Noninvasive prenatal diagnosis of fetal chromosomal
aneuploidy by massively parallel genomic sequencing
of DNA in maternal plasma
Rossa W. K. Chiua,b, K. C. Allen Chana,b, Yuan Gaoc,d, Virginia Y. M. Laua,b, Wenli Zhenga,b, Tak Y. Leunge,
Chris H. F. Foof, Bin Xiec, Nancy B. Y. Tsuia,b, Fiona M. F. Luna,b, Benny C. Y. Zeef, Tze K. Laue, Charles R. Cantorg,1,
and Y. M. Dennis Loa,b,1
aCentre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, Departments ofbChemical Pathology andeObstetrics and
Gynaecology, andfCentre for Clinical Trials, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China;cCenter for the Study of
Biological Complexity anddDepartment of Computer Science, Virginia Commonwealth University, Richmond, VA 23284; andgSequenom, Inc., San Diego, CA
Contributed by Charles R. Cantor, October 22, 2008 (sent for review September 29, 2008)
Chromosomal aneuploidy is the major reason why couples opt for
prenatal diagnosis. Current methods for definitive diagnosis rely on
invasive procedures, such as chorionic villus sampling and amniocen-
been found in maternal plasma but exists as a minor fraction among
a high background of maternal DNA. Hence, quantitative perturba-
overall representation of sequences from that chromosome in ma-
ternal plasma would be small. Even with highly precise single mole-
cule counting methods such as digital PCR, a large number of DNA
molecules and hence maternal plasma volume would need to be
analyzed to achieve the necessary analytical precision. Here we
reasoned that instead of using approaches that target specific gene
loci, the use of a locus-independent method would greatly increase
used massively parallel genomic sequencing to quantify maternal
trisomy 21. Twenty-eight first and second trimester maternal plasma
were correctly identified. Massively parallel plasma DNA sequencing
represents a new approach that is potentially applicable to all preg-
nancies for the noninvasive prenatal diagnosis of fetal chromosomal
Down syndrome ? Solexa sequencing ? trisomy 21
diagnosis. Conventional methods for definitive prenatal diagnosis
of these disorders involve the invasive sampling of fetal materials
the fetus (1). Many workers tried to develop noninvasive ap-
proaches. Methods based on ultrasound scanning and maternal
serum biochemical markers (2) have proved to be useful screening
tests. However, they detect epiphenomena instead of the core
pathology of chromosomal abnormalities. They have limitations
such as a narrow gestational window of applicability and the need
to combine multiple markers, even over different time points, to
arrive at a clinically useful sensitivity and specificity profile.
For the direct detection of fetal chromosomal and genetic
abnormalities from maternal blood, early work focused on the
relatively difficult isolation of the rare fetal nucleated cells from
maternal blood (3–5). The discovery of cell-free fetal nucleic acids
in maternal plasma in 1997 opened up new possibilities (6, 7).
However, the fact that fetal DNA represents only a minor fraction
of total DNA in maternal plasma (8), with the majority being
contributed by the pregnant woman herself, has offered consider-
able challenge. Recently, a number of approaches have been
developed. One strategy targets a fetal-specific subset of nucleic
he testing of fetal chromosomal aneuploidies is the predomi-
nant reason why many pregnant women opt for prenatal
acids in maternal plasma, e.g., placental mRNA (9–11) and DNA
molecules bearing a placental-specific DNA methylation signature
(12–14). The fetal chromosomal dosage is then assessed by allelic
ratio analysis of SNPs within the targeted molecules. These strat-
egies are called the RNA–SNP allelic ratio approach (11) and the
epigenetic allelic ratio approach (14). These allelic ratio-based
methods can be used only for fetuses heterozygous for the analyzed
SNPs. Thus, multiple markers are needed to enhance the popula-
tion coverage of the methods.
To develop a polymorphism-independent method for the detec-
tion of fetal chromosomal aneuploidies from maternal plasma, our
group has recently outlined the principles for the measurement of
RCD aims to measure the total (maternal plus fetal) amount of a
specific locus on a potentially aneuploid chromosome in maternal
plasma, e.g., chromosome 21 (chr21) in trisomy 21 (T21), and
compares it to that on a reference chromosome. Hence, fetal T21
is diagnosed by detecting the small increment in the total amount
as compared with a gene locus on a reference chromosome. The
proportional increment in chr21 sequences is expectedly small
because fetal DNA contributes only a minor fraction of DNA in
maternal plasma (8). To reliably detect the small increase, a large
absolute number of chr21 and reference chromosome sequences of
the loci targeted by the digital PCR assays need to be analyzed and
quantified with high precision. The number of molecules required
for RCD increases by four times, for every twofold reduction in the
which the fractional concentration for circulating fetal DNA is low,
e.g., during early gestation, relatively large volumes of maternal
plasma may be needed. One way is to perform multiplex analysis of
multiple genetic loci. However, the optimization of highly multi-
plexed digital PCR might be challenging. If fluorescence reporters
are used, one would also quickly run out of reporters for distin-
guishing the products from the various loci.
Author contributions: R.W.K.C., K.C.A.C., and Y.M.D.L. designed research; R.W.K.C.,
K.C.A.C., Y.G., V.Y.M.L., W.Z., B.X., N.B.Y.T., and F.M.F.L. performed research; T.Y.L. and
T.K.L. collected clinical samples; R.W.K.C., K.C.A.C., V.Y.M.L., C.H.F.F., B.C.Y.Z., C.R.C., and
Y.M.D.L. analyzed data; and R.W.K.C. and Y.M.D.L. wrote the paper.
Conflict of interest statement: R.W.K.C., K.C.A.C., N.B.Y.T., F.M.F.L., B.C.Y.Z., C.R.C., and
Y.M.D.L. have filed patent applications on the detection of fetal nucleic acids in maternal
plasma for noninvasive prenatal diagnosis. Part of this patent portfolio has been licensed
to Sequenom. C.R.C. is Chief Scientific Officer of and holds equities in Sequenom. Y.M.D.L
is a consultant to and holds equities in Sequenom.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or ccantor@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
December 23, 2008 ?
vol. 105 ?
To overcome the above limitations, we propose to use a method
independent of any particular gene locus to quantify the amount of
chr21 sequences in maternal plasma. When a locus-independent
method is used, potentially every DNA fragment originating from
the amount of that chromosome. Therefore, for any fixed volume
much greater than the number of DNA molecules that could serve
as templates for detection by gene locus-specific assays. Hence,
precise detection of the over- or underrepresentation of sequences
from an aneuploid chromosome could be more readily achieved.
We previously (15) proposed that the recently available massively
parallel genomic sequencing (MPGS) platforms (16, 17) might be
adaptable as an approach to quantify DNA sequences for the
this study, we demonstrate the use of the ‘‘Solexa’’ sequencing
technique (Illumina) (18) for this purpose.
Procedural Framework. The procedural framework of using MPGS
for noninvasive fetal chromosomal aneuploidy detection in mater-
the sequencing-by-synthesis Solexa method (18). As the maternal
plasma DNA (maternal and fetal) molecules were already frag-
end of the clonally expanded copies of each plasma DNA fragment
was sequenced and processed by standard postsequencing bioin-
formatics alignment analysis for the Illumina Genome Analyzer,
which uses the Efficient Large-Scale Alignment of Nucleotide
Databases (ELAND) software. The purpose of the alignment was
to simply determine the chromosomal origin of the sequenced
plasma DNA fragments and details about their gene-specific loca-
tion were not required. The number of sequence reads originating
from any particular chromosome was then counted and tabulated
for each human chromosome. In this study, we counted only
sequences that could be mapped to just one location in the
repeat-masked reference human genome with no mismatch, i.e.,
deemed as a ‘‘unique’’ sequence in the human genome. We termed
these sequences as U0–1–0–0 on the basis of values in a number of
fields in the data output files of the ELAND sequence alignment
software (Illumina) (see Materials and Methods).
We then determined the percentage contribution of unique
count of a specific chromosome by the total number of U0–1–0–0
sequence reads generated in the sequencing run for the tested
sample to generate a value termed % chrN, when the chromosome
belonged to a T21 pregnancy, we calculated the z-score of % chr21
of the tested sample. The z-score refers to the number of standard
deviations from the mean of a reference data set. Hence, for a T21
the mean and standard deviation of % chr21 values obtained from
maternal plasma of euploid pregnancies.
For this procedure to be effective for noninvasive prenatal fetal
chromosomal aneuploidy detection, a number of assumptions need
generate sequence reads for the small fraction of fetal DNA in
maternal plasma alongside the background maternal DNA. Sec-
ond, the pool of plasma DNA fragments captured for sequencing
needs to be a representative sample of the total DNA pool with
similar interchromosomal distribution to that in the original ma-
ternal plasma. Third, there should be no major bias in the ability to
sequence DNA fragments originating from each chromosome.
When these assumptions hold, then the % chrN values should be
reflective of the genomic representation of the maternal and fetal
DNA fragments in maternal plasma. Furthermore, if both the
maternal and the fetal genomes are evenly represented in maternal
plasma, the proportional contribution of plasma DNA sequences
per chromosome should in turn bear correlation with the relative
size of each chromosome in the human genome. If the % chrN
values could be determined precisely enough by sequencing and
counting a large enough pool of plasma DNA sequences, we
hypothesize that we would be able to discriminate perturbations in
the quantitative representation of sequences mapped to the aneu-
ploid chromosomes in a maternal plasma sample from a pregnancy
involving a fetus with the said aneuploidy. We set out to test each
of these assumptions.
Detection of Fetal DNA in Maternal Plasma. If MPGS could sequence
fetal DNA in maternal plasma, one should be able to detect chrY
males and one female) were processed using the beta ChIP-Seq
protocol from Illumina, which included amplification of the adap-
tor-ligated DNA fragments both before and after (i.e., two rounds
of amplification) a gel electrophoresis-based size fractionation step
as described in supporting information (SI) Text.
sively parallel genomic sequencing for the noninvasive prenatal detection of
fetal chromosomal aneuploidy. Fetal DNA (thick red fragments) circulates in
maternal plasma as a minor population among a high background of mater-
nal DNA (black fragments). A sample containing a representative profile of
DNA molecules in maternal plasma is obtained. In this study, one end of each
plasma DNA molecule was sequenced for 36 bp using the Solexa sequencing-
by-synthesis approach. The chromosomal origin of each 36-bp sequence was
each chromosome was counted and then expressed as a percentage of all
unique sequences generated for the sample, termed % chrN for chromosome
N. Z-scores for each chromosome and each test sample were calculated using
the formula shown. The z-score of a potentially aneuploid chromosome is
expected to be higher for pregnancies with an aneuploid fetus (cases E–H
Schematic illustration of the procedural framework for using mas-
Chiu et al.
December 23, 2008 ?
vol. 105 ?
no. 51 ?
The clinical information and sequenced counts for these four
samples are shown in Table S1. The total number of sequences
obtained from each sample was ?9 ? 106. The total U0–1-0–0
counts ranged from ?1.8 ? 106to 2.0 ? 106per case. The
percentages of the U0–1-0–0 counts mapped to each chromosome
are shown in Fig. S1. For the three pregnancies with male fetuses,
i.e., cases 3009, 3034, and 3143, the absolute and fractional (in
parentheses) U0–1–0–0 counts mapped to chrY were 636
(0.032%), 858 (0.048%), and 1,054 (0.056%), respectively. How-
ever, it was unexpected that 177 (0.009%) sequences were also
mapped to chrY in the sample involving a female fetus. Real-time
PCR for the SRY gene (8) was negative for this latter plasma
sample. We next considered that contamination from male se-
quences might occur during the gel electrophoresis.
prepare plasma DNA samples for MPGS whereby the gel electro-
original protocols were compared and denoted as protocols A and
B, respectively. To minimize the chance of bias in the sequencing
results caused by low DNA input, 100 ng of DNA were extracted
processed by either protocol and sequenced in the same manner.
The tested plasma samples included one from a pregnant woman
fetus, and one that was a mixture of plasma from two male
individuals. A mixture was required for the last sample so that 100
ng of DNA could be obtained. The three samples were named
samples 1, 2, and 3, respectively.
The clinical details and sequencing counts for each sample and
each protocol are shown in Table S2. The total U0–1–0–0 counts
ranged from 2.0 ? 106to 2.2 ? 106. The absolute and fractional
U0–1–0–0 counts (in parentheses) mapped to chrY for samples 1,
and 3,523 (0.175%), respectively. The corresponding numbers for
the original protocol were 218 (0.011%), 1,615 (0.077%), and 3,468
(0.169%), respectively. Thus, contamination attributable predom-
inantly to the gel purification and the second amplification steps
could not be substantiated.
We next explored if there might be a bioinformatic explanation.
We used the Basic Local Alignment Search Tool (BLAST) to
analyze each of the U0–1–0–0 sequences mapped to chrY for each
of the three samples and for both protocols. We assessed the
proportion of those DNA sequences that could genuinely be
aligned just to chrY using BLAST. The proportion of sequences
aligned uniquely to chrY by BLAST was comparable for both the
new and the original protocols (Table S3). For the plasma sample
obtained from the pregnancy with a female fetus, only ?30% of
to chrY by BLAST. This was in contrast to samples 2 and 3, where
?90% of the sequences mapped to chrY by ELAND could be
the plasma sample from a pregnancy with a male fetus confirmed
that fetal DNA in maternal plasma could be sequenced by MPGS.
To confirm that there was little mapping error among the
U0–1–0–0 sequences aligned by the ELAND software, we per-
formed a BLAST analysis on 120 randomly selected U0–1–0–0
sequences for each of the other chromosomes for the three plasma
to the corresponding chromosome by BLAST. All 120 chrX se-
quences mapped by ELAND were confirmed by BLAST in sample
1, which was composed of female DNA only. More than 97% of
chrX sequences mapped by ELAND were confirmed by BLAST in
that U0–1–0–0 sequences mapped by the ELAND software were
generally accurate with chrY being the exception.
Distribution of Maternal Plasma DNA Sequences Among the Human
each chromosome among the total U0–1–0–0 sequences were
calculated for samples 1, 2, and 3. To investigate if maternal plasma
we compared the plasma DNA data with the expected genomic
contribution of each chromosome. Our main goal was to analyze
was female. Thus, we calculated the relative genomic representa-
tion, i.e., size, of each chromosome, on the basis of the nucleotide
content of each chromosome within a repeat-masked haploid
reference human genome of a female. The relative size of each
contribution of U0–1–0–0 sequences of the sequenced plasma
protocol, i.e., samples 1A, 2A, and 3A, bore closer resemblances to
the expected genomic representation of each human chromosome
than the corresponding aliquot processed by the original protocol,
i.e., samples 1B, 2B, and 3B. We performed linear regression
analyses to compare the % U0–1–0–0 per chromosome obtained
from both the new and the original protocols against the expected
genomic representation of each chromosome in the human ge-
involving a male fetus (sample 2), and a mixture of plasma from two adult males (sample 3) processed using the new (protocol A) and original (protocol B)
protocols. The percentage of genomic representation of each chromosome as expected for a repeat-masked reference haploid female genome was plotted as
a reference (black bars).
Bar chart of % U0–1–0–0 sequences per chromosome for a maternal plasma sample involving a female fetus (sample 1), a maternal plasma sample
www.pnas.org?cgi?doi?10.1073?pnas.0810641105 Chiu et al.
nome. As shown in Fig. S2, the slopes of the lines obtained from
samples 1A, 2A, and 3A were ?0.95, while those for samples 1B,
for samples 1A, 2A, and 3A but was 0.803, 0.840, and 0.910 for
samples 1B, 2B, and 3B, respectively. These data objectively con-
firmed that the DNA processing protocol with just one PCR
amplification step and the omission of the gel electrophoresis
procedure produced a quantitative profile of sequences that better
resembled the genomic content of each human chromosome than
the original protocol. More importantly, these data suggested that
the overall distribution of DNA molecules in maternal plasma
(inclusive of maternal and fetal DNA) across the human genome
was quite even. The chromosomal distribution of DNA molecules
of adult male plasma (sample 3A). This observation suggested that
it would be unlikely for the maternal and fetal DNA sequences in
maternal plasma to bear significant discrepancies among their
genomic distributions. Otherwise, if the genomic representation of
the maternal DNA differed substantially from that of fetal DNA,
one would expect the overall genomic representation to be dis-
crepant from that of a nonpregnant human plasma DNA sample.
if fetal chromosomal aneuploidy would lead to quantitative per-
turbations in the percentage contribution in aligned sequences for
the aneuploid chromosome. Plasma samples were obtained in the
first and second trimesters of pregnancies from 14 women each
pregnant with a euploid fetus and 14 women each pregnant with a
T21 fetus. The chromosomal status of the fetuses was confirmed by
full karyotyping. Plasma DNAs from the 28 pregnancies (median
sequenced. The clinical details and sequencing counts for each
sample are shown in Table S5. The 28 samples were processed as
The mean number of sequence reads generated per sample was
10.8 ? 106. The mean U0–1–0–0 count was 2.5 ? 106. The
percentage contributions of U0–1–0–0 sequences to each chro-
mosome were plotted against the percentage of genomic represen-
tation per chromosome of the human genome as described above
and are shown in Fig. S3. The data for chr21 and chrX are further
shown in Fig. 3A. The percentage of U0–1–0–0 sequences aligned
to chr21 was slightly higher for all T21 than for euploid cases. The
% chrX was much higher and the % chrY was much lower for all
female than male fetuses.
sequences of the T21 fetuses, we used the data from the 10 euploid
in % U0–1–0–0 per chromosome. The reference population was
restricted to euploid male fetuses so that an expected increase in %
chrX could also be explored in female fetuses. Using these refer-
ence values, we calculated the z-scores for each of the chromo-
3B. All of the T21 cases had a z-score of ?3 (range 5.03–25.11) for
chr21, i.e., at 3 standard deviations above the reference established
from the euploid male fetuses. The cases with female fetuses had
within ?3 for all 28 cases.
Reproducibility of Measuring Percentage of Chromosome Representa-
tion. Among the 28 tested maternal plasma samples, we expected a
difference in % chr21 representation between the T21 and euploid
fetuses and the % chrX representation between the female and
difference in % chr21 representation, which translated to a large
z-score difference but a large absolute difference in % chrX
among the respective cases (Fig. 3). The absolute differences in
chrX counts between female and male fetuses were expected to be
much larger than the difference in chr21 counts between T21 and
euploid fetuses. This was because there was a 2-fold increase in the
dosage of chrX for a female than a male individual, but just a
Furthermore, chrX is much larger than chr21 and contributed to a
a mean of 3.2 ? 104for chr21 in all samples.
expressed as the number of SDs from the mean of a reference data
set, we postulated that the SD was small for the measurement of %
set was in fact reflecting the precision of its measurement, we used
the data from the 10 euploid male fetuses to calculate the coeffi-
cient of variation (CV ? SD/mean ? 100%) of measuring the
percentage of representation of each chromosome. As shown in
Table S6, chr21 had the third lowest CV (0.54%) among all
chromosomes while the CV for the % chrX measurement was
3.10%. As the absolute number of U0–1–0–0 sequences counted
for chrX was threefold higher than that for chr21, the number of
sequences counted could not explain the variation in the precision.
We therefore explored the relationship between the CV in %
U0–1–0–0 counts and the GC content of each chromosome (Fig.
S5). Human chromosomes can be distributed into five groups with
lowest levels while group V chromosomes have the highest levels of
GC content. Interestingly, there was a statistically significant dif-
ference (P ? 0.001, ANOVA) in the CVs for the five groups of
group V was significantly higher (P ? 0.05) than that for the other
four groups. The CV for group IV and group I was each signifi-
cantly higher (P ? 0.05) than for both groups II and III.
We have demonstrated that MPGS can be used as a diagnostic tool
in noninvasive prenatal diagnosis. We have shown that differences
in amounts of chr21 DNA sequences in maternal plasma contrib-
and chromosome X for 28 maternal plasma samples. The sample numbers
correspond to the cases described in Table S5.
Plot of (A) % U0–1–0–0 counts and (B) z-scores for chromosome 21
Chiu et al.
December 23, 2008 ?
vol. 105 ?
no. 51 ?
uted by T21 fetuses compared with euploid fetuses can be unam-
biguously detected. Absolute differences in amounts of chrX and
chrY DNA sequences in maternal plasma contributed by male
The ability of MPGS to differentiate small quantitative perturba-
tions in genomic distributions of chromosomes lies in the very large
number of molecules analyzed, which minimizes the imprecision of
the quantitative measurement. As no specific gene locus was
targeted, all plasma DNA fragments together provide an unprec-
edented number of molecules analyzed per plasma sample.
This approach is in marked contrast to previous methods that
quantified only DNA molecules that could serve as templates for
locus-specific PCR assays, for example, SRY on chrY (8). The gene
locus-specific DNA templates represent only an extremely small
proportion of DNA fragments present in maternal plasma. In fact,
MPGS is such a powerful tool for quantifying the relative genomic
representation of plasma DNA molecules that only an amount
corresponding to just a representative fraction of the human
genome would need to be sequenced. For example, ?10 million
36-bp reads were generated for each plasma sample, which was
this study, only the U0–1–0–0 sequences, representing just ?20%
of all of the reads sequenced from each plasma DNA sample, were
Thus, this is quite unlike some previously described sequencing-
based methods for quantitative nucleic acid profiling that relied on
sequencing at high fold coverage (21), for example, to determine
the relative abundance of RNA species in transcriptome analysis
(21). On the contrary, our present method simply sequences a
of DNA fragments are sequenced once, if at all. The relative
chromosome size is then deduced by counting the relative number
fragments would be of a different nucleotide sequence. In fact, the
pool of DNA sequenced for a sample would vary from run to run.
Despite the randomness of the sequencing, the quantitative
estimation of % chr21 sequences was so precise and robust that the
z-scores for chr21 of the T21 pregnancies were markedly different
from the mean of a reference euploid sample set. In this study, the
median gestational age of the T21 pregnancies (14.1 weeks) was
comparable with the median of the euploid group (15.4 weeks). All
samples from the euploid group were collected before any invasive
procedure in the present pregnancy. Blood samples from 11 of the
T21 pregnancies were collected immediately before pregnancy
termination at a median of 6 days (range: 2–22 days) after invasive
prenatal diagnostic procedure. Our previous study (22) indicated
that there would be no substantial difference in the fetal DNA
theless, blood samples from 3 T21 pregnancies, cases 17, 19, and 25
(Table S5), were collected in the first trimester before chorionic
villus sampling. Increases in their z-scores for chr21 were readily
identifiable (Fig. 3B).
Theoretically, the determination of the presence of quantitative
perturbations in any particular chromosome could be achieved
more precisely, for example, by taking into account the fetal DNA
concentration to estimate the expected degree of chromosomal
perturbation. Fetal DNA concentrations can be readily measured
using either fetal epigenetic markers (23) or paternally inherited
polymorphic markers (24). In this study, the fetal DNA concen-
tration of each case was not required to derive cutoff values for
determining the disease status of each case. First, according to
Table S6, chr21 is one of the chromosomes whose percentage of
representation could be measured at very low imprecision with our
current protocol. Second, when compared to methods like digital
RCD whereby disease cutoff values related to the fetal DNA
measured by sequencing. For digital RCD, we reported that for a
sample with 25% fetal DNA concentration, 7,680 digital PCRs
would need to be performed to achieve a correct classification rate
of 97%. Our previous data also showed that ?20% of the total
number of digital PCRs analyzed, equal to 1,536 chr21 molecules
for a 7,680-well experiment, would contain only the chr21 gene
target and hence be counted as informative. Thus, the number of
chr21 molecules (mean: 3.2 ? 104) analyzed by the sequencing
method is ?20-fold that of the digital RCD method. Hence, the
measurement would be significantly more precise than the present
scale of digital PCR analyses. However, by taking the fetal DNA
concentration into account, the measurement of the percentage of
chromosomal representation could be made more precisely for
some of the other chromosomes or across batches and hence
minimize false diagnoses.
In fact, the precision and accuracy of MPGS for determining the
genomic representation of maternal plasma DNA could be further
improved by a number of postsequencing analysis strategies. For
example, sequences occurring in regions of known copy number
variations (25) could be adjusted for so that the reference range for
euploid pregnancies might be even tighter. Sequences other than
U0–1–0–0, for example, with one or two mismatches to the
reference genome that, in some instances, may represent a poly-
morphic difference between the tested sample and the reference
human genome, may also be used to increase the number of usable
sequence counts. We have also shown that the reproducibility of
measuring the percentage contribution of plasma DNA sequences
varied between chromosomes and the GC content of the chromo-
some may partly explain this variability. Thus, the amount of
sequencing to be done per sample could be varied to ensure that
measurements could be made precisely enough for the detection of
quantitative perturbations of each of the other chromosomes. In
are counted, MPGS may be precise enough to detect quantitative
aberrations involving regions less than a whole chromosome.
We have found a discrepancy in the accuracy of ELAND
mapping for chrY when compared with other chromosomes. This
may be attributable to the known presence of many repetitive
sequences in chrY (26). Even after repeat masking, much of the
remaining chrY sequences are composed of low-copy-number
repeat sequences, which increases the difficulty of aligning such
sequences accurately. Nonetheless, for women carrying female
fetuses, there still appeared to be a small number of sequences
genuinely mapped to chrY. We had confirmed that these plasma
samples were negative for male DNA using a SRY real-time PCR
assay that was widely used in the field (8). The MPGS approach is
potentially much more sensitive than the real-time PCR approach.
Technically, it might mean that an extremely low level of carryover
contamination would be unavoidable in a laboratory environment
Alternatively, the sequences that appeared to be uniquely mapped
to chrY by both ELAND and BLAST may in fact be mappable to
male from the female fetuses just from the % chrY counts in
maternal plasma (Fig. S3F).
We have demonstrated the principle of the plasma DNA se-
same approach can also be used for the other platforms (16, 27).
The main limitation of the method described here is the relatively
high costs. Sequencing reagents alone cost $700 for each sample. A
for the Illumina Solexa platform at present has a throughput of 16
samples per week per instrument. However, it is expected that such
technology will rapidly become more affordable over the next few
years (17). In the interim period, one could potentially reduce the
cost by barcoding individual patients’ samples such that one se-
quencing reaction could generate diagnostic information for mul-
tiple cases. Alternatively, sequencing could be focused just on the
chromosomes of interest by array capture before random sequenc-
www.pnas.org?cgi?doi?10.1073?pnas.0810641105 Chiu et al.
ingofDNAfragmentsoriginatingfromthosechromosomes.Infact, Download full-text
other non-sequencing-based methods of single-molecule analyses
may be suitable for our application and may ultimately prove to be
more cost effective (28). To contain costs of a noninvasive prenatal
diagnostic program, one could also potentially combine the se-
quencing approach with for example, the RNA–SNP allelic ratio
approach (11) such that fetuses that are not heterozygous and thus
uninformative for the RNA–SNP approach could be analyzed by
the more expensive MPGS approach.
Finally, the data reported in this study need to be confirmed by
large-scale clinical trials. Ultimately it is hoped that noninvasive
prenatal diagnosis will make future prenatal testing safer for
use of MPGS for quantitative genomic sequencing in the form of
a diagnostic tool. We expect that the massively parallel plasma
DNA sequencing strategy described in this article could also be
used to analyze the various pathological conditions associated with
aberrations in plasma nucleic acids, e.g., cancer.
While this article was under review, Fan et al. also reported the
use of Solexa sequencing for fetal chromosomal aneuploidy diag-
nosis (29). These authors analyzed 18 maternal plasma samples, of
villus sampling. The median gestational age of the T21 group (18
weeks) was in fact older than that of the euploid group (12 weeks).
As fetal DNA release into the maternal circulation increases
significantly within the immediate period of invasive procedures
(30) and with pregnancy progression, the potential confounding
effects of these factors need to be considered. Nonetheless, the
report by Fan et al. and our study independently demonstrate the
feasibility of MPGS for noninvasive prenatal diagnosis.
Materials and Methods
Details are in the SI Text.
plasma DNA were used for DNA library construction by the beta Chromatin
Immunoprecipitation Sequencing (ChIP-Seq) sample preparation kit (Illumina)
according to the manufacturer’s instructions except when specifically noted
electrophoresis. The selected DNA libraries were then additionally amplified
using a 15-cycle PCR. After the first experiments, all other experiments in this
purification kit (Qiagen).
Sequence Alignment. All 36-bp sequence reads were aligned to the repeat-
masked human genomic reference sequences (NCBI Build 36, version 48) down-
ELAND program in the GAPipeline-0.2.2.5 software package provided by Illu-
on the third field of the output file indicated that the best match found was a
codes 1 in the fourth, 0 in the fifth, and 0 in the sixth fields (hence U0–1–0–0)
indicated that it had just a single exact match in the repeat-masked human
reference genome without any nucleotide mismatch. U0–1–0–0 sequences in
sorted sequences were used for further analysis.
Calculation of the Genomic Representation of Each Chromosome in the Reference
Human Genome. Except for the genomic representation values used as reference
in Fig. S2C (sample 3), the expected chromosome size for a haploid female
genome was calculated as described here. The reference sequences (NCBI Build
the Ensembl Genome Browser (http://www.ensembl.org). All sequences were
subjected to repeat masking and the number of remaining nucleotides was
counted per chromosome. The expected percentage of representation of each
chromosome by the total repeat-masked nucleotide counts of all chromosomes
without including chrY. The reference values used in Fig. S2C were derived by
including the repeat-masked nucleotide counts of chrY to obtain the total
repeat-masked genome size.
ACKNOWLEDGMENTS. We thank Rebecca Chan, Macy Heung, Yongjie Jin, Yu
Kwan Tong, and Dana Tsui for technical assistance. We thank the Information
Technology Services Center of The Chinese University of Hong Kong and the
Center for High Performance Computing at Virginia Commonwealth University
of the Government of the Hong Kong Special Administration Region, China,
under the Areas of Excellence Scheme (AoE/M-04/06) and a sponsored research
scheme of the Li Ka Shing Foundation.
1. Tabor A, et al. (1986) Randomised controlled trial of genetic amniocentesis in 4606
low-risk women. Lancet 1:1287–1293.
2. Malone FD, et al. (2005) First-trimester or second-trimester screening, or both, for
Down’s syndrome. N Engl J Med 353:2001–2011.
3. Bianchi DW, et al. (1990) Isolation of fetal DNA from nucleated erythrocytes in
maternal blood. Proc Natl Acad Sci USA 87:3279–3283.
thalassaemia by analysis of fetal cells in maternal blood. Nat Genet 14:264–268.
5. Bianchi DW, et al. (2002) Fetal gender and aneuploidy detection using fetal cells in
maternal blood: analysis of NIFTY I data. National Institute of Child Health and
Development Fetal Cell Isolation Study. Prenat Diagn 22:609–615.
6. Lo YMD, et al. (1997) Presence of fetal DNA in maternal plasma and serum. Lancet
7. Lo YMD, Chiu RWK (2007) Prenatal diagnosis: progress through plasma nucleic acids.
Nat Rev Genet 8:71–77.
implications for noninvasive prenatal diagnosis. Am J Hum Genet 62:768–775.
Proc Natl Acad Sci USA 100:4748–4753.
10. Oudejans CB, et al. (2003) Detection of chromosome 21-encoded mRNA of placental
origin in maternal plasma. Clin Chem 49:1445–1449.
11. Lo YMD, et al. (2007) Plasma placental RNA allelic ratio permits noninvasive prenatal
chromosomal aneuploidy detection. Nat Med 13:218–223.
12. Chim SSC, et al. (2008) Systematic search for placental epigenetic markers on chromo-
some 21: towards noninvasive prenatal diagnosis of fetal trisomy 21. Clin Chem
13. Old RW, Crea F, Puszyk W, Hulten MA (2007) Candidate epigenetic biomarkers for
non-invasive prenatal diagnosis of Down syndrome. Reprod Biomed Online 15:227–
allelic ratio analysis in maternal plasma: theoretical and empirical considerations. Clin
15. Lo YMD, et al. (2007) Digital PCR for the molecular detection of fetal chromosomal
aneuploidy. Proc Natl Acad Sci USA 104:13116–13121.
16. Margulies M, et al. (2005) Genome sequencing in microfabricated high-density pico-
litre reactors. Nature 437:376–380.
17. Schuster SC (2008) Next-generation sequencing transforms today’s biology. Nat Meth-
18. Dear PH (2003) One by one: single molecule tools for genomics. Brief Funct Genomic
19. Chan KCA, et al. (2004) Size distributions of maternal and fetal DNA in maternal
plasma. Clin Chem 50:88–92.
20. Kel-Margoulis OV, et al. (2003) Composition-sensitive analysis of the human genome
for regulatory signals. In Silico Biol 3:145–171.
21. Reinartz J, et al. (2002) Massively parallel signature sequencing (MPSS) as a tool for
in-depth quantitative gene expression profiling in all organisms. Brief Funct Genomic
22. Lo YMD, et al. (1999) Increased fetal DNA concentrations in the plasma of pregnant
women carrying fetuses with trisomy 21. Clin Chem 45:1747–1751.
23. Chan KC, et al. (2006) Hypermethylated RASSF1A in maternal plasma: a universal fetal
24. Lun FMF, et al. (2008) Microfluidics digital PCR reveals a higher than expected fraction
of fetal DNA in maternal plasma. Clin Chem 54:1664–1672.
25. Redon R, et al. (2006) Global variation in copy number in the human genome. Nature
26. Skaletsky H, et al. (2003) The male-specific region of the human Y chromosome is a
mosaic of discrete sequence classes. Nature 423:825–837.
27. Harris TD, et al. (2008) Single-molecule DNA sequencing of a viral genome. Science
28. Geiss GK, et al. (2008) Direct multiplexed measurement of gene expression with
color-coded probe pairs. Nat Biotechnol 26:317–325.
29. Fan HC, et al. (2008) Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing
DNA from maternal blood. Proc Natl Acad Sci USA 42:16266–16271.
Clin Chem 49:1193–1195.
Chiu et al.
December 23, 2008 ?
vol. 105 ?
no. 51 ?