Advertisement

Conservation Genetics

, Volume 8, Issue 3, pp 733–738 | Cite as

Allelic dropout from a high-quality DNA source

  • Carl D. Soulsbury
  • Graziella Iossa
  • Keith J. Edwards
  • Philip J. Baker
  • Stephen Harris
Technical Note

Abstract

Allelic dropouts are an important source of genotyping error, particularly in studies using non-invasive sampling techniques. This has important implications for conservation biology, as an increasing number of studies are now using non-invasive techniques to study rare species or endangered populations. Previously, allelic dropout has typically been associated with PCR amplification of low quality/quantity template DNA. However, in this study we recorded high levels of allelic dropout (21–57%) at specific loci amplified from a high quality DNA (63.1  ±  7.8 ng/μl) source in the red fox (Vulpes vulpes). We designed a series of experiments to identify the sources of error. Whilst we were able to show that the best method to identify allelic dropout was the dilution of template DNA prior to PCR amplification, our data also showed two specific patterns: (1) allelic dropouts occurred at specific loci; (2) allelic dropouts occurred at specific pair-wise combinations of alleles. These patterns suggest that mechanisms other than low quantity template DNA are responsible for allelic dropout. Further research on the causes of these patterns in this and other studies would further our understanding of genotyping errors and would aid future studies where allelic dropout may be a serious issue.

Keywords

Microsatellites Cross-species Parentage Demography Red fox Vulpes 

Notes

Acknowledgements

We are grateful to the Natural Environment Research Council (CDS), Rotary Foundation of Rotary International, Newby Trust Ltd and the Sir Richard Stapley Educational Trust (GI), the Biotechnology and Biological Sciences Research Council (KJE), the International Fund for Animal Welfare (PJB) and The Dulverton Trust (SH) for financial support. We are also grateful to Jane Coghill of the University of Bristol Transcriptonics unit for processing the MegaBACE samples.

References

  1. Bonin A, Bellemain E, Eidesen PB, Pompanon F, Brochmann C, Taberlet P (2004) How to track and assess genotyping errors in population genetic studies. Mol Ecol 13:3261–3273PubMedCrossRefGoogle Scholar
  2. Broquet T, Petit E (2004) Quantifying genotyping errors in noninvasive population genetics. Mol Ecol 13:3601–3608PubMedCrossRefGoogle Scholar
  3. Buchan JC, Archie EA, Van Horn RC, Moss CJ, Alberts SC (2005) Locus effects and sources of error in noninvasive genotyping. Mol Ecol Notes 5:680–683CrossRefGoogle Scholar
  4. Creel S, Spong G, Sands JL, Rotella J, Zeigle J, Joe L, Murphy KM, Smith D (2003) Population size estimation in Yellowstone wolves with error-prone noninvasive microsatellite genotypes. Mol Ecol 12:2003–2009PubMedCrossRefGoogle Scholar
  5. Dakin EE, Avise JC (2004) Microsatellite null alleles in parentage analysis. Heredity 93:504–509PubMedCrossRefGoogle Scholar
  6. Double MC, Cockburn A, Barry SC, Smouse PE (1997) Exclusion probabilities for single-locus paternity analysis when related males compete for matings. Mol Ecol 6:1115–1166Google Scholar
  7. Flagstad Ø, Hedmark E, Landa A, Brøseth H, Persson J, Andersen R, Segerström P, Ellegren H (2004) Colonization history and noninvasive monitoring of a reestabilshed wolverine population. Conserv Biol 18:676–688CrossRefGoogle Scholar
  8. Francisco LV, Langston AA, Mellersh CS, Neal CL, Ostrander EA (1996) A class of highly polymorphic tetranucleotide repeats for canine genetic mapping. Mamm Genome 7:359–362PubMedCrossRefGoogle Scholar
  9. Gagneux P, Boesch C, Woodruff DS (1997) Microsatellite scoring errors associated with noninvasive genotyping based on nuclear DNA amplified from shed hair. Mol Ecol 6:861–868PubMedGoogle Scholar
  10. Harris S (1978) Age determination in the red fox (Vulpes vulpes) - an evaluation of technique efficiency as applied to a sample of suburban foxes. J Zool 184:91–117CrossRefGoogle Scholar
  11. Hoffman JI, Amos W (2005) Microsatellite genotyping errors: detection approaches, common sources and consequences for paternal exclusion. Mol Ecol 14:599–612PubMedCrossRefGoogle Scholar
  12. Holmes NG, Mellersh CS, Humphreys SJ, Binns MM, Holliman A, Curtis R, Sampson J (1993) Isolation and characterization of microsatellites from the canine genome. Anim Genet 24:289–292PubMedCrossRefGoogle Scholar
  13. Hung C-M, Li S-H, Lee L-L (2004) Faecal DNA typing to determine the abundance and spatial organisation of otters (Lutra lutra) along two stream systems in Kinmen. Anim Conserv 7:301–311CrossRefGoogle Scholar
  14. Jeffery KJ, Keller LF, Arcese P, Bruford MW (2001) The development of microsatellite loci in the song sparrow, Melospiza melodia (Aves) and genotyping errors associated with good quality DNA. Mol Ecol Notes 1:11–13CrossRefGoogle Scholar
  15. Jones AG, Ardren WR (2003) Methods of parentage analysis in natural populations. Mol Ecol 12:2511–2523PubMedCrossRefGoogle Scholar
  16. Klukowska J, Strabel T, Mackowski M, Switonski M (2003) Microsatellite polymorphism and genetic distances between the dog, red fox and arctic fox. J Anim Breed Genet 120:88–94CrossRefGoogle Scholar
  17. Kobayashi N, Tamura K, Aotsuka T (1999) PCR error and molecular population genetics. Biochem Genet 37:317–321PubMedCrossRefGoogle Scholar
  18. Luikart G, England PR (1999) Statistical analysis of microsatellite DNA data. Trends Ecol Evol 14:253–256PubMedCrossRefGoogle Scholar
  19. Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statistical confidence for likelihood-based paternity inference in natural populations. Mol Ecol 7:639–655PubMedCrossRefGoogle Scholar
  20. Miller CR, Joyce P, Waits LP (2002) Assessing allelic dropout and genotype reliability using maximum likelihood. Genetics 160:357–366PubMedGoogle Scholar
  21. Miller CR, Waits LP (2003) The history of effective population size and genetic diversity in the Yellowstone grizzly (Ursus arctos): implications for conservation. Proc Natl Acad Sci 100:4334–4339PubMedCrossRefGoogle Scholar
  22. Ostrander EA, Mapa FA, Yee M, Rine J (1995) One hundred and one new simple sequence repeat-based markers for the canine genome. Mamm Genome 6:192–195PubMedCrossRefGoogle Scholar
  23. Paetkau D (2003) An empirical exploration of data quality in DNA-based population inventories. Mol Ecol 12:1375–1387PubMedCrossRefGoogle Scholar
  24. Pemberton JM, Slate J, Bancroft DR, Barrett JA (1995) Nonamplifying alleles at microsatellite loci: a caution for parentage and population studies. Mol Ecol 4:249–252PubMedGoogle Scholar
  25. Pompanon F, Bonin A, Bellemain E, Taberlet P (2005) Genotyping errors: causes, consequences and solutions. Nat Rev Genet 6:847–859PubMedCrossRefGoogle Scholar
  26. Piggott MP (2004) Effect of sample age and season of collection on reliability of microsatellite genotyping of faecal DNA. Wildl Res 31:485–493CrossRefGoogle Scholar
  27. Piggott MP, Banks SC, Stone N, Banffy C, Taylor AC (2006) Estimating population size of endangered brush-tailed rock-wallaby (Petrogale penicillata) colonies using faecal DNA. Mol Ecol 15:81–91PubMedCrossRefGoogle Scholar
  28. Piggott MP, Taylor AC (2003) Remote collection of animal DNA and its applications in conservation management and understanding the population biology of rare and cryptic species. Wildl Res 30:1–13CrossRefGoogle Scholar
  29. Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225CrossRefGoogle Scholar
  30. Sambrook J, Fritsh EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd Edn. Cold Spring Harbour Laboratory Press, New YorkGoogle Scholar
  31. Smith DA, Ralls K, Hurt A, Adams B, Parker M, Maldonado JE (2006) Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses. Mol Ecol 15:387–406PubMedCrossRefGoogle Scholar
  32. Solberg KH, Bellemain E, Drageset O-M, Taberlet P, Swenson JE (2006) An evaluation of field and non-invasive genetic methods to estimate brown bear (Ursus arctos) population size. Biol Conserv 128:158–168CrossRefGoogle Scholar
  33. Taberlet P, Griffin S, Goossens B, Questiau S, Manceau V, Escaravage N, Waits LP, Bouvet J (1996) Reliable genotyping of samples with very low DNA quantities using PCR. Nucleic Acids Res 24:3189–3194PubMedCrossRefGoogle Scholar
  34. Taberlet P, Waits LP, Luikart J (1999) Noninvasive genetic sampling: look before you leap. Trends Ecol Evol 14:323–327PubMedCrossRefGoogle Scholar
  35. Taberlet P, Camarra J-J, Griffin S, Uhrès E, Hanotte O, Waits LP, Dubois-Paganon C, Burke T, Bouvet J (1997) Noninvasive genetic tracking of the endangered Pyrenean brown bear population. Mol Ecol 6:869–876PubMedCrossRefGoogle Scholar
  36. Wandeler P, Smith S, Morin PA, Pettifor RA, Funk SM (2003) Patterns of nuclear DNA degeneration over time- a case study in historic teeth samples. Mol Ecol 12:1087–1093PubMedCrossRefGoogle Scholar
  37. Xu J, Turner A, Little J, Bleecker E, Meyers D (2002) Positive results in association studies are associated with departure from Hardy–Weinberg equilibrium: hint for genotyping error? Human Genet 111:573–574CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Carl D. Soulsbury
    • 1
  • Graziella Iossa
    • 1
  • Keith J. Edwards
    • 1
  • Philip J. Baker
    • 1
  • Stephen Harris
    • 1
  1. 1.School of Biological SciencesUniversity of BristolBristolUK

Personalised recommendations