WGA and Canine SNPs

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• WGA and Canine SNPs
• Andrea Short
• Gregynog 26-28 April 2004

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Summary

• Identification of potential canine SNPs in a
  number of candidate genes using WAVE.
• Verification of the SNPs by sequencing.
• Genotyping of the breed plate by Taqman.

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Reasons for WGA

• Original breed plate contains 93 samples.
• It is composed of GR, Rott, Shih Tzu, Lab, YT,
  ACS, CKCS, Dob, WHWT, GSD and Beagles.
• Ideally would like to analyse genotyppe
  frequencies on same samples to give a feel for
  breed distribution.
• Finite amount of DNA is available.

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WGA Data Summary to Date

• Amplification of canine DNA from blood
• PCR of WGA DNA
• WAVE of WGA DNA
• Sequencing of WGA DNA
• Preliminary Taqman
• DLA Typing by RSCA

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Results DNA amplification

• M 12Kb ruler
• All other sample names correspond to the well on
  the plate. F12neg, G12pos (billy) and H12WGA
  pos

• The average yield was 710.5ng/ul.
• The lowest yield was 635ng/ul (WGA pos)
• The highest yield was 774ng/ul (C3).

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Results -PCR
Five of the BP PCRs worked well, although the
yield was slightly less than the original PCRs.

• one BP PCR showed a positive band in both the
  negative control (D12) and the WGA positive (H12)

Three of the PCRs appeared to have failed
completely. Further investigations revealed only
one set which did not amplify, and could not be
amplified on the WGA DNA.
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Results - WAVE
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Results - Sequencing.

• 44 sequences were analysed from the eight primer
  sets used to amplify the breed plate.
• 42 sequences were identical.
• 2 sequences varied between original and WGA.

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Results - Taqman and DLA typing

• In a preliminary Taqman assay of 46 original DNAs
  and corresponding 46 WGA DNAs.
• 40 samples gave identical genotypes, while six of
  each sample could not be called.
• Analysis of DLA typing is not yet complete, but
  initial indications are that the original and WGA
  samples are in agreement.

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Summary

• Amplification of 93 samples.
• PCR of nine sets of primers (9x93 samples) - One
  primer set failed completely
• WAVE of eight amplicons (8x93 samples)
• Sequencing of 44 fragments (44x 600bases) - Two
  sequencing patterns varied by a total of five
  bases.
• Taqman genotyping of 46 samples - 6 samples could
  not be called
• DLA typing on three original plates and three WGA
  plates

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Further work

• Microsatellite typing .
• Repeat of PCR and WAVE using DNA from canine
  Buccal swabs.
• Comparison between the Rubicon and Genomphi kit.

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Thank-you

• Masterfoods
• Neale Fretwell
• Prof. Bill Ollier
• Dr. Wendy Thomson
• Annette Barnes
• Francine Jury