Saliva samples collected and stabilized with Oragene •DNA
are a reliable source of DNA for next generation sequencing®

Rafal M. Iwasiow
DNA Genotek Inc., Ottawa, CanadaIntroduction
In recent years we have observed significant advancements in next generation sequencing, decreasing
overall costs and greater acceptance of targeted capture technologies. Together these factors are enabling
the use of next generation sequencing for clinical applications. It is quickly becoming more practical andeconomical to process large numbers of samples for the purpose of genetic analysis. Such analysis requires
the collection of reliable, high quality samples. Saliva collected with Oragene•DNA provides a non-invasive
alternative method to blood samples for collecting large amounts of high quality genomic DNA that issuitable for next generation sequencing applications. Oragene•DNA devices contain a stabilizing reagent thatensures the sample is of high quality and allows long term storage at ambient temperature. In this study weinvestigated DNA extracted from saliva collected using Oragene•DNA for use with Agilent SureSelect HumanAll Exon Kit and paired-end sequencing on the Illumina® Genome Analyzer II.

Using a single lane on the Illumina Genome Analyzer II flow cell we were able to achieve a mean depth of 77x for
both saliva and blood samples that were exome-enriched. In contrast samples that were not enriched only had amean depth of 2.4x. Exome-enriched saliva and blood samples identified a similar mean number of variants: 28,738
for saliva and 28,067 for blood.

To evaluate the performance of saliva samples collected using Oragene•DNA we compared the data against
DNA from blood samples collected from the same individuals. Using the Agilent SureSelect Human All Exon
Kit to capture regions totaling 38Mb = 1.22% of human genomic regions we demonstrate the ability to enricheither blood or saliva DNA samples to investigate genetic variations using next generation sequencing
technologies.
Exome-enriched saliva and blood samples achieved 98.5% coverage of the exon regions targeted by the Agilent
SureSelect All Exon Kit. On average 46% of sequenced bases were located in the exon regions. In contrast, samples
which were not enriched only had 82.4% coverage of the exon regions with much lower depth.

Materials and methods
Collection of saliva using Oragene•DNAFill line

• Saliva was collected according to the instructions provided in the kit.
• Saliva samples were collected from 3 donors.
• Oragene•DNA/saliva samples were purified according to DNA Genotek protocol PD-PR-006.
• DNA was quantified using the Invitrogen Picogreen Quanti-it kit.
• Blood samples were collected from the same 3 donors that donated saliva.
• 8 mL of whole blood was collected using EDTA tubes.
• Blood samples were centrifuged and the buffy coat was collected.
• DNA from buffy coat was purified using the Qiagen Blood mini kit.

Both saliva and blood samples can be used to investigate genetic variations using next generation sequencing
technologies. Here we demonstrate that using DNA from saliva or blood we successfully queried SNPs of interest andobserved a perfect correlation between the sample types. We queried the following previously reported SNPS:
rs2075650 (APOE)1, rs10513025 (SEMA5A)2, rs2736990 (SNCA)3,4, and rs356220 (SNCA)4,5.

rsID• DNA samples were enriched for the human exome using the Agilent SureSelect All Exon kit.
• Samples were sequenced using paired-end sequencing on the Illumina Genome Analyzer II.
• One lane per sample was used.

Bioanalyzer traces of Exome-enriched libraries

GeneChr

Donor 1

Pos

Donor 2

Donor 3

Blood

SalivaBloodSaliva

Blood

Salivars356220

SNCA

490860363

AA

A

A

A

A

rs2075650

APOE

1950087459

RRA

A

A

A

rs2736990

SNCA

490897564

CCCCCCrs10513025 SEMA5A

59676622

TT

T

TT

TConclusions
Saliva is a reliable source of DNA for use with the Agilent SureSelect Human All Exon Kit and sequencing using
paired-end methodology on the Illumina Genome Analyzer II as demonstrated by the coverage (98.7%), depth (74x),and genome aligment (97.3%). Additionally, samples that were not enriched had 91.5% of their sequence align to thereference human genome indicating that saliva can also be used for whole genome sequencing.

Saliva #1

Blood #1

Donor

SampleEnrichment
SourceAResults

BAll samples generated in excess of 6 x 109 bases per lane on the Illumina Genome Analyzer II. Statistically there wasno difference in total bases sequenced between saliva and blood samples or between whole genome or exome
enriched samples. For exome enriched blood and saliva samples in excess of 96.6% of all bases aligned to the humangenome. In comparison, saliva samples not enriched using the Agilent Sureselect kit were observed to have 91.5% ofbases aligned to the human genome.

CAgilent
SureSelect
All Exon
N/A
N/A

Lane Yield (kbases)% Align Genome
Coverage
Variants
Mean Depth
Bases Not In Exon
Bases In Exon
Proportion in Exon

BloodSaliva
BloodSaliva
BloodSaliva
SalivaSaliva

Lane% Align
Yield
Coverage Variants
Genome(kbases)
6,217,788 98.45% 98.50% 27,278
6,434,248 96.74% 98.90% 29,444
6,676,662 99.20% 97.94% 28,983
6,072,430 96.61% 98.55% 27,996
6,447,860 99.08% 98.39% 27,941
6,268,330 98.46% 98.61% 28,773
6,982,002 91.68% 83.02% 2,161
6,818,024 91.23% 81.78% 1,893

MeanDepthkbases Not kbases In Proportion
In ExonExon
in Exon

76.4
74.38
84.78
72.3
78.85
74.72
2.444
2.385

3,253,014 2,845,003
3,416,661 2,781,056
3,461,969 3,139,072
3,147,312 2,693,723
3,433,914 2,932,943
3,359,432 2,785,456
6,308,938 76,708
6,129,287 73,742

46.65%
44.87%
47.55%
46.12%
46.07%
45.33%
1.20%
1.19%

The number of bases provided by the PF reads
The % of PF reads that aligned to the reference
The % of the Exome covered by sequencing reads
The number of variants (non-reference bases) identiﬁed for that sample, after passing quality thresholds
The average depth for all bases covered in the Exome
The number of bases that aligned to the genome but not within a direct exon boundary
The number of bases that fall within an exact exon boundary
The % of bases aligned within an exon versus all bases aligned

References(1) Sudha Seshadri et. al. (2010). Genome-wide Analysis of Genetic Loci Associated With Alzheimer Disease. JAMA. Vol. 303, No. 18, 1832-1840.
(2) Lauren A. Weiss et. al. (2009). A genome-wide linkage and association scan reveals novel loci for autism. Nature. Vol. 461, 802-808.
(3) Javier Simon-Sanchez et. al. (2009). Genome-wide association study reveals genetic risk underlying Parkinson’s disease. Nature Genetics. Vol. 41, No. 12,1308-1312.
(4) The UK Oarkinson’s Disease Consortium and The Wellcome Trust Case Control Consortium 2. (2011). Dissection of the genetics of Parkinson’s disease
identifies an additional association 5’ of SNCA and multiple associated haplotypes at 17q21. Human Molecular Genetics. Vol. 20, No. 2, 345-353.
(5) Taye H Hamza et. al. (2010). Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson’s disease. Nature Genetics. Vol.
42, No. 9, 781-785.

Superior samples
Proven performance

US Patent No. 7,482,116;
Patents issued and pending
MK-00014 Issue 1/2011-03 © DNA Genotek Inc., all rights reserved.
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