Genome-wide scans using archived neonatal dried blood spot samples.

Abstract

Identification of disease susceptible genes requires access to DNA from numerous well-characterised subjects. Archived residual dried blood spot samples from national newborn screening programs may provide DNA from entire populations and medical registries the corresponding clinical information. The amount of DNA available in these samples is however rarely sufficient for reliable genome-wide scans, and whole-genome amplification may thus be necessary. This study assess the quality of DNA obtained from different amplification protocols by evaluating fidelity and robustness of the genotyping of 610,000 single nucleotide polymorphisms, using the Illumina Infinium HD Human610-Quad BeadChip. Whole-genome amplified DNA from 24 neonatal dried blood spot samples stored between 15 to 25 years was tested, and high-quality genomic DNA from 8 of the same individuals was used as reference.
Using 3.2 mm disks from dried blood spot samples the optimal DNA-extraction and amplification protocol resulted in call-rates between 99.15% - 99.73% (mean 99.56%, N = 16), and conflicts with reference DNA in only three per 10,000 genotype calls.
Whole-genome amplified DNA from archived neonatal dried blood spot samples can be used for reliable genome-wide scans and is a cost-efficient alternative to collecting new samples.

Full text

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BioMed Central
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BMC Genomics
Open Access
Methodology article
Genome-wide scans using archived neonatal dried blood spot
samples
Mads V Hollegaard1,2, Jonas Grauholm3, Anders Børglum4, Mette Nyegaard4,
Bent Nørgaard-Pedersen1, Torben Ørntoft3,5, Preben B Mortensen6,
Carsten Wiuf7, Ole Mors8, Michael Didriksen9, Poul Thorsen10 and
David M Hougaard*1
Address: 1Section of Neonatal Screening and Hormones, Statens Serum Institut, Copenhagen, DK-2300, Denmark, 2Department of Epidemiology,
University of Aarhus, Aarhus, DK-8000, Denmark, 3AROS Applied Biotechnology A/S, Aarhus, DK-8000, Denmark, 4Institute of Human Genetics,
University of Aarhus, Aarhus, DK-8000, Denmark, 5Department of Clinical Biochemistry, Skejby Sygehus, Aarhus, DK-8000, Denmark, 6The
National Centre for Register Based Research, University of Aarhus, Aarhus, DK-8000, Denmark, 7Bioinformatics Research Center, University of
Aarhus, Aarhus, DK-8000, Denmark, 8Centre for Psychiatric Research, Aarhus University Hospital Risskov, Aarhus, DK-8000, Denmark, 9Lundbeck
A/S, Taastrup, DK-2630, Denmark and 10Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, USA
Email: Mads V Hollegaard - mvh@ssi.dk; Jonas Grauholm - jg@arosab.com; Anders Børglum - anders@humgen.au.dk;
Mette Nyegaard - nyegaard@humgen.au.dk; Bent Nørgaard-Pedersen - bnp@ssi.dk; Torben Ørntoft - orntoft@ki.au.dk;
Preben B Mortensen - pbm@ncrr.dk; Carsten Wiuf - wiuf@birc.au.dk; Ole Mors - om@psykiatri.aaa.dk;
Michael Didriksen - mdi@lundbeck.com; Poul Thorsen - pt@soci.au.dk; David M Hougaard* - dh@ssi.dk
* Corresponding author
Abstract
Background: Identification of disease susceptible genes requires access to DNA from numerous
well-characterised subjects. Archived residual dried blood spot samples from national newborn
screening programs may provide DNA from entire populations and medical registries the
corresponding clinical information. The amount of DNA available in these samples is however
rarely sufficient for reliable genome-wide scans, and whole-genome amplification may thus be
necessary. This study assess the quality of DNA obtained from different amplification protocols by
evaluating fidelity and robustness of the genotyping of 610,000 single nucleotide polymorphisms,
using the Illumina Infinium HD Human610-Quad BeadChip. Whole-genome amplified DNA from
24 neonatal dried blood spot samples stored between 15 to 25 years was tested, and high-quality
genomic DNA from 8 of the same individuals was used as reference.
Results: Using 3.2 mm disks from dried blood spot samples the optimal DNA-extraction and
amplification protocol resulted in call-rates between 99.15% – 99.73% (mean 99.56%, N = 16), and
conflicts with reference DNA in only three per 10,000 genotype calls.
Conclusion: Whole-genome amplified DNA from archived neonatal dried blood spot samples can
be used for reliable genome-wide scans and is a cost-efficient alternative to collecting new samples.
Published: 4 July 2009
BMC Genomics 2009, 10:297doi:10.1186/1471-2164-10-297
Received: 21 November 2008
Accepted: 4 July 2009
This article is available from: http://www.biomedcentral.com/1471-2164/10/297
© 2009 Hollegaard et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Studies of genetic influence in complex disorders usually
require extensive genome explorations of large cohorts. A
major bottleneck, however, is access to DNA from well-
characterised patients and healthy controls. This may be
circumvented by use of archived residual blood samples
from newborn screening programs, which in several coun-
tries engage the entire population. The blood is usually
collected by heel-prick and applied on special filter paper,
a proven robust and convenient medium for transport
and storage [1]. Storage policies on residual neonatal
dried blood spot samples (DBSS) vary internationally, but
several countries store residuals in repositories for later
research purposes [2-8]. Stored DBSS combined with rel-
evant clinical information from medical registries thus
constitute an ideal resource for large studies. This set-up
enjoys the advantage of representing the entire popula-
tion under a certain age and of avoiding practically any
kind of selection. In addition substantial costs may be
saved.
The Danish Neonatal Screening Biobank (DNSB) con-
tains nearly 2 million DBSS from virtually every Dane
born after 1982. It has recently been updated to meet the
new general guidelines for the establishment and opera-
tion of biobanks [9]. Access to samples for research
requires approval from the Scientific Ethical Committee
System, the Data Protection Agency and the DNSB Steer-
ing Committee. In Denmark, all citizens have a unique
person-identifying number that is used across all public
registration systems, including the DNSB. Denmark also
operates a well-established public health care system
offering treatment to all citizens. Together this makes it
possible to study the "entire country as a cohort" and
makes the DNSB an ideal resource for studying common
and complex genetic diseases in Caucasians [10]. The
major challenge using the DBSS for such studies is how-
ever the small amount of blood available. In theory, the
amount of genomic DNA (gDNA) that can be extracted
from a 3.2 mm punch of a DBSS is about 60 ng [11]. In
general, only one or two 3.2 mm punches per DBSS can
be reserved for each project, which is scarcely enough to
genotype multiple single nucleotide polymorphisms
(SNP). This problem may be overcome by whole-genome
amplification (WGA) of the gDNA. Previous studies have
used whole-genome amplified DNA (wgaDNA) for geno-
typing, and with fair success, but in most cases the
number of polymorphisms that can be tested has been
limited [11-18].
In this study we investigate if a proper combination of
DNA-extraction and WGA procedure can produce
wgaDNA samples suitable for 610,000 SNP genome-wide
scan using the Illumina Infinium HD Human610-Quad
BeadChip. Neonatal DBSS stored for 15 to 25 years in the
DNSB are employed, and as reference is used high-quality
gDNA samples recently obtained from the same individu-
als. Two different WGA methods are tested. The multi-dis-
placement amplification (MDA) method (the REPLI-g kit)
that produces relatively long wgaDNA fragments > 10 kb
[19], and the OmniPlex method (the GPlex2 and the
GPlex4 kits) that produces fragments approximately 500
bp long [20]. We also test the effect of using either one or
three 3.2 mm disks and of extracting proteins from the
disks before the DNA-extraction. Finally, the robustness
of the selected approaches was evaluated.
Methods
Subjects
The investigation comprised 24 subjects born between
1982 and 1992, who all had their residual neonatal DBSS
stored at -24°C in the DNSB. Four subjects were informed
volunteers and 20 were from a genetic study on schizo-
phrenia (ethical approval number: 20020020; data pro-
tection agency number: 2002-41-2059).
DNA-extraction and WGA methods
Reference gDNA was purified from venous blood samples
from the four volunteers and from four subjects from the
schizophrenia study using the Maxwell 16 automatic sys-
tem and the Maxwell® 16 Blood DNA Purification Kit
(Promega). Neonatal DBSS from the eight participants
were retrieved from the DNSB, and DNA was extracted
from one or three DBSS disks, 3.2 mm in diameter, using
Extract-N-Amp Blood PCR Kit (ENA)(extraction volume:
200 mL) (Sigma-Aldrich) or QIAamp DNA Blood Micro
Kit (QIA)(extraction volume: 75 mL) (Qiagen). The DNA
extracts were amplified using the REPLI-g kit (Qiagen),
GenomePlex® Complete WGA Kit (GPlex2, Sigma-
Aldrich), or GenomePlex® Single Cell Whole Genome
Amplification Kit (GPlex4, Sigma-Aldrich). All procedures
were performed according to the manufacturer's instruc-
tions. Prior to DNA-extraction, a subset of disks was
extracted for proteins as described by Skogstrand et al.
2005 [21]. Please consult Additional file 1 for set up. Fur-
thermore, two DBSS disks from 16 other subjects were
extracted for proteins before DNA-extraction using the
ENA kit, and the DNA extracts were amplified using the
REPLI-g and the GPlex4 kits. DNA was quantified using
Quant-iT™ PicoGreen® dsDNA Reagent (Molecular
Probes, Invitrogen).
Genotyping
The gDNA and wgaDNA samples were marked on the Illu-
mina Infinium HD Human610-Quad BeadChip (Illu-
mina) according to the manufacturer's instructions, with
the exception that 240 ng of wgaDNA starting material
was used instead of the prescribed 200 ng. The BeadChips
were scanned using the BeadStation 500GX (Illumina)
with a high-density upgrade and an AutoLoader (Illu-

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mina). The BeadStudio v.3 software (Illumina Corp.) was
used for calculating call- and conflict-rates. In the first part
of the study all calls were made using the reference
Human610-Quadv1B cluster file from Illumina that is
constructed from gDNA. In the second part of the study
two cluster files, each constructed from 16 wgaDNA prep-
arations made by the REPLI-g and GPlex4 kits (tailored
cluster files specific for WGA method), were also used to
analyse the respective samples. Conflict-rates were esti-
mated comparing the wgaDNA samples to their respective
reference gDNA samples. The percentage of conflicts
introduced due to an allelic dropout (eg. AB to AA) was
estimated by re-coding the Illumina data to variables
allowing comparison using STATA v.9.0.
Results
The genotyping performance of the different wgaDNA
preparations is seen in Additional file 1. The ENA DNA-
extraction combined with the REPLI-g kit WGA featured
the highest call-rates (99.30–99.51%) and the lowest con-
flict-rates (0.02–0.03%).
Combining REPLI-g WGA with QIA DNA-extraction was
less successful and the results were highly variable. The
genotyping performance using wgaDNA made by the two
OmniPlex method kits, GPlex2 and GPlex4, was inde-
pendent of the DNA-extraction method, with GPlex4
showing consistently higher call rates than GPlex2 (Wil-
coxon's paired test, p < 0.001). The reference gDNA call-
rates were 99.8–99.9%. Almost all conflicts between
results from the wgaDNA preparations and the reference
gDNA were due to an allelic dropout (data not shown).
Notably, extraction and amplification procedures that
produced high call-rates displayed low conflict-rates with
the reference gDNA and vice versa, which indicates that
genome-wide scans on wgaDNA are reliable when the
call-rates are high [Additional file 1]. This was partially
confirmed by calculating the correlation coefficients
between the call- and conflict-rates of the three WGA kits
[Additional file 1]. It made no significant difference
whether one or three DBSS disks were used for extraction.
No systematic differences in genotyping performance
were related to sample age.
Based on the results displayed in Additional file 1, the
combinations of DNA extraction by the ENA kit and WGA
by the REPLI-g and GPlex4 kits were selected for further
evaluation. For this, 16 new subjects were employed. After
DNA-extraction, WGA and subsequent genome-wide
scans (GWS), the results were analysed using both a stand-
ard Human610-Quadv1B Cluster, provided by Illumina,
and WGA kit specific tailored cluster files. The rationale
for the tailored cluster files is demonstrated in Figure 1.
Generally, the wgaDNA samples cluster nicely, but not
always in the area defined by the Illumina Human610-
Quadv1B cluster file, which is based on gDNA samples. By
creating tailored WGA-specific cluster files and using these
for analysis, the genotype call-rates of both set-ups
(REPLI-g and GPlex4) improved significantly (Wilcoxon
paired test, p < 0.001) as seen in Table 1. Comparison of
the call-rates indicated that the REPLI-g samples had a sig-
nificantly higher call-rate than the GPlex4 samples (Wil-
coxon's paired test, p < 0.001). Comparison of the
Plot of the normalized values measure for the A allele and B allele
Figure 1
Plot of the normalized values measure for the A allele and B allele. The same 16 samples were amplified using the
GPlex4 and the REPLI-g WGA kits. The "Illumina cluster" plot shows how the GPlex4 (blue dots) and the REPLI-g (green dots)
wgaDNA genotypes compare to the Illumina cluster file. The "GPlex4 cluster" plot shows how a custom-made cluster file
based on GPlex4 samples (blue dots) improves both fit and call-rate of the loci. The "REPLI-g cluster" plot shows how a cus-
tom-made cluster file based on REPLI-g samples (green dots) improves both fit and call-rate of the loci.

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amount of wgaDNA amplified by each kit revealed no sig-
nificant difference (Wilcoxon's paired test, p > 0.050).
Discussion
We demonstrate that wgaDNA, made from 3.2 mm disks
of DBSS that have been stored at -24°C for more than 20
years is well suited for reliable genotyping of 610,000
SNPs, with call-rates comparable to those obtained using
gDNA. The accuracy of genotype calls using wgaDNA
from stored DBSS has been of some concern. The issue has
been addressed several times, using both low and
medium throughput genotyping platforms, and overall
with good success [11,12,14-18]. In this study we took the
usage of DBSS one step further by conducting GWS. More-
over the accuracy of genotype calls from wgaDNA was
assessed by comparing the results with results from high-
quality reference gDNA from the same individuals. Ini-
tially, we tested two commercial DNA-extraction proce-
dures, three WGA procedures, the effect of number of 3.2
mm disks used, and the effect of protein extraction prior
to the gDNA extraction. The efficiency and reliability of
the GWS were highly dependent on the employed DNA-
extraction and WGA method. Interestingly, call- and con-
flict-rates were inversely related; indicating that genome
scan of wgaDNA is highly reliable when the call-rates are
close to 100%. However because only few samples were
available to calculate the correlation coefficient, we can-
not clearly define a cutoff for the call-rate that would
ensure reliable genotyping. In general, the OmniPlex
method performed more constantly than the MDA
method, producing fairly the same call-and conflict-rates
independently of the other variables tested. Of the two
OmniPlex based kits the GPlex4 kit performed the best,
showing high call-rates and low error-rates. The MDA
method performed excellent using the ENA extraction kit
and poorly when using the QIA extraction kit. In general,
it appeared unimportant whether one or three DBSS disks
were used for extraction. This was surprising since the
amount of input gDNA for the WGA reactions is supposed
to be critical, and in our set-up it was often below the
lower limit of 10 ng that is recommended by the manufac-
turer. Moreover, the preceding protein extraction ofthe
disks did not impair the genotyping of the produced
wgaDNA, which is in accordance with similar observa-
tions from our laboratory [17].
Because the investigation focuses ondifferent combina-
tions of wgaDNA preparation, it suffers from the weak-
ness that the number of samples in each group is limited.
In addition, only samples from the DNSB were used.
The combination of the ENA DNA-extraction with either
the REPLI-g or the Gplex4 WGA kit were selected to see if
the procedures were robust enough for GWA studies
employing numerous samples. Both set-ups produced
wgaDNA from 16 DBSS stored for 15 to 25 years that per-
formed well with constant high call-rates. Corresponding
reference gDNA samples were not available. Notably,
when calling genotypes of wgaDNA preparations with the
BeadStudio software, albeit clusters were nice and tight for
Table 1: Robustness of the GPlex4 and REPLI-g WGA kits.
ID GPlex4REPLI-g
Call-rate A1
Call-rate B2
WGAout3
Call-rate A1
Call-rate B2
WGAout3
1
2
3
4
5
6
7
8
9
97.74%
97.41%
98.21%
97.80%
97.90%
98.04%
97.83%
97.62%
97.56%
97.30%
98.17%
98.13%
96.62%
97.55%
96.75%
96.53%
99.24%
99.30%
99.42%
99.38%
99.41%
99.42%
99.38%
99.29%
99.29%
99.26%
99.32%
99.38%
99.08%
99.35%
99.17%
99.16%
4.86
4.85
5.06
4.93
5.20
4.92
5.00
5.01
5.15
5.33
5.41
5.49
5.15
5.48
5.14
4.13
99.42%
99.56%
98.54%
99.14%
99.00%
99.73%
99.72%
99.49%
99.55%
95.91%
99.43%
99.38%
99.43%
99.22%
98.97%
98.49%
99.65%
99.64%
99.33%
99.56%
99.51%
99.73%
99.73%
99.66%
99.64%
99.15%
99.60%
99.56%
99.60%
99.63%
99.53%
99.43%
6.55
7.22
3.53
3.00
4.47
3.74
6.02
6.00
7.64
7.86
7.48
1.56
6.71
1.40
4.98
2.72
10
11
12
13
14
15
16
Median
Std. Dev.
97.68%
0.53%
99.31%
0.10%
5.10
0.33
99.40%
0.92%
99.60%
0.15%
5.49
2.18
1Call-rate (percent) using the Illumina Human610-Quadv1B cluster file.
2Call-rate (percent) using the WGA kit custom cluster file.
3wgaDNA (mg) produced per reaction.

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some loci they did not fit well into the standard cluster
positions. This is because the BeadStudio software calls
the genotypes of a given locus by comparing the observed
values with the expected values, defined by the
Human610-Quadv1B Cluster file, which is based on
gDNA samples [22,23]. In such cases, data fit and call-
rates can be improved by adjusting the cluster positions to
match the observed data [23]. Cluster files tailored for the
OmniPlex and MDA method were hence created from the
samples available, and the call-rates were significantly
improved for both wgaDNA preparations. They were in
fact comparable to call-rates obtained using high-quality
gDNA, indicating that the approach is robust.
Eighteen WGA reactions, each producing ~5 mg of
wgaDNA, can be made per ENA DNA extraction. As the
Illumina Infinium HD Human610-Quad BeadChip uses
240 ng of wgaDNA, one WGA reaction is enough to run
20 chips. Thus one to three 3.2 mm disks from a DBSS are
sufficient to make repeated GWS as well as fine-mapping
genotyping, if required. We have briefly tested the per-
formance of the two wgaDNA preparations on the
Affymetrix platform and found that wgaDNA produced by
the OmniPlex method was unsuitable, whereas wgaDNA
produced by the MDA method gave results comparable to
those obtained by the Illumina platform. In addition to
being used for GWS, DBSS can also be used for multiplex
protein measurements [21], quantitative RNA micro
arrays detecting up to 3000 genes [24], and quantitative
DNA methylation analysis [25].
Conclusion
The results demonstrate that residual DBSS from neonatal
screening that have been stored for several years in
biobanks can be used for GWS and hence for large
genome-wide association studies. Using DBSS instead of
collecting new samples may, in a cost-efficient way, reveal
important correlations between genotypes, environment
and human diseases. Both the OmniPlex and the MDA
method performed excellently in combination with the
ENA extraction, and we recommend to test which of the
two WGA procedures is most suitable for a given task.
Authors' contributions
MVH prepared the DNA samples, participated in the
design of the study and did the major part of data analysis
and drafting the manuscript. JG conducted the Illumina
GWS and participated in the data analysis. AB, MN, BNP,
TFO, PMB, CW, OM, MD and PT initiated the study and
participated in its design and the interpretation of results.
DMH initiated the study, participated in its design, the
interpretation of results and drafting the manuscript. All
authors were involved in a critical revising and approved
the final manuscript.
Additional material
Acknowledgements
The study was supported by grants from the Danish Strategic Research
Council (2101-07-0059) Stanley Medical Research Institute, the Danish
Medical Research Council (271-06-0368), and the Novo Nordisk Founda-
tion. None of the sponsors participated in any part of this manuscript or
study.
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Performance of different wgaDNA preparations. 1 Year the DBSS was
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Infinium Genotyping Data Analysis Illumina TechNote [http:/
/www.illumina.com/downloads/GTDataAnalysis_TechNote.pdf]
Infinium HD DNA Analysis BeadChips Illumina TechNote
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