US Geological Service PIERC BRD, Kilauea Field Station, Hawaii National Park, HI 96718, USA, Department of Zoology and Physiology, University of Wyoming, 1000 E. University Avenue, Laramie, Wyoming 82071, USA.
Mol Ecol Resour. 2008 Mar;8(2):282-9. doi: 10.1111/j.1471-8286.2007.01992.x.
In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and 'comparative method') in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters (Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7-32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method.
在非侵入性遗传采样中，如果基因分型错误率较高且重捕率较低，个体的错误识别可能导致种群数量的高估。因此，估计基因分型错误率至关重要。然而，在多个位点进行多次聚合酶链反应（PCR）既耗时又昂贵。为了解决关于获得共识基因型所需的最小 PCR 数量的争议，我们比较了两种基因分型方案（多管和“比较法”）在基因分型成功率和错误率方面的表现。我们从 2003 年在怀俄明州采集的 48 个水獭（Lontra canadensis）粪便样本以及从五只圈养水獭的血液样本中获得的结果，这些样本使用了四种不同的引物进行扩增，表明使用比较基因分型方案可以最小化每个位点的 PCR 数量。除了一个位点的五个样本外，与多管方法相比，该方案使用更少的 PCR 和更低的错误率获得了相同的共识基因型。这一发现令人放心，因为即使在血液和毛发等组织中，基因分型错误率也可能相对较高。此外，我们发现，易于扩增且产生共识基因型的位点，仍可能表现出较高的错误率（7-32%），并且不同引物的扩增会导致不同类型和速率的错误。因此，基于多个位点的单个 PCR 分配基因型可能会导致个体的错误识别。我们建议，应修改旨在统计分配共识基因型的方案，以允许比较法固有的杂合子和纯合子的不同处理。
