Clinical Chemistry 56:8
`1279–1286 (2010)
`
`Molecular Diagnostics and Genetics
`
`Analysis of the Size Distributions of Fetal and Maternal
`Cell-Free DNA by Paired-End Sequencing
`H. Christina Fan,1 Yair J. Blumenfeld,2 Usha Chitkara,2 Louanne Hudgins,3 and Stephen R. Quake1*
`
`BACKGROUND: Noninvasive prenatal diagnosis with
`cell-free DNA in maternal plasma is challenging be-
`cause only a small portion of the DNA sample is de-
`rived from the fetus. A few previous studies provided
`size-range estimates of maternal and fetal DNA, but
`direct measurement of the size distributions is difficult
`because of the small quantity of cell-free DNA.
`
`METHODS: We used high-throughput paired-end se-
`quencing to directly measure the size distributions of
`maternal and fetal DNA in cell-free maternal plasma
`collected from 3 typical diploid and 4 aneuploid male
`pregnancies. As a control, restriction fragments of ␭
`DNA were also sequenced.
`
`RESULTS: Cell-free DNA had a dominant peak at ap-
`proximately 162 bp and a minor peak at approximately
`340 bp. Chromosome Y sequences were rarely longer
`than 250 bp but were present in sizes of ⬍150 bp at a
`larger proportion compared with the rest of the se-
`quences. Selective analysis of the shortest fragments
`generally increased the fetal DNA fraction but did not
`necessarily increase the sensitivity of aneuploidy detec-
`tion, owing to the reduction in the number of DNA
`molecules being counted. Restriction fragments of ␭
`DNA with sizes between 60 bp and 120 bp were prefer-
`entially sequenced, indicating that the shotgun se-
`quencing work flow introduced a bias toward shorter
`fragments.
`
`CONCLUSIONS: Our results confirm that fetal DNA is
`shorter than maternal DNA. The enrichment of fetal
`DNA by size selection, however, may not provide a
`dramatic increase in sensitivity for assays that rely on
`length measurement in situ because of a trade-off be-
`tween the fetal DNA fraction and the number of mol-
`ecules being counted.
`© 2010 American Association for Clinical Chemistry
`
`Traditional methods of prenatal diagnosis of genetic
`disorders use materials obtained by amniocentesis or
`chorionic villus sampling, invasive procedures that
`carry a small but clear risk of miscarriage (1 ). The dis-
`covery of cell-free fetal nucleic acids in the plasma of
`pregnant mothers has led to the development of several
`noninvasive prenatal diagnostic techniques in the past
`decade (2 ). The detection of fetal aneuploidy and au-
`tosomal recessive disorders with cell-free nucleic acids
`is particularly challenging because only a small portion
`of the cell-free nucleic acids in maternal plasma is
`derived from the fetus. We recently demonstrated
`noninvasive detection of fetal aneuploidy by high-
`throughput shotgun sequencing of cell-free DNA (3 ),
`and an independent group quickly reproduced our re-
`sults (4, 5 ). For almost all prenatal diagnostic assays,
`the background of maternal DNA provides a practical
`limit on sensitivity, and therefore the fraction of fetal
`DNA present in the maternal plasma is a critical pa-
`rameter. There is evidence that fetal DNA is shorter on
`balance than maternal DNA, and therefore substantial
`effort has been invested in developing methods to en-
`rich for fetal DNA (6, 7 ). Extracting fractions of lower
`molecular weight DNA with electrophoretic tech-
`niques or the use of smaller PCR amplicons could in-
`crease the fraction of fetal DNA, and such methods
`have been used to improve the detection of fetal point
`mutations and the determination of fetal genotypes
`(8 –12 ).
`Paired-end sequencing is a technique that obtains
`sequence information for both ends of each DNA mol-
`ecule. By finding the coordinates of the 2 sequences on
`the genome through sequence alignment, one can de-
`duce the length of the DNA fragment. A single se-
`quencing experiment yields sequence and size infor-
`mation for tens of millions of DNA fragments. In this
`study, we used high-throughput paired-end sequenc-
`ing of cell-free DNA in maternal plasma to study the
`length distributions of
`fetal and maternal DNA.
`Paired-end sequencing enabled us to directly measure
`
`1 Department of Bioengineering, Stanford University and Howard Hughes Medical
`Institute, Stanford, CA; 2 Division of Maternal-Fetal Medicine, Department of
`Obstetrics and Gynecology, Stanford University, Stanford, CA; 3 Division of
`Medical Genetics, Department of Pediatrics, Stanford University, Stanford, CA.
`* Address correspondence to this author at: Department of Bioengineering,
`
`Stanford University and Howard Hughes Medical Institute, 318 Campus Dr.,
`Clark Center, Room E300, Stanford, CA 94305. Fax 650-736-1961; e-mail
`quake@stanford.edu.
`Received January 28, 2010; accepted May 10, 2010.
`Previously published online at DOI: 10.1373/clinchem.2010.144188
`
`1279
`
`00001
`
`EX1059
`
`

`

`the size distributions of maternal DNA and fetal DNA
`with single-base resolution from cell-free DNA col-
`lected from women carrying male fetuses, without the
`need to pool samples and with much higher precision
`than can be obtained by gel electrophoresis or via the
`PCR. Our data confirm that fetal DNA is shorter than
`maternal DNA and is predominantly within the size
`range of a mononucleosome. We demonstrated that
`the shotgun sequencing work flow introduces a bias
`toward shorter fragments, a phenomenon that effec-
`tively enriches the fetal DNA fraction. Finally, by
`selectively analyzing only the shortest fragments, we
`showed that there is a delicate trade-off in sensitivity in
`fetal aneuploidy detection between the fetal DNA frac-
`tion and the number of molecules counted.
`
`Materials and Methods
`
`SAMPLE PROCESSING
`Blood samples were collected at the Lucile Packard
`Children’s Hospital (Stanford University), with in-
`formed consent obtained under an institutional review
`board–approved study. Maternal blood samples from
`7 pregnancies with male fetuses, including 2 cases of
`trisomy 21, a case of trisomy 13, and a case of trisomy
`18, were selected for this study. These samples were
`collected at gestational ages of 12–23 weeks. Plasma
`was first separated from the blood cells by centrifuga-
`tion at 1600g at 4 °C for 10 min. The plasma was then
`centrifuged at 16 000g for 10 min at room temperature
`to remove residual cells. DNA was extracted from 1.6 –
`2.4 mL of cell-free plasma with the NucleoSpin Plasma
`F Kit (Macherey-Nagel; purchased from E&K Scien-
`tific). To measure the quantity of cell-free DNA, we
`performed real-time TaqMan PCR assays specific for a
`chromosome 1 locus and a chromosome Y locus (3 ).
`To investigate the fragment length– dependent se-
`quencing bias, we prepared a restriction digest of ␭
`DNA (Invitrogen). ␭ DNA was digested with AluI, a
`4-bp cutter, for 2 h at 37 °C. Thedigest was then heated
`at 65 °C for 20 min to inactivate the enzyme. The digest
`was purified with the aid of a QIAquick PCR Purifica-
`tion Kit (Qiagen), and 5 ng of the purified DNA was
`used to construct the sequencing library.
`
`SEQUENCING LIBRARY CONSTRUCTION
`A combination of the protocols detailed in Kozarewa et
`al. (13 ) and Fan et al. (3 ) were used to construct Illu-
`mina sequencing libraries. To preserve the original
`length of plasma DNA, we performed no fragmenta-
`tion procedures. Full-length paired-end sequencing
`adaptors were ligated directly onto end-polished,
`A-tailed double-stranded plasma DNA. The adaptors
`were purified by HPLC and treated with T4 polynucle-
`otide kinase to phosphorylate the 5⬘ ends. The final
`
`1280 Clinical Chemistry 56:8 (2010)
`
`concentration of the adaptors in the ligation reaction
`was 800 pmol/L. The libraries were amplified with 12
`cycles of the PCR. No agarose gel purification was per-
`formed. A Bioanalyzer (Agilent Technologies) and the
`High Sensitivity DNA Kit were used to analyze the li-
`braries. The libraries were quantified by traditional
`real-time TaqMan PCR assays with human-specific
`primers and by digital PCR (Fluidigm) with a universal
`template assay (14 ) designed for paired-end libraries.
`Details of the library-preparation protocols and adap-
`tor sequences can be found in the Data Supplement
`files that accompany the online version of this article at
`http://www.clinchem.org/content/vol56/issue8.
`
`SEQUENCING
`Libraries were sequenced on the Genome Analyzer II
`(Illumina) according to the manufacturer’s instruc-
`tions. Thirty-two bases at each end were sequenced.
`
`SEQUENCE ALIGNMENT
`Image analysis, base calling, and alignment were per-
`formed with Illumina’s Pipeline software (version
`1.4.0). For the plasma DNA libraries, we used the
`ELAND_PAIR option to map the first 25 bases of each
`sequenced end to the reference human genome (NCBI
`Build 36).
`For the alignment of ␭DNA digest, the first 2 cy-
`cles on both ends were omitted because they corre-
`sponded to the restriction site sequences and because
`the domination of certain bases in the first cycle caused
`calibration problems in the image analysis software.
`The sequences were mapped to the genome of ␭DNA
`(GenBank accession no. J02459).
`The Pipeline software outputs files that provide
`information that included the sequence of a read, the
`chromosome, the coordinate on the forward strand to
`which the 5⬘ end of a read mapped with at most 2 mis-
`matches, and the coordinate offset if the paired read
`also mapped to the same chromosome.
`
`DATA ANALYSIS
`Custom Python and MATLAB scripts were written for
`further analysis of the data. The absolute value of the
`coordinate offset plus 25 bases was interpreted as the
`length of the sequenced DNA fragment. We used only
`reads that had one end mapped to the forward strand
`and one end mapped to the reverse strand. In addition,
`for paired reads with the first read mapped to the for-
`ward strand, the offset value in principle should be ⬎0,
`whereas for paired reads with the first read mapped to
`the reverse strand, the offset value should be ⬍0 (see
`Fig. 1 in the online Data Supplement). Reads that did
`not follow this rule were filtered out.
`For ␭DNA sequences, we counted the number of
`reads mapped to each restriction site and ignored sites
`
`00002
`
`

`

`Paired-End Sequencing of Maternal Plasma DNA
`
`fit
`data
`
`100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400
`Fragment size (bp)
`
`3
`
`2.5
`
`2
`
`1.5
`
`1
`
`0.5
`
`0
`
`0
`
`Number of reads
`
`Fig. 1. Effect of library preparation and sequencing on the length distribution of DNA.
`␭ DNA was digested with AluI and then paired-end sequenced. The number of sequenced fragments is plotted against length.
`Each black dot represents the mean number of reads in every 20-bp bin. The red line is a locally weighted logistic regression
`fit.
`
`with restriction fragment lengths of ⬍30 bp (because
`25 bp was used for alignment). The data were divided
`into 20-bp bins from 30 bp to 2500 bp. For each 20-bp
`bin, we calculated the number of reads for all restric-
`tion digest fragments falling within the 20-bp bin and
`divided it by the number of restriction digests within
`the bin. We fitted the data by locally weighted logistic
`regression.
`To measure the length distributions of maternal
`and fetal DNA, we tallied the number of reads that had
`sizes between 30 bp and 510 bp in 20-bp intervals for
`each chromosome. We applied weighting to each data
`point by using the fitted data of ␭ DNA to correct for
`the length-dependent sequencing bias. For each 20-bp
`bin, we calculated the -fold increase in fetal DNA frac-
`tion as:
`
`fi冒冘
`ti冒冘
`
`i
`
`fi
`
`ti
`
`,
`
`i
`
`where fi is the count of fetal (chromosome Y) sequences
`within the ith bin and ti is the count of all sequences
`within the ith bin.
`
`As in our previous study (3 ), we observed a GC
`bias in read coverage. To reduce the effect of such bias,
`we followed the procedures outlined by Fan and Quake
`(15 ). Overrepresentation and underrepresentation of
`chromosomes were measured, and the fetal fraction
`was estimated from the depletion of chromosome X
`sequences and/or the overabundance of chromosome
`18, 13, or 21, as described in our previous study (3 ).
`
`Results
`
`ANALYSIS OF LENGTH-DEPENDENT BIAS OF ILLUMINA
`
`SEQUENCING
`We used the restriction digest of ␭ DNA to study the
`effects of library preparation and sequencing on the
`length distribution of DNA. We prepared a sequencing
`library from AluI-digested ␭DNA that had a total DNA
`amount similar to that of the plasma DNA samples.
`Sequencing on a single lane of the flow cell yielded ap-
`proximately 14 ⫻ 106 paired-end reads, 97% of which
`were mapped to restriction sites with the predicted
`fragment length and used for subsequent analysis (see
`Table 1 in the online Data Supplement). Fig. 1 is a plot
`of the number of reads vs restriction fragment length.
`Bins with 60 –120 bp had the most reads. The number
`
`Clinical Chemistry 56:8 (2010) 1281
`
`00003
`
`

`

`P48
`
`100 200 300 400 500
`
`P59
`
`100 200 300 400 500
`
`P73
`
`100 200 300 400 500
`
`P75
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`P52
`
`100 200 300 400 500
`
`P64
`
`100 200 300 400 500
`
`P74
`
`100 200 300 400 500
`
`autosomes
`chrY
`chrX
`
`100 200 300 400 500
`
`Fragment length (bp)
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`Percent of reads
`
`Fig. 2. Length distributions of fetal (chromosome Y)
`and total DNA sequenced from 7 samples of maternal
`plasma.
`chrY, chromosome Y.
`
`vided the reads into 3 groups by size: 30 –150 bp, 150 –
`170 bp, and 170 – 600 bp. Each group represented ap-
`proximately one-third of the total paired reads.
`The fetal DNA percentage was calculated for all
`samples from the underrepresentation of chromosome
`X and/or the overrepresentation of trisomic chromo-
`somes for all reads and for each size fraction, after cor-
`rection for GC bias (Table 1). The fetal DNA percent-
`age for the fraction of ⬍150 bp was generally higher (by
`a factor of approximately 1.2–2) than the overall fetal
`DNA percentage (when all reads were taken into ac-
`count), whereas for the fractions ⬎150 bp, the fetal
`DNA percentage was lower than the overall value (Fig.
`3). Thus, selecting reads of ⬍150 bp was able to enrich
`the fetal DNA fraction.
`We calculated the z statistic, a measure that reflects
`the confidence in the deviation of the representation of
`
`of reads decreased rapidly as the fragment size in-
`creased. Very few fragments ⬎1 kb were sequenced.
`
`SIZE DISTRIBUTION OF TOTAL AND FETAL DNA IN MATERNAL
`
`PLASMA DETERMINED BY PAIRED-END SEQUENCING
`With real-time PCR, we determined the concentra-
`tions of cell-free plasma DNA in the 7 sequenced sam-
`ples to be within 0.7–5.6 ␮g/L plasma (assuming 6.6
`pg/genome). DYS14, a chromosome Y–specific se-
`quence, was detected in all samples from male fetus
`pregnancies and was not detected in a female genomic
`DNA control.
`Table 2 in the online Data Supplement presents
`statistics for the paired-end sequencing run and details
`of the plasma samples. The mean number of total reads
`was approximately 19 ⫻ 106, with about 52% (i.e.,
`10 ⫻ 106 reads) having both ends mapped to 2 unique
`locations on a single chromosome with no more than 2
`mismatches. Paired-end reads mapped to the forward
`and reverse strands in equal proportions. We filtered
`out reads that had ends mapped to the same strand and
`reads that did not have reasonable offset values (i.e.,
`values that were too large compared with the upper
`limit of the amplicon size for a PCR reaction). The
`remaining reads (approximately 99.5% of all paired
`reads) were used for downstream analyses.
`The mean number of chromosome Y reads was
`approximately 13 000, which is equivalent to approxi-
`mately 0.1% of the total paired-end reads. Fig. 2 pre-
`sents the size distribution of sequenced cell-free DNA
`according to the chromosomes. Sizes ranged from 30 to
`510 bp in 20-bp bins. The median length was 162 bp.
`We applied weighting to the length distribution by us-
`ing values of the Loess fit from Fig. 1. The dominant
`peak was approximately 162 bp, approximately the size
`of a monochromatosome. A minor peak at approxi-
`mately 340 bp, approximately the size of a dichroma-
`tosome, was also observed.
`We observed that the size distribution for chro-
`mosome Y was shifted for most samples toward the
`shorter end, compared with the other chromosomes
`(Fig. 2). Very few chromosome Y sequences had the
`length of a dichromatosome. Additionally, there were
`slightly more chromosome Y sequences with lengths
`shorter than that of a monochromatosome. One can
`enrich the fraction of fetal DNA by a factor of approx-
`imately 1.5 by targeting sequences shorter than 150 bp
`(Fig. 3).
`
`FETAL DNA FRACTION AND ANEUPLOIDY DETECTION IN
`
`DIFFERENT SIZE FRACTIONS
`Because chromosome Y sequences appeared to be
`shorter (Fig. 2), we investigated whether selecting reads
`that had shorter lengths would increase the fetal DNA
`fraction and improve aneuploidy detection. We di-
`
`1282 Clinical Chemistry 56:8 (2010)
`
`00004
`
`

`

`Paired-End Sequencing of Maternal Plasma DNA
`
`50
`
`100
`
`150
`
`200
`
`250
`
`300
`
`350
`
`400
`
`450
`
`500
`
`Fragment size (bp)
`
`2
`
`1.8
`
`1.6
`
`1.4
`
`1.2
`
`1
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`
`0
`0
`
`Relative enrichment of fetal (chrY) DNA
`
`Fig. 3. Relative increase or decrease in the fetal DNA fraction from 30 bp to 510 bp at 20-bp intervals.
`We used locally weighted logistic regression to visualize the trend (solid line). Each patient sample is represented by a different
`symbol and a differently colored solid line. The vertical dashed lines represent the size cutoffs used to divide the reads into 3
`portions with approximately equal numbers of reads. chrY, chromosome Y.
`
`a chromosome from normal. Because the statistic
`also depends on the number of reads being consid-
`ered, we randomly selected a third of the total reads
`within a sample for comparison. This random selec-
`tion of reads had fragment sizes that represented the
`overall length distribution in the cell-free DNA sam-
`ple. Although the fetal fraction and relative chromo-
`
`some copy number were highest for the fraction of
`⬍150 bp, as observed by the increase in the deviation
`of the relative copy number of chromosome X and
`trisomic chromosomes from 1.0 (Fig. 4A), the mag-
`nitude of the z statistic was not always the highest. In
`4 of the 7 cases, the sensitivity was highest when all
`fractions were used (Fig. 4B).
`
`Table 1. Size distribution of fetal and total DNA in maternal plasma and fetal DNA percentages in different
`size fractions.
`
`Median length, bp
`
`Fetal DNA estimated from
`chrX, %
`
`Fetal DNA estimated from trisomic
`chr, %
`
`Sample
`
`Karyotype
`
`Non-chrYa
`
`ChrY
`
`P48
`P52
`P59
`P64
`P73
`P74
`P75
`
`46XY
`47XY⫹21
`47XY⫹18
`47XY⫹13
`46XY
`47XY⫹21
`46XY
`
`a chrY, chromosome Y.
`
`164
`161
`165
`164
`159
`164
`164
`
`163
`153
`162
`155
`148
`159
`152
`
`All
`reads
`
`6.64
`21.06
`42.20
`6.78
`24.72
`16.44
`15.16
`
`0–150
`bp
`
`150–170
`bp
`
`170–600
`bp
`
`5.72
`30.30
`46.90
`13.08
`37.46
`16.52
`25.32
`
`4.00
`13.32
`36.88
`2.68
`14.38
`11.48
`6.92
`
`8.28
`15.04
`38.34
`2.76
`12.20
`14.32
`6.44
`
`All
`reads
`
`—
`21.46
`42.34
`12.72
`—
`16.82
`—
`
`0–150
`bp
`
`150–170
`bp
`
`170–600
`bp
`
`—
`32.08
`52.48
`22.88
`—
`22.18
`—
`
`—
`14.58
`41.54
`9.22
`—
`16.20
`—
`
`—
`15.16
`34.72
`5.12
`—
`10.16
`—
`
`Clinical Chemistry 56:8 (2010) 1283
`
`00005
`
`

`

`1.3
`
`1.2
`
`1.1
`
`1
`
`0.9
`
`0.8
`
`A
`
`Relative chromosome copy number
`
`0.7
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`-10
`
`-20
`
`-30
`
`-40
`
`-50
`
`-60
`
`-70
`
`B
`
`z-Statistic
`
`P73 (46X Y) (150-–170 bp)
`
`P59 (47X Y+18) (150–170 bp)P64 (47X Y+13) (170–600 bp)
`
`P52 (47X Y+21) (150–170 bp)P52 (47X Y+21) (170–600 bp)
`P59 (47X Y+18) (170–600 bp)
`P64 (47X Y+13) (150–170 bp)
`P74 (47X Y+21) (150–170 bp)
`P73 (46X Y) (170–600 bp)
`P75 (46X Y) (150–170 bp)
`P75 (46X Y) (170–600 bp)
`P74 (47X Y+21) (30–150 bp)
`P52 (47X Y+21) (30–150 bp)
`P64 (47X Y+13) (30–150 bp)
`P75 (46X Y) (30–150 bp)
`P73 (46X Y) (30–150 bp)
`P59 (47X Y+18) (30–150 bp)
`P48 (46X Y) (30–150 bp)
`P52 (47X Y+21) (all)
`P48 (46X Y) (all)
`P59 (47X Y+18) (all)
`P64 (47X Y+13) (all)
`P73 (46X Y) (all)
`P75 (46X Y) (all)
`P74 (47X Y+21) (all)
`P59 (47X Y+18) (rando m)
`P74 (47X Y+21) (170–600 bp)
`P64 (47X Y+13) (rando m)
`P52 (47X Y+21) (rando m)
`P74 (47X Y+21) (rando m)
`P73 (46X Y) (rando m)
`P75 (46X Y) (rando m)
`P48 (46X Y) (rando m)
`P48 (46X Y) (150–170 bp)
`P48 (46X Y) (170–600 bp)
`
`Sample (karyotype) (length of sequences used for analysis)
`
`chr1
`chr2
`chr3
`chr4
`chr5
`chr6
`chr7
`chr8
`chr9
`chr10
`chr11
`chr12
`chr13
`chr14
`chr15
`chr16
`chr17
`chr18
`chr19
`chr20
`chr21
`chr22
`chrX
`
`chr1
`chr2
`chr3
`chr4
`chr5
`chr6
`chr7
`chr8
`chr9
`chr10
`chr11
`chr12
`chr13
`chr14
`chr15
`chr16
`chr17
`chr18
`chr19
`chr20
`chr21
`chr22
`chrX
`alpha = 0.001
`
`P52 (47X Y+21) (150–170 bp)
`P64 (47X Y+13) (150–170 bp)
`P74 (47X Y+21) (150–170 bp)
`P52 (47X Y+21) (170–600 bp)
`P64 (47X Y+13) (170–600 bp)
`P74 (47X Y+21) (170–600 bp)
`P59 (47X Y+18) (150–170 bp)
`P59 (47X Y+18) (170–600 bp)
`P52 (47X Y+21) (30–150 bp)
`P64 (47X Y+13) (30–150 bp)
`P74 (47X Y+21) (30–150 bp)
`P59 (47X Y+18) (30–150 bp)
`
`P48 (46X Y) (150–170 bp)P48 (46X Y) (170–600 bp)
`P75 (46X Y) (150–170 bp)
`P73 (46X Y) (30–150 bp)
`P73 (46X Y) (170–600 bp)
`P73 (46X Y) (150–170 bp)
`P75 (46X Y) (30–150 bp)
`P48 (46X Y) (30–150 bp)
`P52 (47X Y+21) (all)
`P74 (47X Y+21) (all)
`P59 (47X Y+18) (all)
`P64 (47X Y+13) (all)
`P48 (46X Y) (all)
`P75 (46X Y) (all)
`P73 (46X Y) (all)
`P59 (47X Y+18) (rando m)
`P64 (47X Y+13) (rando m)
`P74 (47X Y+21) (rando m)
`P73 (46X Y) (rando m)
`P52 (47X Y+21) (rando m)
`P75 (46X Y) (rando m)
`P48 (46X Y) (rando m)
`P75 (46X Y) (170–600 bp)
`
`Sample (karyotype) (length of sequences used for analysis)
`
`Fig. 4. Copy number for each chromosome relative to the diploid condition and confidence in the measurement of
`chromosome representation in the different size fractions.
`(A), Copy number of each chromosome relative to the diploid condition in different size fractions. Chromosome X (chrX) is
`underrepresented for all 7 male pregnancies. Chromosomes 13, 18, and 21 are overrepresented in the corresponding cases of
`trisomy 13, 18, and 21. The degree of deviation from the typical diploid representation is generally larger when analyzing reads
`⬍150 bp, compared with randomly selecting the similar number of reads from all reads, whereas the deviation decreases as
`the length of the analyzed reads increases. (B), Confidence in the measurement of chromosome representation in different size
`fractions. The horizontal dashed lines represents the 99.99% confidence level (2-tailed) for the detection of over- or
`underrepresentation of chromosomes. In 4 of the 7 cases, the sensitivity is the highest when all fractions are used for analysis,
`although the fetal DNA percentage is generally higher in the size fraction of ⬍150 bp.
`
`1284 Clinical Chemistry 56:8 (2010)
`
`00006
`
`

`

`Paired-End Sequencing of Maternal Plasma DNA
`
`Discussion
`
`We have demonstrated the direct measurement of the
`size distributions of maternal and fetal DNA in mater-
`nal plasma. A few recent studies also used traditional
`sequencing and 454 pyrosequencing to study the size
`distributions and profiles of cell-free DNA in healthy
`individuals and cancer patients (16 –18 ). We also at-
`tempted to measure the size distribution of maternal
`cell-free DNA with 454 pyrosequencing (3 ). The re-
`sults of these studies were in agreement that cell-free
`DNA has a peak size of 160 –180 bp and that this DNA
`derives mostly from apoptotic cells. In this study, we
`used paired-end sequencing on the Illumina platform,
`which has a much higher throughput than the 454 plat-
`form with respect to the number of reads sequenced.
`The large number of reads enabled us to characterize
`the size distribution of not only maternal DNA but also
`chromosome Y sequences, which constituted only ap-
`proximately 0.1% of all the sequences in a maternal
`plasma DNA sample. Our sequencing approach, how-
`ever, could measure only the distribution in the lower
`molecular weight range because higher molecular
`weight species (⬎1 kb) undergo attrition in the current
`preparation of sequencing samples. Because previous
`experiments with the PCR and gel electrophoresis have
`shown that the majority of fetal DNA had sizes ⬍500
`bp, the current measurement approach should capture
`the size distribution of most fetal DNA. We noted that
`Southern blots of maternal plasma DNA revealed the
`presence of DNA with sizes ⬎20 kb (7 ). Future exper-
`iments with the newly developed mate pair sample-
`preparation technique, which allows an insert size of
`⬎2 kb(19 ), should give a detailed size estimate of the
`higher molecular weight species.
`In our previous study, the estimates of the fraction
`of fetal DNA obtained from sequencing data ranged
`from 8% to 40%, higher than the estimates from our
`own digital PCR measurements before sequencing li-
`brary preparation and the estimates of ⬍10% observed
`by others (20 ). Our explanations at that time had 2
`components: (a) According to the studies of Li et al. (7 )
`and Chan et al. (6 ), fetal DNA in maternal plasma is
`shorter than the maternal DNA counterpart; and (b)
`our sequencing method involved PCR amplifications
`with universal primers during library preparation and
`cluster generation. The PCR is known to have a higher
`efficiency for lower molecular weight species. We spec-
`ulated that the increased fraction of fetal DNA mea-
`sured from the sequencing data was an artifact of the
`sequencing method we chose to use.
`In this study, we experimentally validated both
`components of our argument. By sequencing restric-
`tion digests of ␭DNA, we discovered that lower molec-
`ular weight species were overrepresented. In addition,
`
`our sequencing measurements of the size distributions
`of maternal and fetal DNA (Fig. 2) agreed with previ-
`ous findings that fetal DNA is mostly shorter than 300
`bp, whereas a portion of maternal DNA is ⬎300 bp in
`size (6, 7 ). These observations suggested that the pro-
`cess of sequencing maternal plasma DNA with the Il-
`lumina platform increased the representation of the
`shorter fetal DNA species, thereby increasing the fetal
`DNA fraction.
`Since the discovery that fetal DNA is generally
`shorter than maternal DNA in maternal plasma, a
`number of techniques have been developed to enrich
`fetal DNA fraction by size selection. These techniques
`have included traditional gel electrophoresis (7, 21 ),
`combinations of PCR assays with amplicons of differ-
`ent lengths (11 ), and microchip separation (22 ). Be-
`cause the length bias of the shotgun sequencing reads
`was suspected to derive from the PCR, one could po-
`tentially ligate universal adaptors to the ends of plasma
`DNA and then perform PCR amplification against the
`universal sequences to enrich the fetal DNA fraction. In
`using these 2 approaches, it is important that the
`plasma DNA not be fragmented by nebulization or
`sonication so that the original size distribution of the
`DNA can be preserved.
`The sensitivity of fetal aneuploidy detection via the
`counting of single DNA molecules depends on both the
`fetal DNA fraction and the number of molecules
`counted. Aneuploidy is more confidently detected if
`the fetal DNA fraction is high and a large number of
`molecules are counted. Our results show that although
`the fetal DNA fraction is increased in the shortest frag-
`ments (Fig. 4A), the fact that the total number of mol-
`ecules being counted is smaller negatively affects the
`confidence of detection (Fig. 4B). Therefore, “infor-
`matic” enrichment of length fragments by digital size
`selection in samples such as those collected early dur-
`ing the gestation when fetal DNA fraction is generally
`low, whether by paired-end sequencing or by digital
`PCR (11, 23 ), may not yield any appreciable gain in the
`sensitivity of aneuploidy detection and should be used
`with caution. Whether one can gain sensitivity in an-
`euploidy detection depends on the initial fetal DNA
`fraction, the magnitude of the increase in the fetal DNA
`fraction obtained by size selection, and the number of
`molecules retained after size selection. One situation in
`which we can imagine digital size selection being quite
`useful is when samples have been obtained subopti-
`mally. For instance, if the processing of blood samples
`is delayed, maternal lymphocytes will lyse and artifi-
`cially decrease the fetal fraction by contaminating the
`sample with longer fragments of maternal genomic
`DNA. These longer fragments potentially can be ex-
`cluded without reducing the number of fetal fragments
`used in the analysis.
`
`Clinical Chemistry 56:8 (2010) 1285
`
`00007
`
`

`

`In conclusion, we have shown that paired-end se-
`quencing allows the direct measurement of the length
`distribution of cell-free fetal DNA in maternal plasma,
`with single-base resolution. The process of Illumina
`sequencing introduced a bias in the length distribution
`of the sequenced sample that increased the representa-
`tion of fetal DNA. Selecting sequenced reads with
`lengths ⬍150 bp could further increase the fetal DNA
`fraction but would not necessarily increase the sensi-
`tivity of aneuploidy detection by single-molecule
`counting. We envision that the rapid advances in se-
`quencing and related technologies will enable the real-
`ization of many novel techniques for the study of cell-
`free nucleic acids, not only for prenatal diagnosis but
`also for early cancer diagnosis.
`
`or revising the article for intellectual content; and (c) final approval of
`the published article.
`
`Authors’ Disclosures of Potential Conflicts of Interest: Upon
`manuscript submission, all authors completed the Disclosures of Poten-
`tial Conflict of Interest form. Potential conflicts of interest:
`
`Employment or Leadership: None declared.
`Consultant or Advisory Role: S.R. Quake, Fluidigm Corporation,
`Helicos Biosciences Corporation, and Artemis Health.
`Stock Ownership: S.R. Quake, Fluidigm Corporation, Helicos Bio-
`sciences Corporation, and Artemis Health.
`Honoraria: None declared.
`Research Funding: The work was supported by the Wallace H.
`Coulter Foundation and the National Institutes of Health Director’s
`Pioneer Award. H.C. Fan, a Stanford Graduate Fellowship and an
`award from the Siebel Scholars Foundation.
`Expert Testimony: None declared.
`
`Role of Sponsor: The funding organizations played no role in the
`design of study, choice of enrolled patients, review and interpretation
`of data, or preparation or approval of manuscript.
`
`Author Contributions: All authors confirmed they have contributed to
`the intellectual content of this paper and have met the following 3 re-
`quirements: (a) significant contributions to the conception and design,
`acquisition of data, or analysis and interpretation of data; (b) drafting
`
`Acknowledgments: We thank the Division of Perinatal Genetics and
`General Clinical Research Center of Stanford University for patient
`recruitment and enrollment. We also thank Norma Neff for her help
`in performing sequencing experiments.
`
`1. American College of Obstetricians and Gynecol-
`ogists. ACOG Practice Bulletin No. 88, December
`2007. Invasive prenatal testing for aneuploidy.
`Obstet Gynecol 2007;110:1459 – 67.
`2. Dennis Lo YM, Chiu RW. Prenatal diagnosis:
`progress through plasma nucleic acids. Nat Rev
`Genet 2007;8:71–7.
`3. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L,
`Quake SR. Noninvasive diagnosis of fetal aneu-
`ploidy by shotgun sequencing DNA from maternal
`blood. Proc Nat Acad Sci U S A 2008;105:16266 –
`71.
`4. Chiu RW, Chan KC, Gao Y, Lau VY, Zheng W,
`Leung TY, et al. Noninvasive prenatal diagnosis
`of fetal chromosomal aneuploidy by massively
`parallel genomic sequencing of DNA in maternal
`plasma. Proc Nat Acad Sci U S A 2008;105:
`20458 – 63.
`5. Chiu RW, Sun H, Akolekar R, Clouser C, Lee C,
`McKernan K, et al. Maternal plasma DNA analysis
`with massively parallel sequencing by ligation for
`noninvasive prenatal diagnosis of trisomy 21. Clin
`Chem 2010;56:459 – 63.
`6. Chan KC, Zhang J, Hui AB, Wong N, Lau TK,
`Leung TN, et al. Size distributions of maternal and
`fetal DNA in maternal plasma. Clin Chem 2004;
`50:88 –92.
`7. Li Y, Zimmermann B, Rusterholz C, Kang A, Hol-
`zgreve W, Hahn S. Size separation of circulatory
`DNA in maternal plasma permits ready detection
`of fetal DNA polymorphisms. Clin Chem 2004;50:
`1002–11.
`8. Li Y, Di Naro E, Vitucci A, Grill S, Zhong XY,
`Holzgreve W, Hahn S. Size fractionation of cell-
`free DNA in maternal plasma improves the de-
`
`References
`
`tection of a paternally inherited beta-thalassemia
`point mutation by MALDI-TOF mass spectrome-
`try. Fetal Diagn Ther 2009;25:246 –9.
`9. Li Y, Holzgreve W, Page-Christiaens GC, Gille JJ,
`Hahn S. Improved prenatal detection of a fetal
`point mutation for achondroplasia by the use of
`size-fractionated circulatory DNA in maternal
`plasma— case report. Prenat Diagn 2004;24:
`896 – 8.
`10. Li Y, Wenzel F, Holzgreve W, Hahn S. Genotyping
`fetal paternally inherited SNPs by MALDI-TOF MS
`using cell-free fetal DNA in maternal plasma:
`influence of size fractionation. Electrophoresis
`2006;27:3889 –96.
`11. Lun FM, Tsui NB, Chan KC, Leung TY, Lau TK,
`Charoenkwan P, et al. Noninvasive prenatal di-
`agnosis of monogenic diseases by digital size
`selection and relative mutation dosage on DNA in
`mate