Present address for Matt Smith: U.S. Fish and Wildlife Service, Abernathy Fish Technology Center, Conservation Genetics Laboratory, 1440 Abernathy Creek Road, Longview, WA 98632.
Multiplex preamplification PCR and microsatellite validation enables accurate single nucleotide polymorphism genotyping of historical fish scales
Article first published online: 17 FEB 2011
© 2011 Blackwell Publishing Ltd
Molecular Ecology Resources
Special Issue: SNP Development in Non-Model Organisms
Volume 11, Issue Supplement s1, pages 268–277, March 2011
How to Cite
SMITH, M. J., PASCAL, C. E., GRAUVOGEL, Z., HABICHT, C., SEEB, J. E. and SEEB, L. W. (2011), Multiplex preamplification PCR and microsatellite validation enables accurate single nucleotide polymorphism genotyping of historical fish scales. Molecular Ecology Resources, 11: 268–277. doi: 10.1111/j.1755-0998.2010.02965.x
- Issue published online: 17 FEB 2011
- Article first published online: 17 FEB 2011
- Received 14 June 2010; accepted 18 November 2010
- genotype error;
Incorporating historical tissues into the study of ecological, conservation and management questions can broaden the scope of population genetic research by enhancing our understanding of evolutionary processes and anthropogenic influences on natural populations. Genotyping historical and low-quality samples has been plagued by challenges associated with low amounts of template DNA and the potential for pre-existing DNA contamination among samples. We describe a two-step process designed to (i) accurately genotype large numbers of historical low-quality scale samples in a high-throughput format and (ii) screen samples for pre-existing DNA contamination. First, we describe how an efficient multiplex preamplification PCR of 45 single nucleotide polymorphisms (SNPs) can generate highly accurate genotypes with low failure and error rates in subsequent SNP genotyping reactions of individual historical scales from sockeye salmon (Oncorhynchus nerka). Second, we demonstrate how the method can be modified for the amplification of microsatellite loci to detect pre-existing DNA contamination. A total of 760 individual historical scale and 182 contemporary fin clip samples were genotyped and screened for contamination. Genotyping failure and error rates were exceedingly low and similar for both historical and contemporary samples. Pre-existing contamination in 21% of the historical samples was successfully identified by screening the amplified microsatellite loci. The advantages of automation, low failure and error rates, and ability to multiplex both the preamplification and subsequent genotyping reactions combine to make the protocol ideally suited for efficiently genotyping large numbers of potentially contaminated low-quality sources of DNA.