Reports of Major Impact
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`www.AJOG.org
`
`Noninvasive detection of fetal trisomy 21 by sequencing
`of DNA in maternal blood: a study in a clinical setting
`
`Mathias Ehrich, MD; Cosmin Deciu, MSc; Tricia Zwiefelhofer; John A. Tynan, DPhil; Lesley Cagasan, MSc; Roger Tim, DPhil;
`Vivian Lu; Ron McCullough, DPhil; Erin McCarthy; Anders O. H. Nygren, DPhil; Jarrod Dean; Lin Tang, DPhil;
`Don Hutchison, MSc; Tim Lu, DPhil; Huiquan Wang, DPhil; Vach Angkachatchai, DPhil; Paul Oeth, MSc;
`Charles R. Cantor, DPhil; Allan Bombard, MD; Dirk van den Boom, DPhil
`
`OBJECTIVE: We sought to evaluate a multiplexed massively parallel
`shotgun sequencing assay for noninvasive trisomy 21 detection using
`circulating cell-free fetal DNA.
`
`STUDY DESIGN: Sample multiplexing and cost-optimized reagents
`were evaluated as improvements to a noninvasive fetal trisomy 21 de-
`tection assay. A total of 480 plasma samples from high-risk pregnant
`women were employed.
`
`RESULTS: In all, 480 prospectively collected samples were obtained
`from our third-party storage site; 13 of these were removed due to in-
`sufficient quantity or quality. Eighteen samples failed prespecified as-
`say quality control parameters. In all, 449 samples remained: 39 tri-
`
`somy 21 samples were correctly classified; 1 sample was misclassified
`as trisomy 21. The overall classification showed 100% sensitivity (95%
`confidence interval, 89 –100%) and 99.7% specificity (95% confi-
`dence interval, 98.5–99.9%).
`
`CONCLUSION: Extending the scope of previous reports, this study dem-
`onstrates that plasma DNA sequencing is a viable method for noninva-
`sive detection of fetal trisomy 21 and warrants clinical validation in a
`larger multicenter study.
`
`Key words: circulating cell-free fetal DNA, massively parallel shotgun
`sequencing, maternal blood, NIPD, noninvasive prenatal diagnosis
`
`Cite this article as: Ehrich M, Deciu C, Zwiefelhofer T, et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical
`setting. Am J Obstet Gynecol 2011;204:205.e1-11.
`
`T risomy 21 is the most common
`
`chromosomal aneuploidy in live
`born infants. The overall incidence of
`trisomy 21 is approximately 1 in 800
`births in the general population,1 but
`this risk increases to 1 in 35 term births
`for women 45 years of age.2-4 Advanced
`maternal age is only one factor contrib-
`uting to increased risk. When other fac-
`tors, such as positive serum screening re-
`sults, fetal ultrasound abnormality, or
`family history are included, the odds of
`being affected given a positive result of
`
`Down syndrome can be as high as 1 in 9
`using the integrated test.5 For women in
`this high-risk group, an invasive diag-
`nostic procedure is currently the only
`way to confirm the diagnosis of trisomy
`21, commonly by means of a fetal karyo-
`type. Although the safety of the invasive
`procedures, specifically genetic amnio-
`centesis and chorionic villus sampling
`(CVS), has improved greatly since their
`introduction, there remains a well-rec-
`
`From Sequenom Inc (Drs Ehrich, Cantor, Bombard, and van den Boom), and Sequenom
`Center for Molecular Medicine LLC (Mr Oeth, Mr Deciu, Ms Zwiefelhofer, Ms Cagasan, Ms
`V. Lu, Ms McCarthy, Mr Dean, Mr Hutchison, and Drs Tynan, Tim, McCullough, Nygren,
`Tang, Lu, Wang, and Angkachatchai), San Diego, CA.
`
`Received Sept. 30, 2010; revised Dec. 28, 2010; accepted Dec. 28, 2010.
`
`Reprints: Dirk van den Boom, DPhil, Sequenom Inc, 3595 John Hopkins Ct., San Diego, CA
`92121. dvandenboom@sequenom.com.
`
`DISCLOSURE: All authors of this article are employees and shareholders of Sequenom, Inc. or its
`subsidiaries, and therefore, a potential conflict of interest exists.
`
`0002-9378/free • © 2011 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2010.12.060
`
`For Editors’ Commentary, see Table of Contents
`
`See related editorial, page 183
`
`ognized risk of iatrogenic fetal loss. Tests
`that could better identify those women
`who would most benefit from confirma-
`tory invasive diagnostic tests are of great
`public health interest. Since the initial
`seminal work by Merkatz et al,6 contin-
`uous efforts have focused on increasing
`the specificity of primary screening
`methods; eg, by including more serum
`protein markers or through the addition
`of ultrasound findings suggestive of fetal
`aneuploidy. Consequently,
`screening
`tests have greatly improved in their clin-
`ical sensitivity and specificity over the
`last 2 decades. These developments have
`also led to contemporary testing pro-
`grams involving a variety of potential
`screening algorithms.7 Accurate gesta-
`tional dating through ultrasound is a
`critical element to achieve high accuracy,
`but may not be readily available to all
`pregnant women.
`A new approach to detect fetal aneu-
`ploidy analyzes fetal DNA itself rather
`than the surrogate biochemical or ultra-
`sound markers in current maternal se-
`rum screening protocols. In 1997, Lo et
`al8 reported that circulating cell-free
`(ccf) fetal (ccff) DNA is present in the
`plasma of pregnant women. DNA of fetal
`
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`origin ranges between 2% and 40% with
`a mean around 10% of the total ccf DNA
`across varying gestational ages.9-12 The
`ccff DNA is cleared from the maternal
`bloodstream within hours after birth;
`thus, misdiagnosis from carryover con-
`tamination from a previous pregnancy is
`unlikely.13 A noninvasive ccff method
`for prenatal Rhesus D testing in Europe
`has already been widely adopted.14
`In comparison to Rhesus D testing, an-
`euploidy detection from ccf DNA is far
`more challenging. In principle, aneuploidy
`detection could be enabled through a vari-
`ety of methods including the analysis of
`single nucleotide polymorphisms,15 DNA
`methylation,16,17 or fetally expressed RNA
`transcripts.18,19 The most convincing data
`to date for a generally applicable test, how-
`ever, have been generated through mas-
`sively parallel shotgun sequencing (MPSS)
`of ccf DNA. Two groups have indepen-
`dently shown that MPSS can unambigu-
`ously identify plasma samples
`from
`women carrying a trisomy 21 fetus20-23
`compared to samples from women with
`euploid fetuses. These studies were per-
`formed with small numbers of clinical
`samples and, while these preliminary re-
`sults are very promising, the true clinical
`performance remains to be established. As
`originally described in 2008, the overall
`cost of a sequencing-based test was prohib-
`itive in terms of potential deployment in
`clinical practice. However, next-genera-
`tion sequencing methods such as MPSS
`are rapidly evolving with concomitant de-
`clines in reagent and instrument costs.
`We have implemented several process
`improvements in MPSS for noninvasive
`aneuploidy detection using ccf DNA.
`These modifications provide an afford-
`able testing procedure with the potential
`for widespread utilization. Because such
`a test, first and foremost, has to be safe
`and efficacious we designed a blinded
`study that tested a total of 480 plasma
`samples collected from pregnant woman
`at high risk for fetal aneuploidy.
`
`M A T E R I A L S AND M E THOD S
`Study design
`The study was set up to include at least 40
`trisomy 21 samples, a design chosen to
`achieve a lower 95% confidence bound
`
`of 91% when all trisomy 21 cases are cor-
`rectly identified. We matched trisomy 21
`samples with euploid samples at a ⬃1:11
`ratio, slightly higher than the more typi-
`cal prevalence in a high-risk group of 1
`in 15.
`Patients at increased risk for fetal
`Down syndrome and other chromo-
`somal aneuploidies were asked to partic-
`ipate in this prospective study. Risks in-
`cluded a positive serum biochemical
`screening test; advanced maternal age
`(ⱖ35 years at the estimated date of deliv-
`ery); a fetal ultrasound finding sugges-
`tive of Down syndrome; or a personal/
`family history of Down syndrome.
`Patient informed consent was obtained
`for peripheral blood sampling and for
`the inclusion of karyotype results from
`an already scheduled, subsequent inva-
`sive diagnostic procedure. Fetal karyo-
`types or quantitative fluorescent PCR re-
`sults were obtained as part of regular
`clinical care on either CVS or genetic
`amniocentesis samples. These data were
`unknown to the investigators prior to
`unblinding. The sample demographics
`were representative for pregnant women
`at high risk for fetal trisomy 21 (Table 1).
`Samples were blinded to the investiga-
`tors and prospectively collected, pro-
`cessed, and stored at an independent,
`contracted, third-party location (Bio-
`storage Technologies Inc [BST], Indian-
`apolis, IN). All information was kept
`within an independent, third-party data-
`base (Pharmaceutical Research Associ-
`ates Inc [PRA], Raleigh, NC). A total of
`480 samples were requested from PRA
`and provided by BST for analysis at Se-
`quenom Center for Molecular Medicine,
`San Diego, CA. Karyotype results were
`unknown to the investigators and data
`analysts until after completion of all
`sample testing and submission for re-
`view. The MPSS results were sent to an
`independent, third-party biostatistician
`who had all clinical information includ-
`ing confirmatory karyotypes. The data
`were matched and unblinded by this
`third-party biostatistician and the con-
`cordance of the results was reported.
`
`Sample collection
`For the study presented here, samples
`were collected at clinical practices active
`
`in the treatment of patients undergoing
`invasive prenatal diagnosis by CVS (first
`trimester) and genetic amniocentesis
`(second trimester) and, for some of the
`cases, from pregnancy termination cen-
`ters. Eight samples were collected for
`research purposes under Food and Drug
`Administration approval (FDA Estab-
`lishment Identifier no. 3005208435). All
`remaining samples were collected under
`institutional review board (IRB) ap-
`proval (Western Institutional Review
`Board [WIRB] no. 20091396, WIRB no.
`20080757, Compass IRB no. 00351). All
`samples, demographics, and karyotype
`results were completely blinded to the
`laboratory investigators by the third-
`party clinical
`research organization
`(PRA) and the BST facility. Patients were
`approached during their genetic coun-
`seling sessions and, if they gave their
`informed consent, the study protocol
`dictated that phlebotomy was to be per-
`formed prior to their invasive procedure.
`The vast majority of samples were col-
`lected after August 2009 and none were
`collected before May 2009; therefore, the
`oldest samples in the study were ⬃10
`months old. Samples were all collected at
`specifically contracted processing cen-
`ters operating under study-specific pro-
`tocols. None of the samples were ob-
`tained and analyzed as fresh samples; ie,
`all were processed and frozen before
`shipment to the central, independent
`biostorage
`facility (http://biostorage-
`.com/; a full description of the indepen-
`dent nature of this widely used biostor-
`age company is detailed on their World
`Wide Web site).
`All samples were collected and pro-
`cessed under the same protocol: 10 mL of
`maternal whole blood was drawn into an
`EDTA-K2 spray-dried Vacutainer (Bec-
`ton Dickinson, Franklin Lakes, NJ),
`stored, and transported to the processing
`laboratory on wet ice. Within 6 hours of
`the blood draw, the maternal whole
`blood was
`centrifuged (Eppendorf
`5810R plus swing out rotor) chilled
`(4°C) at 2500g for 10 minutes and the
`plasma was collected. The plasma was
`centrifuged a second time (Eppendorf
`5810R plus fixed angle rotor) at 4°C at
`15,000g for 10 minutes. After the second
`spin, the plasma was removed from the
`
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`pellet that formed at the bottom of the
`tube and distributed into 4-mL plasma
`bar-coded aliquots. In this study, only a
`single 4-mL plasma aliquot from each
`patient was used for DNA isolation.
`
`MPSS aneuploidy detection
`DNA was prepared from 4 mL of mater-
`nal plasma. The short lengths of ccf DNA
`afford direct use in preparing the librar-
`ies of DNA fragments that were se-
`quenced. In practice, 4 different libraries
`each containing a synthetic oligonucleo-
`tide sequence as a bar code were mixed
`and analyzed together (multiplexing).
`The bar code revealed which library each
`sequence read represented. Eight sepa-
`rate mixtures of 4 libraries were analyzed
`in parallel. One MPSS process required
`about 2 days and yielded 36 bases of se-
`quence from each DNA fragment. Ap-
`proximately 5 million 36-base fragments
`were sequenced from each library. These
`represented about 6% of the human ge-
`nome in each sample. As is standard in
`MPSS, the 36-base reads were processed
`to exclude poor-quality data and then
`matched to a reference human genome
`to determine their chromosome origin.
`The fraction of reads is proportionate to
`chromosome size. Thus, typically 8.5%
`of all reads are from chromosome 1,
`while only about 1.2% are from chromo-
`some 22.20,21
`A fetus with trisomy 21 contributes
`additional genetic material to the total
`pool of ccf DNA. Consequently, in com-
`parison to women carrying a euploid fe-
`tus, a slightly larger contribution of se-
`quence reads mapping to chromosome
`21 is observed in a plasma sample of a
`woman carrying a fetus with Down syn-
`drome. Ccf DNA in plasma from a preg-
`nant woman with a euploid fetus shows
`an average 1.35% of all aligned sequence
`reads located on chromosome 21. A va-
`riety of analytical methods have been
`published to detect an overabundance of
`genetic material from chromosome 21 in
`trisomic pregnancies.20,21,23 These use
`some form of normalization to calibrate
`the results against a known set of euploid
`reference samples. Contributions greater
`than the reference range are then indica-
`tive of additional genetic material from
`chromosome 21 and in many cases can
`
`be interpreted as a fetal trisomy 21. In
`this study, a modification of a method
`used by Chiu et al20 was used for classi-
`fication. Prior to the main study a set of
`known euploid reference samples was
`used to calculate the mean and standard
`deviation (SD) of the representation of
`chromosome 21 (percentage of reads ob-
`tained from chromosome 21). Then, for
`every test sample, the distance, measured
`in SD, from the mean in the euploid ref-
`erence set was calculated. A fixed cutoff
`of 2.5 SD was used to identify samples
`with an overrepresentation of chromo-
`some 21 material.
`
`Assay design
`Compared to previously published stud-
`ies,20,21 3 important modifications were
`made to the sequencing protocol. We
`used custom purified enzymes in the li-
`brary generation process to achieve a re-
`duction in assay cost. We employed the
`latest sequencing biochemistry available
`for the GAIIx sequencer (Illumina Inc,
`San Diego, CA) in combination with
`the manufacturer’s analysis
`software
`CASAVA version 1.6. These changes in-
`creased the number of sequence reads
`from approximately 13 to 20 million per
`lane. We also used indexing primers dur-
`ing library amplification to allow analy-
`sis of multiple samples in a single se-
`quencing reaction (“multiplexing” vs
`“monoplexing”). In this study, 4 samples
`were analyzed per lane (“4-plex” or “tet-
`raplexing”), which equates to approxi-
`mately 3 to 5 million available sequence
`reads per sample. The combination of
`these modifications enabled 4 times
`higher throughput at about 4 times
`lower cost.
`
`DNA extraction
`The Qiagen ccf nucleic acid kit (Qiagen,
`Hilden, Germany) was used according to
`the manufacturer’s specifications. The
`resulting DNA was eluted in 55 ␮L of
`buffer AVE (part of the Qiagen kit).
`
`Quality control of extracted DNA
`The quantity of the extracted DNA was
`determined with an assay that uses si-
`multaneous quantification of fetal and
`total ccf DNA. This fetal quantifier assay
`(FQA) was recently published10 and uses
`methylation-sensitive
`restriction en-
`
`zymes to eliminate the maternal contri-
`bution of genomic regions that are
`methylated in fetal DNA and unmethyl-
`ated in maternal DNA. The remaining
`nondigested fetal DNA is coamplified in
`the presence of a known amount of syn-
`thetic oligonucleotide to permit compet-
`itive polymerase chain reaction (PCR).
`This synthetic oligonucleotide has an
`identical sequence to the target genomic
`DNA, apart from 1 nucleotide that can
`be targeted by single-base extension and
`quantitative matrix-assisted laser de-
`sorption/ionization time-of-flight mass
`spectrometric analysis. To ensure accu-
`rate results, the assay comprises multiple
`markers in 4 different categories. Three
`markers are used to measure total DNA
`amounts. Three markers are used to
`measure chromosome Y copy numbers;
`2 markers interrogate the efficiency of
`the methylation-specific digestion reac-
`tion, and 5 markers are used to measure
`fetal DNA amounts.
`Methylation-based DNA discrimina-
`tion was performed using 10 ␮L of eluted
`DNA per reaction. All reagents and ap-
`paratus were obtained from Sequenom
`Inc, San Diego, CA, unless stated other-
`wise. Digestion of plasma DNA was per-
`formed for 30 minutes at 41°C by adding
`25 ␮L of a mixture containing 3.5X PCR
`buffer, 2.22 mmol/L MgCl2, 10 U HhaI
`(New England Biolabs, Ipswich, MA), 10
`U HpaII (New England Biolabs), and 10
`U ExoI (New England Biolabs). Exonu-
`clease was added to eliminate single-
`stranded DNA that would escape diges-
`tion and overestimate the fetal fraction.
`After the restriction was complete, the
`enzymes were inactivated and the DNA
`denatured by heating the mixture for 10
`minutes at 98°C. All steps following the
`restriction reaction were performed ac-
`cording to Nygren et al.10
`
`Library preparation
`The extracted ccf DNA was used for li-
`brary preparation without further frag-
`mentation or size selection, because ccf
`DNA is already naturally fragmented,
`having an average length of approxi-
`mately 160 base pairs. Low binding Ep-
`pendorf tubes were used to store 55 ␮L of
`DNA eluent at 4°C following extraction
`until the library preparation had started.
`
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`FIGURE 2
`Robust versus externally
`referenced z-scores
`
`20
`
`15
`
`10
`
`5
`
`0
`
`0
`
`5
`
`10
`
`15
`
`20
`
`Relationship between z-scores calculated based
`on 24 external samples using mean and SD (x-
`axis) and using median and median absolute de-
`viation (y-axis). The blue line indicates the em-
`pirically derived z-score cutoff of 2.5, which is
`comparable to the cutoff of 3 (red line) using the
`robust method.
`Ehrich. Noninvasive detection of fetal trisomy 21. Am J
`Obstet Gynecol 2011.
`
`was mixed with 1X Phusion MM, Illu-
`mina Inc PE 1.0 and 2.0 primers, and 1 of
`12 index primers for a total PCR reaction
`volume of 50 ␮L. The sample was ampli-
`fied in a 0.65-mL PCR tube using a MJ
`Research (Bio-Rad, Hercules, CA)
`Model PTC-200 thermal cycler. The
`PCR conditions were an initial denatur-
`ation at 98°C for 30 seconds, 15 cycles of
`denaturation at 98°C for 10 seconds, an-
`nealing at 65°C for 30 seconds, and ex-
`tension at 72°C for 30 seconds. A final
`extension at 72°C for 5 minutes was fol-
`lowed by a 4°C hold. The PCR products
`were cleaned with MinElute columns
`and the libraries eluted in 17 ␮L of EB.
`
`Quality control of generated
`sequencing library
`The libraries were quantified via SYBR
`Green quantitative PCR (qPCR) analysis
`as outlined by Meyer et al.24 Each library
`was diluted 1:108 and quantified against
`a library standard using Power SYBR
`Green PCR Master Mix (ABI, Foster
`City, CA).
`Each sample or standard was assayed
`in triplicate, including triplicate non-
`template control reactions. The sample
`was gently inverted or pipetted up and
`
`FIGURE 1
`Histogram of z-scores calculated using 24 reference samples
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0.0
`
`Relative frequency
`
`0
`
`5
`
`10
`
`15
`
`A red vertical line is drawn at z ⫽ – 0.6 and corresponds to the median z-score for all samples; its
`expected position, under the assumption that reference samples have same distribution of chromo-
`some 21 representation as all euploid samples in this study, is at z ⫽ 0.
`Ehrich. Noninvasive detection of fetal trisomy 21. Am J Obstet Gynecol 2011.
`
`Storage times ranged from 24-72 hours.
`The library preparation was carried out
`according to the manufacturer’s specifi-
`cations (Illumina Inc) with some modi-
`fications. Enzymes and buffers were
`sourced from Enzymatics (End Repair
`Mix –LC; dNTP Mix [25 mmol/L each];
`Exo(-) Klenow polymerase; 10X Blue
`Buffer; 100 mmol/L dATP; T4 DNA Li-
`gase; 2X Rapid Ligation Buffer) and
`New England Biolabs (Phusion MM).
`Adapter oligonucleotides, indexing oli-
`gonucleotides, and PCR primers were
`obtained from Illumina Inc.
`Library preparation was initiated by
`taking 40 ␮L of ccf DNA for end repair,
`retaining 15 ␮L for QC by FQA. End re-
`pair was performed with a final concen-
`tration of 1X End Repair buffer, 24.5
`␮mol/L each dNTPs, and 1 ␮L of End
`Repair enzyme mix. The end repair reac-
`tion was carried out at room tempera-
`
`ture for 30 minutes and the products
`were cleaned with Qiagen Qiaquick col-
`umns, eluting in 36 ␮L of elution buffer
`(EB). 3= mono-adenylation of the end-
`repaired sample was performed by mix-
`ing it with a final concentration of 1X
`Blue Buffer, 192 ␮mol/L dATP, and 5 U
`of Exo(-) Klenow Polymerase. The reac-
`tion was incubated at 37°C for 30 min-
`utes and cleaned up with Qiagen Min-
`Elute columns, eluting the products in
`14 ␮L of EB. Adapters were ligated to the
`fragments by incubating for 10 minutes
`at room temperature with 1X Rapid Li-
`gation buffer, 48.3 nmol/L Index PE
`Adapter Oligos, and 600 U T4 DNA Li-
`gase. The ligation reaction was cleaned
`up with QiaQuick columns, and the sam-
`ple eluted in 23 ␮L of EB. The adapter-
`modified sample was enriched by ampli-
`fying with a high-fidelity polymerase.
`The entire 23 ␮L eluent of each sample
`
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`
`FIGURE 3
`Distributions of quality control parameters
`
`Unique sequencing counts
`
`Total DNA
`
`0e+00
`
`Relative frequency
`
`0.0e+00
`
`1.0e+07
`
`2.0e+07
`
`3.0e+07
`
`0
`
`5000
`
`10000
`
`15000
`
`Counts
`
`Genome equivalents
`
`Fetal fraction
`
`Library concentration by qPCR
`
`0.00000.00100.00200.0030
`
`Relative frequency
`
`0.0e+00
`
`6
`
`5
`
`4
`
`3
`
`2
`
`1
`
`0
`
`Relative frequency
`
`Relative frequency
`
`0.0
`
`0.1
`
`0.2
`
`0.3
`
`0.4
`
`0.5
`
`0
`
`200
`
`400
`
`600
`
`800
`
`Fetal fraction
`
`nM
`
`Histograms showing distribution of quality control parameters (each histogram has optimal number of
`bins, as calculated with Scott’s rule). Very high values of unique sequence counts (⬎10 million) are
`mainly obtained from samples that were analyzed in monoplex.
`Ehrich. Noninvasive detection of fetal trisomy 21. Am J Obstet Gynecol 2011.
`
`by all sequence reads excluding sequence
`reads from chromosomes X and Y. The
`fractional genomic representation was
`then standardized by subtracting the
`mean of a control group and dividing by
`the standard deviation (SD) of that same
`control group. Using a set of known eu-
`ploid samples as a control group, this
`method determines the distance in SD of
`the tested sample to the mean of the eu-
`ploid reference group. This metric, stan-
`dardized fractional genomic representa-
`tion (the so-called z-scores), is the metric
`used to classify samples as euploid or tri-
`somy 21. Details of this procedure are
`outlined in Chiu et al.20
`Ideally, the standardization process
`would be based on the true mean and
`true SD as calculated from a very large
`set of euploid samples. In the absence
`
`of such a large set, a control group of 24
`euploid samples from a previous ex-
`periment was used. Given both the
`limited sample size and the latent dif-
`ferences between the 2 different exper-
`iments, this control group may pro-
`duce biased estimates of the true mean
`and true SD. In the design of the cur-
`rent study, similar to an expected clin-
`ical setting, it was anticipated that the
`majority of the samples would be eu-
`ploid. Therefore the distribution of the
`z-scores should have a large normally
`distributed component centered on 0
`and with SD close to 1. Any significant
`departure from this situation would be
`an indicator of improper standardiza-
`tion. Alternatively, a robust standard-
`ization can be employed given the data
`from the current experiment, by using
`
`MARCH 2011 American Journal of Obstetrics & Gynecology 205.e5
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`down to mix, then spun down. In the re-
`action 2 ␮L of the 1:108 dilution are
`added to a reaction mix containing 9 ␮L
`of Ultrapure Water, 12.5 ␮L 2x Power
`Mix, 0.5 ␮L of each forward (GAT ACG
`GCG ACC ACC GAG AT) and reverse
`(CAA GCA GAA GAC GGC ATA CGA
`G) primer at 10 ␮mol/L, and 0.5 ␮L of 1
`U/␮L uracil-N-glycosylase. Amplifica-
`tion was performed on an ABI 7500 (Ap-
`plied Biosystem, Foster City, CA). The
`cycling protocol began with a 2-minute
`uracil-N-glycosylase decontamination
`step at 50°C, this was followed by Hot-
`Start activation 95°C for 10 minutes. The
`program cycling was commenced and
`continued through 46 cycles of 15 sec-
`onds denaturation at 95°C followed by 1
`minute of annealing/extension at 60°C.
`The final step was a 15-second denatur-
`ation at 95°C.
`
`Clustering and sequencing
`Clustering and sequencing were per-
`formed according to standard Illumina
`Inc protocols. Individual libraries were
`normalized to a 5-nmol/L concentration
`and then clustered in 4-plex format to a
`final flow cell loading concentration of
`1.75 pmol/L per sample or 7 pmol/L per
`flow cell lane. The cBOT instrument and
`v4 Single-Read cBOT reagent kit (Illu-
`mina Inc) were used. Thirty-six cycles
`of single-read multiplexed sequencing
`were performed on the Genome Ana-
`lyzer IIx with Paired-End module using
`v4 SBS reagent kits and supplemental
`Multiplex Sequencing Primer kits (Illu-
`mina Inc). Image analysis and base call-
`ing were performed with RTA1.6/SCS2.6
`software (Illumina Inc). Sequences were
`aligned to the UCSC hg19 human refer-
`ence genome (nonrepeat-masked) using
`CASAVA version 1.6 (Illumina Inc).
`
`Data analysis
`Sequence reads unique to a chromosome
`were counted, up to 1 mismatch (U1
`counts), and the chromosome 21-spe-
`cific genomic representation was calcu-
`lated based on these unique sequence
`reads. The fractional genomic represen-
`tation of chromosome 21 (also referred
`to as the percentage of chromosome 21)
`was determined by dividing the number
`of sequence reads from chromosome 21
`
`00005
`
`

`

`Reports of Major Impact
`
`www.AJOG.org
`
`TABLE 1
`Demographics of 449 analyzed samples
`
`Demographic
`
`Median
`
`Range
`
`(18–47)
`37
`Maternal age, y (n ⫽ 448)
`..............................................................................................................................................................................................................................................
`Gestational age, wk (n ⫽ 448)
`16
`(8–36)
`..............................................................................................................................................................................................................................................
`Maternal weight, lb (n ⫽ 425)
`153
`(96–314)
`..............................................................................................................................................................................................................................................
`Variable
`Percent
`(Naffected/Ntotal)
`..............................................................................................................................................................................................................................................
`Indication for testinga
`.....................................................................................................................................................................................................................................
`Positive serum screening
`30.2
`(133/441)
`.....................................................................................................................................................................................................................................
`Advanced maternal age
`68.3
`(306/448)
`.....................................................................................................................................................................................................................................
`Ultrasound abnormality
`12.9
`(57/441)
`.....................................................................................................................................................................................................................................
`Positive family history
`5.2
`(23/441)
`.....................................................................................................................................................................................................................................
`Not specified
`10.2
`(45/441)
`..............................................................................................................................................................................................................................................
`Procedure
`.....................................................................................................................................................................................................................................
`CVS
`19
`(84/442)
`.....................................................................................................................................................................................................................................
`Genetic amniocentesis
`81
`(358/442)
`..............................................................................................................................................................................................................................................
`Confirmation
`.....................................................................................................................................................................................................................................
`Karyotype
`59.9
`(269/449)
`.....................................................................................................................................................................................................................................
`FISH
`2.9
`(13/449)
`.....................................................................................................................................................................................................................................
`Both
`35.6
`(160/449)
`.....................................................................................................................................................................................................................................
`QF-PCR
`1.6
`(7/449)
`..............................................................................................................................................................................................................................................
`For some patients not all information was available. Number of patients used to calculate statistics is indicated for each
`parameter.
`CVS, chorionic villus sampling; FISH, fluorescent in situ hybridization; QF-PCR, quantitative fluorescent polymerase chain
`reaction.
`
`a Some patients had ⬎1 indication.
`
`Ehrich. Noninvasive detection of fetal trisomy 21. Am J Obstet Gynecol 2011.
`
`the median and median absolute devi-
`ation for the calculation of z-scores.
`Twenty-four known euploid sam-
`ples from a previous study were used to
`determine mean and SD of the percent
`of
`chromosome 21 representation
`needed for calculating the z-scores for
`the set of 480 samples. The determina-
`tion of the mean and SD of the distri-
`bution of z-scores was performed by
`applying an iterative censoring ap-
`proach. In each iteration we excluded
`the most extreme values (outside of 3
`SD) and recalculated mean and SD.
`The values for mean and SD ap-
`proached a stable value after 10 itera-
`tions. Using this method, we estimate
`the true mean to be – 0.6 and the SD to
`be 1.03. Based on these values the em-
`pirically derived z-score cutoff was set
`to 2.5 (z-score cutoff ⫽ mean ⫹ 3 SD).
`This distribution of the z-scores is dis-
`played in Figure 1. The z-score cutoff
`
`was derived and applied to the data be-
`fore unblinding.
`We used the same 24 reference sam-
`ples and sequenced them in monoplex
`format. The resulting data were used to
`calculate mean and SD for this monoplex
`reference dataset. For the set of 10 sam-
`ples that were run in monoplex we did
`not have enough data available for an
`equivalent bias estimate. Consequently,
`we applied the same z-score cutoff to the
`entire dataset.
`The empirically derived threshold of
`z ⫽ 2.5 correlates well with the cutoff
`of z ⫽ 3, when z-scores are calculated
`using the rob