Using Whole Genome Amplified (wga) Dna Samples In The Goldengateо ...
Illumina® SNP Genotyping
TECHNICAL NOTE
Using Whole-Genome Amplified (WGA) DNA
Samples in the GoldenGate® Genotyping Assay
INTRODUCTION
Illumina recognizes the need of some investigators to
use whole-genome amplified (WGA) DNA because of
limited sources of genomic DNA (gDNA) for their genetic
studies. An increasing number of publications are being
generated on the viability of WGA products. This docu-
ment provides information from publications and cus-
tomer feedback that will assist researchers considering
the use of WGA DNA samples for their genetic studies
using the GoldenGate genotyping assay.
PUBLICATIONS USING THE ILLUMINA PLATFORM
AND WGA PRODUCTS
The use of WGA products on the Illumina platform was
to note that the average starting concentration of the
first described by Barker et. al1. In this study, high quality,
genomic DNA input into the WGA reaction was clearly
intact DNA was used to assess the reliability of two meth-
lower in the failed samples (5.9 ng/μl) than in the 104
ods of WGA using Illumina’s GoldenGate assay. Multiple
samples which were successful (17.4 ng/μl). No mention
displacement amplification (REPLI-g®, Qiagen®, Inc.) and
was made as to the quality or age of the samples used for
OmniPlex® (Rubicon Genomics®) technologies were tested
the WGA reactions. While concordance rates and genotype
in five Centre d'Etudes du Polymorphisme Humain (CEPH)
call rates were not directly addressed, the authors did
DNA samples from Coriell Cell Repositories. Both meth-
demonstrate a crude error rate of 0.025% using the
ods yielded >99.8% concordance relative to gDNA samples
WGA-derived samples.
and >98% genotype call rates.
A later study by Pask et. al2 examined a larger cohort
IMPORTANT SUGGESTIONS FOR USING WGA SAMPLES
of 86 gDNA samples and their corresponding WGA prod-
ON THE ILLUMINA GOLDENGATE PLATFORM
ucts. In this study, WGA products were generated using
Based on the publications described above as well as
Φ29 polymerase Multiple Displacement Amplification.
customer feedback, Illumina has compiled the following
A concordance rate of 98.8% between the WGA and gDNA
list of important considerations when deciding to use
samples was observed, as well as a genotype call rate
WGA samples on the Illumina GoldenGate platform:
of 99.8%.
While both of these studies provide positive indicators
1. Know your DNA. The quality of DNA used in the
for using WGA products on the Illumina platform, it must
WGA reaction appears to be one of the most impor-
be noted that both studies used high quality, intact DNA
tant factors for the success of samples used with the
within the recommended ranges of the manufacturer’s
GoldenGate chemistry. High-quality, intact DNA has
specifications.
been shown to generate the best results. Sample
A more recent study also examined the use of WGA
preparation, storage conditions, length of storage,
samples on the Illumina platform. Sawcer et. al3 used 508
source of DNA, as well as a host of other factors can
WGA samples as part of their study. While only 104 of the
affect quality of DNA.
508 samples generated genotypes, the study was clear
making sense out of life
Illumina® SNP Genotyping
2. Use sufficient amounts of input DNA. Although
Staging, Inc. It is now sold under the same name by
some WGA methods describe successful whole
Qiagen (www.qiagen.com), catalog #59043.
genome amplification with smaller amounts of
The OmniPlex method is available as a service
starting DNA, Illumina suggests using a minimum
from Rubicon Genomics (www.rubicongenomics.com).
of 50 ng of intact, high quality DNA for input into
The kit is available for purchase from Sigma-Aldrich®
the WGA reaction. DNA suspected or known to have
(www.sigmaaldrich.com/genomeplex) under the
undergone degradation should be used in minimum
name GenomePlex®.
quantities of 100-200 ng. These suggestions are not
Illumina strongly suggests testing samples with
only supported by the above publications, but also
these kits prior to performing a complete study, as
by Illumina customer feedback.
formulary changes may have occurred when these
products were transferred to new companies or as
3. Accurately quantify your DNA prior to addition to the
part of the product’s evolution.
WGA reaction. Since the quality and quantity of DNA
used in WGA reactions is an important contributing
2. Do I need to clean WGA products prior to using them
factor to sample success on the Illumina platform,
in GoldenGate assays? Most importantly, Illumina
accurate quantification of input DNA is essential.
suggests that customers refer to the manufacturer’s
Samples should be quantified using
protocols for the kit they are using. If a purification
a DNA-specific method or reagent such as PicoGreen®.
step is not included in the protocol, customers can
Products from the WGA reaction should also be quan-
then decide whether it is a cost-efficient step to add
tified using the same method prior to entry into the
one or not. Illumina customer feedback has suggested
GoldenGate procedures, as recommended in current
that cleaning WGA products will not cause WGA
BeadLab and BeadStation protocols. For accurate quan-
samples that would not normally work to now
titation of highly degraded DNA, particularly formalin-
perform well. Anecdotally, however, customers have
fixed, paraffin-embedded (FFPE) samples, the use of
stated they have seen slightly improved genotyping
real-time PCR with the ALU Yb8 subfamily may be use-
call rates. Of note, the Barker et. al1 implemented a
ful4.
purification step using QIAquick® (Qiagen) columns.
4. Test your samples. Regardless of the WGA method
3. How can I tell if my DNA samples are intact
used, Illumina strongly suggests testing a small num-
or degraded? Prior to WGA, molecular weight and
ber of samples prior to initiating a study that partially
sample degradation can be assessed by electrophore-
or completely utilizes WGA samples. This is not only
sis through agarose. For the purposes of WGA,
important for WGA kits that are new to the market
samples with high molecular weights (20 kb and
and for which prior information is not available, but
above) can be considered relatively intact. Samples
also warranted for the methods described in the publi-
with low molecular weights (lower than 20 kb)
cations above. Formulary changes in kits can adverse-
should be considered degraded.
ly affect the successful generation of results. Testing
12-16 samples side-by-side on an array with gDNA
4. Can I use optical density (OD260) to quantify DNAs that
samples using a well-performing oligo pool (OPA) is
will be added to the WGA reaction? Use of OD260 is
highly recommended before undertaking any projects
strongly discouraged as it is not a direct measurement
that include WGA samples. When possible, include
of DNA quantity, and is subject to a variety of influ-
family DNAs in these tests to increase the confidence
ences that can falsely elevate calculated DNA concen-
level of the test.
tration. Samples should be quantified using a
DNA-specific method (i.e., RediPlate™ 96 PicoGreen®
FREQUENTLY ASKED QUESTIONS
dsDNA Quantitation Kit, Molecular ProbesTM, catalog
1. Where can I get more information and order the kits
#R-21495, www.invitrogen.com). Products of the WGA
described in the publications summarized above?
reaction should also be quantified using the same
The REPLI-g kit is no longer available from Molecular
method prior to entry into the GoldenGate procedures,
as recommended in current BeadLab and BeadStation
protocol. The use of WGA samples with the Infinium
protocols.
product is currently being explored by the Illumina
Research and Development team.
5. Can results from WGA samples be analyzed with
gDNA results? For many loci, WGA samples will
cluster well with gDNA samples in GenCall. However,
for some loci a slight shift in intensity or theta values
may be observed. Therefore, it is recommended that
samples from WGA not be analyzed concurrently with
non-WGA derived DNA samples. Including family DNA
samples when possible can also increase confidence
in the clustering and calling of genotypes by estimat-
ed Mendelian error rates.
6. Can GenTrain files be used for analyzing the
results for WGA samples?
GenTrain files that were created with WGA samples
can certainly be used to increase efficient data pro-
cessing. However, GenTrain files generated using
gDNA samples should not be used for WGA projects.
GenTrain files for standard products, such as the
Linkage IVb panels, have been generated from gDNA
samples and therefore are not recommended for use
with WGA samples.
7. Is there allelic bias observed for WGA samples
on the Illumina platform? Amplification and allelic
bias is a concern with in any WGA product. Pask et. al2
found that potential allelic bias, as demonstrated by
inheritance errors in their family DNAs, was nearly
identical and occurred for the same loci between
the Illumina and TaqMan® (Applied Biosystems®, Inc.)
platforms. This demonstrates that any allelic bias
most likely stems from the WGA reaction and is not
platform-specific. In addition, insufficient DNA added
to WGA reactions can be a major source of allelic
bias. Starting with sufficient, high-quality DNA will
reduce these effects.
8. Can WGA samples be used with the Infinium™ Assay?
The GoldenGate and Infinium assays use
different chemistries and could potentially perform
differently with WGA DNA samples. Additionally,
the Infinium Assay includes a WGA step as part of
the protocol. As such, any potential amplification bias
that may be present in WGA samples could theoreti-
cally be enhanced in the WGA reaction of the Infinium
Illumina® SNP Genotyping
REFERENCES
(1)
Barker, D.L., Hansen, M.S.T., Faruqi, A.F., Giannola, D., Irsula, O.R., Lasken, R.S.,
Latterich, M., Makarov, V., Oliphant, A., Pinter, J.H., Shen, R., Sleptsova, I., Ziehler,
W., and Lai, E. (2004) Two Methods of Whole-Genome Amplification Enable Accurate
Genotyping Across a 2320-SNP Linkage Panel. Genome Res 14, 901-907.
(2)
Pask, R., Rance, H., Barratt, B., Nutland, S., Sebastian, D.M., Twells, R.C.J., Smith, A.,
Lam, A.C., Walker, J.L., and Todd, J.A. (2004). Investigating the utility of combining
Φ29 whole genome amplification and highly multiplexed single nucleotide poly-
morphism BeadArray genotyping. BMC Biotechnol 4, 15.
(3)
Sawcer, S., Ban, M., Maranian, M., Yeo, T.W., Compston, A., Kirby, A., Daly, M.J.,
DeJager, P.L., Walsh, E., Lander ES, Rioux JD, Hafler DA, Ivinson A, Rimmler J,
Gregory, S.G., Schmidt, S., Pericak-Vance, M.A., Akesson, E., Hillert, J., Datta, P.,
Oturai, A., Ryder, L.P., Harbo, H.F., Spurkland, A., Myhr, K.M., Laaksonen, M., Booth,
D., Heard, R., Stewart, G., Lincoln, R., Barcellos, L.F., Hauser, S.L., Oksenberg, J.R.,
Kenealy, S.J., Haines. J.L.; for the International Multiple Sclerosis Genetics
Consortium. (2005). A high-density screen for linkage in multiple sclerosis. Am J
Hum Genet 77, 454-467.
(4)
Walker, J.A., Kilroy, G.E., Xing, J., Shewale, J., Sinha, S.K., Batzer, M.A. (2003). Human
DNAquantitation using Alu element-based polymerase chain reaction. Anal
Biochem 315, 122-8.
ADDITIONAL INFORMATION
For more information about WGA amplification or
Illumina’s genotyping products and services, contact us.
Illumina, Inc.
Customer Solutions
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San Diego, CA 92121-1975
1.800.809.4566 (toll free)
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© 2005 Illumina, Inc.
Illumina, BeadArray, Sentrix, Array of Arrays, GoldenGate, Infinium, DASL and Making Sense Out of Life, are
trademarks of Illumina. All other names and marks are the property of their respective owners.
Pub. No. 370-2005-011 29Sep05
making sense out of life
