title: Highly efficient multiplex PCR of noninvasive DNA does not require pre-amplification.
abstract: Among the key issues determining success of a study employing molecular genetics tools in wildlife monitoring or research is a large enough set of highly informative genetic markers and a reliable, cost effective method for their analysis. While optimized commercial genotyping kits have been developed for humans and domestic animals, such protocols are rare in wildlife research. We developed a highly optimized multiplex PCR that genotypes 12 microsatellite loci and a sex determination locus in brown bear (Ursus arctos) faecal samples in a single multiplex PCR and a single sequencer run. We used this protocol to genotype 1053 faecal samples of bears from the Dinaric population, and obtained useful genotypes for 88% of the samples, a very high success rate. The new protocol outperformed the multiplex pre-amplification strategy used in a previous study of 473 faecal samples with a 78.4% success rate. On a subset of 182 samples we directly compared the performance of both approaches, and found no advantage of the multiplex pre-amplification. While pre-amplification protocols might still improve PCR success and reliability on a small fraction of low-quality samples, the higher costs and workload do not justify their use when analysing reasonably fresh non-invasive material. Moreover, the high number of multiplexed loci in the new protocol makes it comparable to commercially developed genotyping kits developed for domestic animals and humans.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia Fish and Wildlife Resources, University of Idaho, Moscow, Idaho, ID 83844-1136, USA.
authors.forename: Tomaž
authors.initials: T
authors.lastname: Skrbinšek
authors.forename: Maja
authors.initials: M
authors.lastname: Jelenčič
authors.forename: Lisette
authors.initials: L
authors.lastname: Waits
authors.forename: Ivan
authors.initials: I
authors.lastname: Kos
authors.forename: Peter
authors.initials: P
authors.lastname: Trontelj