Enabling genome discovery in sheep: Superior DNA collection
from nasal samples using Performagene™
J.O. Niles, C.M. James, E.J. Maclean, and R.M. Iwasiow
DNA Genotek Inc., Ottawa, Canada

Introduction

Figure 2: Representative image of visual QC check

Genome discovery in sheep has been vastly accelerated by the introduction of the Illumina
OvineSNP50 BeadChip. A reliable DNA sample must be collected in order to facilitate this genetic
analysis. In order to enable large sets of DNA samples to be collected, an easy-to-use collection
method is preferred. To date the source of DNA for parentage and genotyping on sheep has beenblood, semen or ear tissue. These samples can be costly and inconvenient for the producer to obtainand are often difficult and inefficient to process in the lab. The reliable collection of genomic DNA
from sheep nasal samples is demonstrated using the Performagene™ DNA collection kit developed
by DNA Genotek, Inc. Nasal sample collection is an easy-to-use and reliable alternative that provides
high quantity, high quality genomic DNA from sheep. Our study samples were collected from thenostrils of 18 young lambs and adult ewes. Paired blood samples were collected from 6 of theanimals for SNP concordance analysis. Fluorescent quantification is used to demonstrate that thePerformagene DNA collection method yields a median 8.2 μg of DNA per sample and medianconcentration of 82.2 ng/μL compared with blood samples which yield a median concentration of
25.8 ng/μL. Agarose gel analysis of sheep DNA isolated from nasal and blood samples demonstrates
that high molecular weight genomic DNA is consistently obtained. Analysis from genotyping shows
the suitability of nasal samples on the OvineSNP50 BeadChip with median call rates of 99.8% andgenotype concordance for paired nasal and blood samples was 100%.

• Nine of the nasal samples did not pass the visual QC test
due to a distinct yellow colour.
• Regardless of the sample colour all samples were
subsequently analyzed using the OvineSNP50 BeadChip.
• Visual QC is a subjective assessment of quality. The colour
observed in nasal samples does not appear to impact
performance on the OvineSNP50 BeadChip.
• Further purification of the samples beyond alcohol
precipitation can be used to eliminate the material
contributing to coloured samples.

Figure 3: High molecular weight QC agarose gel of nasal and blood
DNA samples
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Materials and methods
Figure 1: Nasal sample collection using Performagene• 2 µL of each sample was run on an agarose gel to evaluate presence of high molecularweight DNA, with lanes 2-19 corresponding to nasal samples and lanes 20-25 corresponding
to blood samples.
• All samples were observed to contain high molecular weight DNA.
• As a constant volume was loaded on the gel, some lanes appear overloaded while someare under loaded. This is a reflection of the concentration differences between blood andnasal samples.

DO NOTSPILL• 18 nasal samples were collected according to the DNA Genotek protocol PD-PR-099
from 18 different sheep.
• Blood was drawn from 6 of the sheep into a standard EDTA blood tube.
• Nasal samples were shipped at ambient temperature by standard mail to DNA Genotek forprocessing.
• Blood samples were shipped on ice using standard precautions to DNA Genotek for processing.
• A 0.5 mL aliquot of the nasal samples was purified using PG-L2P followed by alcohol precipitationaccording to the DNA Genotek purification protocol PD-PR-083.
• Four 200 μL aliquots of blood where purified using the Qiagen Blood Mini Kit. The four elutions
were pooled and reprecipitated.
• All of the purified DNA was quantified using Quant-It™ PicoGreen® and shipped to DNA LandMarksfor QC check and analysis on the OvineSNP50 BeadChip.
• A visual QC was performed on the DNA samples and a high molecular weight test was conducted.
All samples were processed on the OvineSNP50 BeadChip.

Results
Table 1: Quantification results for nasal and blood samples
Animal ID

Nasal – DNA
concentration(ng/μL)

Blood – DNAConcentration(ng/μL)

SA6
SA34
SA36
SA38
SA39
SA42
SA44
SA46
SA47
SA48
SA49
SA51
SA55
SHL91
S7841
S11274
S11275
S11282

43.5
47.9
99.8
100.8
96.0
70.6
121.7
69.4
121.0
68.2
64.3
117.2
93.9
33.8
111.3
40.8
137.3
40.2

29.5
40.0

Median82.2

25.8• A 500 µL aligout of a nasal sample collectedfrom sheep using Performagene and purified
using PG-L2P gave a median concentrationof 82.2 ng/µL and a minimum concentrationof 33.8 ng/µL.
• The median amount of DNA collected using
the nasal swab was 8.2 µg. This is an amountof DNA well in excess of requirements for theOvineSNP50 BeadChip.

Table 2: Summary of results from Illumina OvineSNP50 BeadChip
Animal ID

Nasal sample
call rate

Blood sample
call rate

Saliva/blood
concordance

SA6
SA34
SA36
SA38
SA39
SA42
SA44
SA46
SA47
SA48
SA49
SA51
SA55
SHL91
S7841
S11274
S11275
S11282
Median99.89%
99.73%
99.29%
99.89%
99.90%
99.93%
99.92%
99.75%
98.63%
99.69%
98.63%
99.89%
99.88%
44.25%
99.39%
99.84%
99.56%
99.80%
99.80%

99.87%
99.58%

100.00%100.00%99.91%
99.76%
99.02%

100.00%100.00%99.63%
99.70%

100.00%100.00%* sample SHL91 was removed from the median calculation due to sample or chip

• The median call rate for
gDNA from nasal samples
was 99.8%.
• One of the 18 nasal samples
did not perform well on theOvineSNP50 BeadChip
despite having sufficient
DNA that was assessed tobe of high molecularweight. At this time itremains unclear if thefailure was due to sample orprocedure or BeadChip.
Further work including
repurification andre-running on theOvineSNP50 BeadChip will
be performed to evaluate
the issue.
• The median call rate for
gDNA from blood samples
was 99.7%.
• The genotype concordancefor paired nasal and blood
samples was 100%.

failure.

Conclusions

23.5
28.1
23.1

• Performagene provides a quick and easy method for collecting large amountsof high quality DNA from sheep using non-invasive methods.
• DNA collected using Performagene can be used on the OvineSNP50 BeadChip.
• DNA collected from nasal samples is the same as DNA from blood as shown throughthe100% genotyping concordance rates.

21.6

Superior Samples
Proven Performance

Performagene™ is a trademark of DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc.
All other brands and names contained herein are the property of their respective owners.
Patent (www.dnagenotek.com/legalnotices)
MK-00012 Issue 2/2013-05