Comparison of high density genotyping results from saliva and blood
samples on Affymetrix GeneChip® GenomeWide SNP 6.0 arrays
J.D. Reynolds, T.T. Do, D.B. Hongo, I.K. Kuramoto, W.H. Biggs III, C.K. French.
Affymetrix Clinical Services Laboratory, Affymetrix, Inc., West Sacramento, CA.
Nsp

Nsp

RE Digestion

QC

ComplexityReduction

CallRate(%)

Hyb & Wash

Fragmentationand Labeling

AA BB AB

Figure 2. Overview of the Affymetrix GWS6.0 process. The WGSA protocol
requires 500 ng (250ng for each Nsp and Sty reactions) of purified genomic
DNA . The GWS6.0 array is used in conjunction with Affymetrix Genotyping
Console to determine genotypes at each of 909,000 loci.

DNA was successfully extracted from blood samples using theautomated extraction procedure and also from saliva samples
using a manual extraction technique. The paired samples
demonstrated similarities in concentration, integrity, size andpurity when analyzed on the NanoDrop ND1000
spectrophotometer [Figure 3, Table 1] as well as on a 1% agarosegel [Figure 4].
a.)

blood

2.00

35.00

1.80
1.60

saliva25.00ugng/ul

200.00

40.00

30.00

300.00250.00

Average A260/A280

Average Yield

20.00

150.0015.00

100.00

10.00

50.00

5.00

0.00

0.00

blood
saliva

blood

Blood
Saliva96.45+/‐ 0.39

Median(Saliva)

95.40+/‐ 0.30

100%

AA %

BB %

99.65+/‐ 0.03

25.87
37.18
+/‐ 0.12 +/‐ 0.07

36.36+/‐ 0.06

99.74+/‐ 0.02

25.91
37.25
+/‐ 0.17 +/‐ 0.09

36.51+/‐ 0.08

99.44+/‐ 0.04

25.75
37.25
+/‐ 0.18 +/‐ 0.10

36.43+/‐ 0.09

GenderConc.

Percent (%)

100%

99.65 +/‐ 0.31100.00
98.00
96.00
94.00
92.00
90.00
88.00
86.00
84.00

QC CallRate
Birdseed
Call RatesalivaBloodSaliva

Figure 5. Graphical representation of the average QC and Birdseed call rates for theentire set of samples as well as the individual averages for the blood and saliva
samples. The red lines indicate the minimum call rate percentages for QC ( 86%) andBirdseed (97%).

0.60
0.40
0.20
0.00

Figure 3. Average quality control metrics analyzed by the NanoDrop ND1000
spectrophotometer for blood and saliva samples. a.) The average concentration(ng/μl) for the blood and saliva samples. b.) Average total yield for the bloodand saliva samples. c.) Average A260/A280 ratios for the blood and salivasamples. Red lines indicate minimums/maximums.

A260/A280+/‐ stdev
Acceptable range:
1.5‐2.0
1.63 +/‐ 0.09
1.79 +/‐ 0.08

Median(Blood)

AB %

All

1.40
1.20
1.00
0.80

96.26
+/‐ 0.25

Concordance

CallRate(%)

Average QC and Birdseed Call Ratesc.)b.)
Average Concentration

Median(All)

BIRDSEED

GenderConc.

Table 2: GWS6.0 analysis QC and Birdseed call rates for the average of all
samples, average blood and average saliva. All of the call rates were above thestandard passing minimum rates. Reproducibility averages in excess of 99%
demonstrate the similarity between the paired blood and saliva samples.

Results

350.00

Paired EDTA‐stabilized whole blood samples and DNA
Genotek Oragene saliva samples were collected from 66
IRB‐approved volunteer donors.

AdaptorLigation

PCR: One Primer
Amplification

400.00Materials and Methods

The paired blood and saliva samples were all successfully
run on the GWS6.0 arrays and passed the QC call rate
thresholds. The samples were analyzed using the Birdseedalgorithm yielding an excellent average call rate for each setof samples, as well as successful concordance between thepaired blood and saliva samples. Reproducibility was almost
100% between the paired samples.

Nsp

Percent (%)

Currently, EDTA‐stabilized whole blood is the mostcommon sample type used for high density genotyping.
Blood has proven a very consistent and reliable source ofgenetic material for many avenues of testing andresearch, but it can also be a time consuming, expensive
and invasive collection method‐ especially for long termor broad range studies. Finding a comparable source ofgenetic material, such as saliva, that is more cost effective,
more stable and less invasive would be extremely
beneficial to the scientific community.
This experiment used the Beckman‐Coulter Biomek
NXP platform with Agencourt chemistry to extract
genomic DNA from blood samples. DNA from the paired
saliva samples were extracted using the manual
extraction method provided by DNA Genotek. The study
compared not only the DNA quality and quantity, butalso the microarray call and concordance rates (CR) toindicate salivaʹs suitability for genetic association studies.
All samples were prepared and run simultaneously on
the Genome‐Wide SNP6.0 arrays (GWS6.0).
GeneChip® microarrays consist of small DNA
fragments (or probes), that are synthesized to specificlocations on a coated array quartz surface. Millions ofprobes can be contained on one array. All the samples arescanned and analyzed using Affymetrix GeneChip®
Genotyping Console (AGGC). AGGC performs a
multiple‐chip analysis fitting probe effects to increase
precision on signal estimates for the two alleles of each
SNP, followed by a Bayesian classification approach tomake genotype calls.

250 ng genomic DNA per array

A 2 60 /A 2 8 0Introduction

Concentration(ng/ul) +/‐ stdev
Acceptable range:> 50
149.34 +/‐ 59.67
138.57 +/‐ 83.81

Yield (ug)+/‐ stdev
Acceptable range:>2
5.97 +/‐ 2.39
5.54 +/‐ 3.35

Table 1. The average metrics of DNA extracted from blood and saliva samples
and their respective standard deviations. Also listed are the Affymetrix rangesfor each metric that are considered acceptable for downstream applications.

a.)Conclusions
Concentration and purity QC metrics have demonstrated
that DNA extracted from saliva is of similar quality andquantity to that extracted from the paired blood sample. The
quality of the genomic DNA from saliva samples
demonstrated their suitability for genotyping studies. The
paired blood and saliva samples were run on the GWS6.0
arrays, analyzed and then compared to internal standards andto each other. Call rates and reproducibility percentages inexcess on 99% verifies that saliva can be used successfully as
an alternative source of genomic DNA for use in high density
genotyping.
These results demonstrate that genomic DNA extracted fromsaliva may be used in a clinical and research environment as acomparable source of genetic material for high density
genotyping studies. The suitability of saliva samples as asource of genomic DNA allows for the flexibility of acollection method in the clinical and research environmentthat is more cost effective, less invasive and more suitable forlong term and/or broad range studies.

Literature citedb.)

1.) Kennedy, G.C., et. al. Large‐scale genotyping of complex
DNA. Nat Biotechnol 21:1233‐7 (2003).
2.) Matsuzaki, H., et. al. Parallel genotyping of over 10,000 SNPs
using a one‐primer assay on a high density oligonucleotide
array. Genome Res 14:414‐25 (2004).

c.)3.) Agencourt® Genfind™ V2 Blood and Serum Genomic DNA
Isolation Kit protocol.
4.) DNA Genotek Laboratory Protocol for Manual Purification ofDNA from 0.5 mL of Oragene®∙DNA/salivad.)5.)Affymetrix Genome‐Wide Human SNP Nsp/Sty 6.0 Users
Guide

Acknowledgments
The blood and saliva sample donors from Affymetrix.
Figure 1: Overview of the extraction procedures and QC for
paired blood and saliva samples. All samples were analyzed forconcentration (ng/μL), purity (A260/A280 ratios) and yield (μg)
on a NanoDrop ND‐1000 spectrophotometer as well as for
integrity on a 1% agarose gel.

Figure 4. Gel electrophoresis. a.) 1% agarose gel of blood genomic DNA. b.) 1%
agarose gel of saliva genomic DNA. c.) 2% agarose gel of PCR purification forboth blood and saliva samples. d.) 4% agarose gel of fragmentation for bloodand saliva samples.

For further informationwww.affymetrix.com*Data is from a single lab at Affymetrix and is not a
supported protocol for arrays in the field.