably identify

fetal aneuploidies. We first performed statistical analysis based on the Poisson distribution to evaluate the

required number of target DNA molecules and the total number of droplets for a ddPCR assay. Next, we

designed two sets of primers and probes to quantify cfDNA from chromosomes 21 and 18 and then

determined the disease status of a sample. Finally, we evaluated our multiplex ddPCR assay with 60

clinical plasma samples. All of the 60 clinical samples were correctly identified. The accessibility and

cost-effectiveness of our multiplex ddPCR-based NIPT make it a competitive prenatal testing method in

clinical use.

Introduction

In humans, the most common aneuploidies are trisomies,which are trisomies 21, 18, and 13 (T21, T18, and T13).1 Thegold standard for diagnosing fetal aneuploidies is karyotypingof fetal materials obtained via chorionic villus sampling oramniocentesis.2,3 These invasive procedures may cause iatro-genic fetal loss and uterine infection.4,5 Hence, the standardprenatal care uses maternal serum screening and ultrasono-graphic measurement to identify women with high-risk preg-nancies, who are then offered a diagnostic test. However, inva-sive sampling is often performed for normal fetuses due to thehigh proportion of false positives (about 5%) in the screeningtest. Furthermore, the detection rate of these screeningmethods is relatively low (50%–95%).6,7 For these reasons,researchers worldwide are working on developing a non-inva-sive and accurate prenatal testing method.

The discovery of cell-free fetal DNA (cffDNA) in maternalblood has opened up new possibilities for prenatal testing.8

However, the low percentage of cffDNA in maternal plasmahas posed challenges for developing successful prenatal tests.On average cffDNA accounts for 10%–20% of maternal plasmaDNA between 10 and 21 weeks of gestation.9,10 The develop-ment of next-generation sequencing (NGS) has advanced thefield of non-invasive prenatal testing (NIPT).11,12 NGS-basedNIPT was introduced into clinical practice in 2011, and it hasbecome the most sensitive and specific screening option toidentify aneuploidies (T21, T18, and T13).13–16

As a method that enables absolute quantification of nucleicacids, digital PCR (dPCR) has been used for non-invasivedetection of fetal aneuploidies since 2007.17–21 In dPCR,22–24

template DNA molecules are distributed across multiple repli-cate reactions; some of the reactions contain template DNAwhile the others do not. After PCR amplification, the reactionscontaining template DNA produce a positive signal while thosewithout template DNA present a negative signal. Finally, thenumber of template DNA molecules in the initial reactions canbe quantified from the proportion of positive reactions usingPoisson statistics. Although dPCR-based NIPT has great poten-tial to be more straightforward, rapid and cost-effective thanNGS-based NIPT, there is an obstacle that prevents dPCR-based NIPT from performing in clinical practice: a largenumber of PCR-positive reactions are required to achievereliability for clinical use (due to the low fraction of cffDNA inmaternal plasma).17,18,25

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c8an02018c‡These authors contributed equally to the work.

aDepartment of Biomedical Engineering, School of Medicine,

Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,

Tsinghua University, Beijing 100084, China. E-mail: yongguo@tsinghua.edu.cn;

Tel: +86-10-6278 3960bNational Research Institute for Health and Family Planning, Beijing 100081, China.

E-mail: zhulingxiang@nrifp.org.cn; Tel: +86-10-6211 2945cTargetingOne Corporation, Beijing 100190, China

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To overcome this obstacle, El Khattabi et al.19 and Leeet al.21 performed octuplex droplet digital PCR (ddPCR) toidentify T21. El Khattabi et al. performed 8–16 replicates(eight replicates can be processed per ddPCR run) for eachsample to increase the number of positive droplets while Leeet al. additionally used the size selection method to increasethe fraction of cffDNA. Although their results indicated thatthe ddPCR assay is feasible to non-invasively detect fetal aneu-ploidies, these additional procedures increased the complexityof ddPCR-based NIPT, which may hinder its wider application.Performing higher multiplexing in ddPCR can provide ade-quate positive droplets without adding procedures or increas-ing the amount of input DNA. However, primer and probedesign for multiplex ddPCR becomes more challenging whenthe number of PCRs increases progressively. Furthermore, acorrespondingly large number of probes are required toachieve higher multiplexing, which may produce a high back-ground fluorescence and therefore affects the detection of thepositive signal. Hence, a high level of multiplex ddPCR-basedNIPT has not previously been reported.

Here, we developed a multiplex ddPCR assay (40-plex) usinguniversal locked nucleic acid (LNA) probes to reliably dis-tinguish between euploid and aneuploid samples, using T21and T18 as a model. Furthermore, we performed statisticalanalysis based on Poisson distribution to provide the idealconditions for achieving reliable results, guiding the develop-ment of a multiplex ddPCR assay. We then evaluated our assaywith 60 clinical plasma samples (30 samples for assay vali-dation and 30 euploid samples for the training test).

ExperimentalSample collection and preparation

All experiments were performed in accordance with theDeclaration of Helsinki and the guidelines of the NationalResearch Institute for Health and Family Planning (Beijing,China). All experiments were approved by the Ethics Committeeof the National Research Institute for Health and FamilyPlanning. Informed consents were obtained from human par-ticipants of this study. All blood samples were collected fromBeijing Guowei Reproductive Health Hospital (Beijing, China). Atotal of 60 plasma samples were collected from pregnant womenwith the gestational age ranging from 12 weeks to 25 weeks.Cell-free DNA (cfDNA) was isolated from 2 ml plasma with aMagMAX Cell-Free DNA Isolation kit (Thermo Fisher Scientific,MA) following the manufacturer’s instructions. cfDNA was resus-pended in a final volume of 30 μl and stored at −80 °C until use.Normal female genomic DNA (purchased from Promega, WI;catalog number G1521) was sheared into ∼200 bp fragmentsusing a Covaris M220 sonicator (Thermo Fisher Scientific, MA)in a 100 μL AFA Fiber Snap-Cap microtube with the followingprogram: a duty factor of 20%, a peak incident power of 50 Wwith 200 cycles per burst, a treatment time of 160 s, and a temp-erature of 20 °C. Both DNA samples were quantified using aQubit 3.0 fluorometer (Thermo Fisher Scientific, MA).

Design of primers and probes

Primer and probe sets were designed to quantify DNA mole-cules from chromosome 21 (chr21) and chromosome 18(chr18). First, the target sequences were selected in conservedregions that did not coincide with common single-nucleotidepolymorphisms or copy number variants. The primersand universal probes were designed using the UniversalProbeLibrary Assay Design Center (https://qpcr.probefinder.com/organism.jsp). Second, the primers with the same probewere chosen and were screened for potential secondary struc-tures using AutoDimer software.26 To minimize undesiredprimer–primer interactions in the multiplex PCR, in silicomultiplexing optimization was performed. The reverse comp-lement of each primer sequence was locally aligned to otherprimers using the Biostrings function pairwiseAlignment inR version 3.0, and the primer-specific alignment score wascalculated. Third, all of the candidate primers and probeswere examined by quantitative real-time PCR (see the ESI†).The specific universal probes for chr21 and chr18 were LNA-based oligonucleotides labeled with 6-FAM (λex 494 nm/λem522 nm) and VIC (λex 528 nm/λem 554 nm), respectively (syn-thesized by Integrated DNA Technologies, IL). The probeswere further modified to 10 nt long to enhance the detectedfluorescent signals. Then, the corresponding primers wereselected. All of the candidate primers and probes weretested using a RainDrop Digital PCR System (RainDanceTechnologies, MA). Finally, 20 primer pairs for chr21 andanother 20 pairs for chr18 were selected for furtherexperiments.

Droplet digital PCR

The digital PCR system utilized for the detection was theRainDrop Digital PCR System. A ddPCR mix of 30 μL was pre-pared for each sample with 15 μL TaqMan Universal MasterMix (Thermo Fisher Scientific, MA) containing 0.75 μL of40 mmol L−1 deoxynucleotide (dNTP) solution mix, 1.2 μLdroplet stabilizer (RainDance Technologies, MA), FAM and VICprobes at a final concentration of 0.05 μM each, and primersat a final concentration of 0.1 μM each. Samples wereloaded onto the RainDrop Source instrument (RainDanceTechnologies, MA) to produce water-in-oil emulsion dropletsby following the manufacturer’s operating guidelines.Following droplet generation, the samples were amplifiedusing the following PCR conditions: 25 °C for 10 min, 95 °Cfor 10 min, 40 cycles of 94 °C for 20 s and 60 °C for 60 s, 72 °Cfor 5 min, 98 °C for 10 min, and finally held at 12 °C.Thermally cycled samples were loaded onto the RainDropSense instrument (RainDance Technologies, MA). Finally, datafrom cluster plots were analyzed using the RainDrop Analystdata analysis software to investigate the quantity, integrity, andfluorescent signals of the droplets. Each 30 μL sample wasemulsified into approximately 4–5 million droplets, includingat least 98% of intact droplets. Data from samples that did notmeet these standards were considered invalid and excludedfrom the analysis.

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Statistical models

In a ddPCR assay, the number of DNA molecules in eachdroplet (x) can be modeled as a Poisson process, and the prob-ability of observing k DNA molecules in a droplet is given bythe following equation (x = k, k = 0, 1, 2, 3, …):

pðx¼kÞ¼ λk

k!e�λ: ð1Þ

The proportion of positive droplets (P) is

P ¼ 1� pðx¼0Þ ¼ 1� e�λ: ð2Þ

Note that both the mean and variance of the Poisson distri-bution equal λ. Thus, the average number of DNA molecules ineach droplet is

λ¼� lnð1� PÞ¼ � ln 1� HN

� �; ð3Þ

where H is the number of positive droplets and N is the totalnumber of droplets. The ratio of the total copy number ofchr21 to the total copy number of chr18 (R21:18) is

R21:18¼Nλ21Nλ18

¼ λ21λ18

: ð4Þ

In addition, the confidence interval (CI) of the estimatednumber of DNA molecules should be considered. The 95%confidence level is commonly used in ddPCR assays. Asdescribed by Dube et al.,27 the lower and upper bounds of P, λ,and R21:18 are given by the following equations:

Pmin; Pmax¼P + z1�α

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiPð1� PÞ

N

rð5Þ

λmin;max ¼ �lnð1� Pmin;maxÞ ð6Þ

For a 95% confidence, z1−α = 1.96. The measurement pre-cision of R21:18 is defined as follows:

Precision ¼maxðjR21:18 � R21:18;maxj; jR21:18 � R21:18;minjÞR21:18

; ð9Þ

referring to the definition of precision in the study byMajumdar et al.28 A narrower CI indicates better precision.

Results and discussionThe procedural framework of multiplex ddPCR-based NIPT

The procedural framework of using multiplex ddPCR for non-invasive fetal aneuploidy detection is illustrated in Fig. 1. First,cfDNA is extracted from maternal plasma (which containsboth maternal and fetal cfDNA) and is mixed with ddPCRreagents. There are two sets of primers and probes (containing20 primer pairs and one universal probe in each set) for target-ing DNA molecules from chr21 and chr18. Second, water-in-oildroplets are produced using a droplet generation system.Third, PCR amplification is performed, where each droplet isan independent reaction. The target DNA molecules fromchr21 and chr18 are amplified, resulting in the emission offluorescent signals from the probes (labeled with FAM forchr21 and VIC for chr18). Following PCR amplification, theFAM and VIC fluorescence intensity per droplet is read andanalyzed to determine the number of target DNA molecules.Finally, R21:18 is calculated as follows:where f is the fraction of cffDNA in maternal plasma. Thetheoretical R21:18 value should be 1 and higher than 1 for aeuploid fetus and T21 fetus, respectively. The degree of overre-presentation depends on the fraction of cffDNA in maternalplasma. For example, the theoretical ratio becomes 1.05 whenthe maternal plasma contains 10% of cffDNA from a T21 fetus.Moreover, the z-score is calculated to determine the diseasestatus of a test sample. The z-score indicates the number ofstandard deviations from the mean of euploid sample data-sets. Therefore, a z-score cutoff of three standard deviationscan be used to classify a sample as euploid or aneuploid.

Statistical analysis

In ddPCR-based NIPT for fetal aneuploidy detection, themeasurement precision for R21:18 would significantly affect theaccuracy of the results due to the low fraction of cffDNA in thematernal plasma. To ensure the reliability of our ddPCR assay,

R21:18;min ¼λ21λ18 �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiλ212λ182 � ðλ21 � λ21;minÞ2 � λ212

� � ðλ18;max � λ18Þ2 � λ182� �q

λ182 � ðλ18;max � λ18Þ2ð7Þ

R21:18;max¼λ21λ18 �

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiλ212λ182 � ðλ21;max � λ21Þ2 � λ212

� � ðλ18 � λ18;minÞ2 � λ182� �q

λ182 � ðλ18 � λ18;minÞ2: ð8Þ

R21:18¼ðnumber of chr21fetalÞ�ðf Þþðnumber of chr21maternalÞ�ð1� f Þðnumber of chr18fetalÞ�ðf Þþðnumber of chr18maternalÞ�ð1� f Þ ð10Þ

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we studied the measurement precision for R21:18 and calcu-lated the requirements based on the Poisson distribution.

To investigate the relationships between N, P, and λ, andthe measurement precision for R21:18, we plotted theoretical95% confidence bounds for R21:18 = 1 for a range of P(λ) acrossan increasing N (Fig. 2A), followed by calculating the corres-ponding precision (Fig. 2B). The precision improved as P(λ)increased within a range. When P was ∼0.8 (λ ≈ 1.61), thehighest degree of precision was achieved. Furthermore, theprecision improved as N increased. Regardless of N, the great-est precision was always achieved at P ≈ 0.8 (λ ≈ 1.61).

To illustrate the effect of measurement precision on testresults, we plotted theoretical 95% confidence bounds for aeuploid sample (R21:18 = 1) and a T21 sample containing 10%of cffDNA (R21:18 = 1.05) for a range of the proportion of posi-tive droplets for chr18 (P18) (λ18) (Fig. 2C). The red dashed

lines indicate the points of intersection of the upper bound ofthe euploid sample and the lower bound of the T21 sample,where ∼26% and ∼98% of the droplets (N = 50 000) were foundto be positive. At these points, the probabilities of themeasured value of a euploid sample being lower than thecrossover value and the measured value of a T21 sample beinghigher than the crossover value were both 97.5%. Therefore,the joint probability was 0.975 × 0.975 ≈ 95%, suggesting thatwe could accurately classify the sample as euploid or T21 witha 0.95 probability. The measurement precision for R21:18 = 1 atP18 ≈ 0.26 (λ18 = 0.30) and P18 ≈ 0.98 (λ18 = 3.91) was nearlyequal (∼2.48% and ∼2.43%, respectively). Moreover, accordingto our calculation, the precisions at the points of intersectionfor different N were also the same (data not shown). Theseresults suggested that a certain level of precision is required toclassify a sample as euploid or aneuploid accurately.

We next calculated the target DNA molecule number of areference chromosome that corresponded to the required levelof precision to accurately classify a sample as euploid or aneu-ploid with 0.95 probability for a range of N (Fig. 2D). Thedashed lines indicate the minimum value of the required N. Atleast 20 000–120 000 droplets are required to reliably test thesamples containing 10%–4% of cffDNA. Since a decrease in Nled to a decrease in precision, the required number of targetDNA molecules increased as N decreased. In addition, therequired number of target DNA molecules increased as thefraction of cffDNA decreased. Interestingly, the increase in thenumber of target DNA molecules improved the resolution if

Fig. 1 The procedural framework of using multiplex ddPCR for non-invasive fetal aneuploidy detection. Fetal (red) and maternal (black)cfDNA was extracted from maternal plasma and mixed with ddPCRreagents (which contained 40 primer pairs and two universal probes).Following droplet generation, PCR amplification and fluorescencedetection, R21:18 and the z-score were calculated to determine thedisease status of a sample.

Fig. 2 The results of statistical analysis based on Poisson distribution.(A) Theoretical 95% confidence bounds for R21:18 = 1 for a range of P (λ)across an increasing N. (B) The measurement precision correspondingto the confidence interval of (A). (C) Theoretical 95% confidence boundsfor a euploid sample (R21:18 = 1) and a T21 sample containing 10% ofcffDNA (R21:18 = 1.05). Red dashed lines indicate the positions of thepoint of intersection of the upper bound for the euploid sample andlower bound for the T21 sample. (D) The required number of target DNAmolecules from a reference chromosome for a range of N. Dashed linesindicate the minimum value of the required N.

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and only if P was less than ∼0.8 (λ ≤ 1.61) since the greatestprecision was achieved at this point.

We calculated the amount of input DNA that met therequired number of target DNA molecules (using one primerpair per chromosome) for a total of 5 million droplets (thetotal number of droplets we generated in this study). Details ofthe calculation are described in the ESI.† A large amount ofinput DNA (∼41–252 ng) is needed to identify an aneuploidsample containing a low fraction of cffDNA (10%–4%), seeTable S1.† It is not practical in a clinical procedure becauseonly 1–7 ng of cfDNA can be extracted from 1 ml of blood.

Since cfDNA is fragmented and the molecules that originatefrom the same chromosome were dispersed into different dro-plets, multiple primer pairs targeting multiple genes on thesame chromosome could be used to increase the detectednumber of target DNA molecules without increasing theamount of input DNA. Assuming that 1 to 2 ml of blood isused for testing, the amount of input DNA is less than 14 ng.Based on the 14 ng of input DNA, we calculated the requirednumber of primer pairs to reliably test the samples containing4% of cffDNA, which is 18 pairs. Details of the calculation aredescribed in the ESI.† Hence, we designed 40 primer pairs toquantify cfDNA from chr21 and chr18 (20 pairs for eachchromosome). Furthermore, we used the RainDrop DigitalPCR System for the detection because it can generate as manydroplets as needed (up to 10 million droplets).

Validation of multiplex ddPCR

To develop a practical ddPCR-based NIPT method, we selected20 loci on chr21 and 20 loci on chr18 (Fig. 3A) and designedthe corresponding primer pairs and probes (Table S2†). Thelength of the primers ranged from 18 to 27 nt, the ampliconlength ranged from 60 to 100 bp, and the distance betweeneach amplicon was greater than 1 × 105 bp. To reduce the back-ground fluorescence, we designed two universal probes for tar-geting chr21 and chr18, which were LNA-based oligonucleo-tides labeled with fluorescein. The length of the probes wasonly 10 nt, shorter than the probes that are commonly used inddPCR (15–25 nt). Therefore, it was easier to find the samesequence on multiple regions of a chromosome or even ondifferent chromosomes. To improve the specificity andbinding affinity of the short probes, they were substituted withLNA. LNA is a type of nucleic acid analog that contains a 2′-O,4′-C methylene bridge in the ribose moiety. When LNA wasincorporated into a probe, the thermal stability of the probeincreased, leading to an increase in melting temperature(Tm).

29,30

We used 10 ng fragmented normal genomic DNA andvarious numbers of primer pairs (1, 5, 10, and 20 pairs eachfor chr21 and chr18) to validate the relationship between thenumber of primer pairs and the number of target DNA mole-cules detected by ddPCR. Eight replicates were performed foreach group. As shown in Fig. 3B and C, the number of DNAmolecules detected was highly dependent on the number ofprimer pairs, as shown by the linear fitting results (R2 > 0.998).These data demonstrated that the increase in the number of

primer pairs was able to increase the detected number oftarget DNA molecules, given a certain amount of input DNA.The strategy of targeting multiple genes on the same chromo-some to reduce the amount of input DNA was feasible so thatthe ddPCR-based NIPT method was practical in a clinicalprocedure.

Validation of the relationship between the number of targetDNA molecules and the measurement precision for R21:18

To validate the relationship between the number of target DNAmolecules and the measurement precision for R21:18, we per-formed ddPCR experiments with (1) various amounts of frag-mented normal genomic DNA (1, 2, 5, and 10 ng) and (2)various numbers of primer pairs (2, 10, 20, and 40 pairs). Fortyprimer pairs and 10 ng of fragmented normal genomic DNAwere used in experiment (1) and (2), respectively. Eight repli-cates were performed for each group. We then calculated R21:18and the precision (95% CI) using eqn (4) and (9), respectively.As expected, both the increase in the amount of input DNA(Fig. 4A) and the numbers of primer pairs (Fig. 4B) improvedthe precision of the results (Fig. 4A and B, inset), suggestingthat the increase in the number of target DNA molecules wasable to improve the precision. Therefore, a reliable ddPCR-based detection method could be achieved.

Fig. 3 The selected loci and validation of multiplex ddPCR. (A) Theselected loci on chr21 and chr18 are plotted as vertical lines (accordingto the human genome assembly version hg19/GRCh37). The relationshipbetween the number of primer pairs (1, 5, 10, and 20 pairs) and thedetected number of DNA molecules from chr21 (B) and chr18 (C). Thelines show a linear fit of experimental points. The error bar representsthe standard deviation, n = 8.

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Validation of clinical sample detection when using multiplexddPCR

To validate the accuracy and clinical applicability of multiplexddPCR-based NIPT, we used our 40-plex ddPCR assay to testthe plasma DNA samples from 30 pregnant women, including16 male fetuses and 14 female fetuses. The average gestationalage was 17.5 weeks (range 12–25.3 weeks).

All of the samples were classified for trisomy risk statususing NGS (26 cases classified as euploid and 4 cases classifiedas T21). There was a complete consistency between the ddPCRtest results and NGS test results (Table 1). The R21:18 values ofeuploid samples ranged from 0.9715 to 1.0054, with a mean of0.9934 and a standard deviation of 0.0089. The R21:18 values ofT21 samples ranged from 1.0349 to 1.1083, with a mean valueof 1.0655 and a standard deviation of 0.0325. Euploid samplesand T21 samples were well separated (Fig. 5A).

We further investigated the confidence level of the resultsby calculating the z-score (Table 1 and Fig. 5B). The mean andstandard deviation of the euploid samples were calculatedusing another 30 euploid samples and were 0.9843 and 0.0161(Table S4†), respectively. The z-scores of the euploid sampleswere close to 0 (range 0.0381–1.2752). In contrast, the z-scoresof T21 samples were greater than 3 (range 3.2910–8.2960),showing that the samples were aneuploid and these results

exceeded the 99% confidence level. These results indicatedthat our multiplex ddPCR-based NIPT was able to detect fetalaneuploidies accurately.

The design guidelines for multiplex ddPCR-based NIPT

The design guidelines for multiplex ddPCR-based NIPT are asfollows:

(1) Perform statistical analysis to assess the number of dro-plets and target DNA molecules required to achieve reliabilityfor clinical use, and the amount of input DNA and the fractionof cffDNA should be considered in the analysis.

(2) Design a sufficient number of primer pairs and univer-sal probes and select an appropriate ddPCR platform accord-ing to the results of the analysis.

(3) The interactions between the primer pairs should beavoided, the length of the universal probes should be short,and the short probes should be substituted with LNA.

(4) Evaluate the feasibility of the primer pairs and the uni-versal probes and further optimize them.

(5) Prepare standard solutions with fragmented normalgenomic DNA and use the standard solutions to validate theddPCR assay.

(6) Use clinical samples to evaluate clinical applicability.The advantage of our approach is that we can detect eight

samples in one ddPCR run without any additional process.Compared to NGS-based NIPT, ddPCR-based NIPT has the fol-lowing significant advantages: (1) the workflow and data ana-lysis of the multiplex ddPCR assay are easier, and the require-ments for equipment are relatively less, which should be easierto implement in genetic testing laboratories, (2) the multiplexddPCR assay takes only ∼4.5 hours to complete a test, and (3)ddPCR-based NIPT is expected to achieve lower-cost detectionsince the cost of equipment and reagents required for ddPCRis lower than that for NGS.

In this study, using T21 and T18 as a model, we chosechr21 and chr18 as the reference chromosome for each otherto develop a method for simultaneously detecting T21 andT18. Although it may give a false negative result for a fetuswith T21 and T18 concurrent, this chromosomal aneuploidy israrely reported. Also, conceptions with double/multiple aneu-ploidies are unlikely to have a clinically recognized pregnancyor live birth.31,32 To further improve the reliability of ourmethod, we designed an alternative reference primer set fortargeting DNA molecules from chr1 to chr5, chr10, and chr11(20 primer pairs, see Table S3†). We used 10 ng of fragmentednormal genomic DNA to test the validity and reliability of thisprimer set. The detected ratio was 0.9956, and the detectednumber of DNA molecules was similar to the results of usingthe chr18 primer set (Fig. S1†), indicating that this referenceprimer set was effective. In order to include three primer sets(chr21, chr18, and chr1 to chr11) within a single reaction, mul-tiple fluorescent barcodes can be added in ddPCR amplifica-tion or a third optical channel can be equipped in the ddPCRplatform. In future studies, this test should be further opti-mized based on the development of ddPCR technology toachieve better robustness.

Fig. 4 The relationship between the number of target DNA moleculesand the measurement precision for R21:18. We used (A) various amountsof fragmented normal genomic DNA (1, 2, 5, and 10 ng) and (B) variousnumbers of primer pairs (2, 10, 20, and 40 pairs) in ddPCR experiments.The error bar represents the standard deviation, n = 8. The insets showthe trend of the precision vs. the number of target DNA molecules.

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As the number of clinical samples was relatively small andthere was no T18 sample, a trial with a larger sample size isrequired to further verify the specificity and sensitivity of themethod. To further improve the accuracy and reliability of thisapproach, the number of primer pairs can be increased toincrease the detected number of target DNA molecules. Inaddition, the fetal fraction should be evaluated to check thequality of clinical samples and to interpret the results pre-cisely. Y-chromosomal sequences9,33 and hypermethylated

RASSF1A34 can be served as fetal DNA markers in ddPCR toestimate the fetal fraction.

Furthermore, the concept of multiplex ddPCR-based NIPTcan be extended to detect other chromosomal aneuploidies(T13 and sex chromosome aneuploidy), so that we can developa ddPCR-based NIPT for simultaneous detection of multipleaneuploidies.

Conclusions

In this study, we developed multiplex ddPCR-based NIPT forthe detection of fetal aneuploidies. Using higher multiplexingin ddPCR-based NIPT could simplify the process of detectionand improve its practicability. Importantly, we performed stat-istical analysis based on the Poisson distribution to determinethe requirements for accurate and reliable test results. Becauseof the advantages of our multiplex ddPCR-based NIPT, it isexpected to become a competitive prenatal testing method inroutine prenatal care.

Conflicts of interest

There are no conflicts to declare.

Table 1 Information of clinical samples and the results of multiplex ddPCR-based T21 detection

Sample Gestational age Gender Input DNA (ng)

Total copy number

R21:18 ddPCR result NGS result z-Scorechr21 chr18

1 21w + 6 F 4.050 17 397 16 178 1.0753 T21 T21 4.08382 16w + 6 F 3.294 29 204 28 239 1.0342 T21 T21 5.82793 14w M 2.506 14 951 13 771 1.0857 T21 T21 3.29104 13w M 4.061 20 186 18 213 1.1083 T21 T21 8.29605 18w + 1 M 4.061 21 906 21 909 0.9999 E E 0.89876 15w + 3 M 6.361 32 192 32 232 0.9988 E E 0.82347 12w + 5 M 2.398 12 902 12 905 0.9998 E E 0.89228 16w M 4.050 22 844 22 892 0.9979 E E 0.76519 16w F 4.817 28 414 28 583 0.9941 E E 0.504810 19w + 1 F 7.290 35 849 35 948 0.9972 E E 0.720311 17w M 8.554 47 255 47 299 0.9991 E E 0.844612 17w + 4 M 8.986 51 256 51 404 0.9971 E E 0.711713 15w + 4 F 10.498 48 290 48 362 0.9985 E E 0.806514 24w M 8.165 44 614 45 891 0.9722 E E 0.989715 19w F 8.402 44 816 44 668 1.0033 E E 1.134016 19w + 4 F 8.284 44 573 45 268 0.9846 E E 0.139017 16w + 5 F 9.482 50 382 50 766 0.9924 E E 0.392218 18w + 4 F 6.826 40 688 41 880 0.9715 E E 1.033019 16w F 9.245 43 210 44 141 0.9789 E E 0.530420 12w M 7.009 38 668 39 212 0.9861 E E 0.038121 25w + 2 M 7.538 41 476 41 900 0.9899 E E 0.217922 18w M 8.824 48 186 47 928 1.0054 E E 1.275223 18w + 4 M 5.864 24 100 24 340 0.9901 E E 0.235624 13w + 1 F 7.279 23 452 23 802 0.9853 E E 0.094825 18w + 2 M 4.655 22 644 22 741 0.9957 E E 0.617226 17w M 4.201 23 486 23 413 1.0031 E E 1.120727 19w + 4 M 6.296 34 300 34 437 0.996 E E 0.636828 20w F 5.389 24 939 25 130 0.9924 E E 0.389729 16w + 1 F 5.173 24 551 24 570 0.9992 E E 0.855330 19w + 3 F 8.586 44 839 44 888 0.9989 E E 0.8336

“F”: Female, “M”: Male, “T21”: Trisomy 21, “E”: Euploidy.

Fig. 5 The results of 30 clinical samples detected using ddPCR. (A) Boxplot of calculated R21:18. (B) Box plot of z-scores calculated using the 30euploid samples. The dashed line indicates the z-score cutoff of three.

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Acknowledgements

This work was financially supported by the National NaturalScience Foundation of China (Grant No. 81572083 and81772288), the Central Public Interest Scientific InstitutionBasal Research Fund (Grant No. 2016GJZ01), and the Fundfrom TargetingOne Corporation (Grant No. 20162000009).

Notes and references

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