Multiplex preamplification PCR and microsatellite validation allows accurate single nucleotide polymorphism (SNP) genotyping of historical fish scales

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 preexisting 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 preexisting 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 preexisting 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. Preexisting contamination in 21% of the historical samples was successfully identified by screening the amplified microsatellite loci. The potential for 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.

将历史组织样本纳入生态学、保护生物学与资源管理相关研究，可通过加深对自然种群进化过程及人为活动影响的认知，拓展种群遗传学研究的范畴。针对历史样本与低质量样本的基因分型长期面临模板DNA含量极低、样本间存在预先DNA污染等挑战。本文报道一套两步流程，旨在达成两大目标：（1）以高通量方式精准对大量历史低质量鳞片样本进行基因分型；（2）筛查携带预先DNA污染的样本。首先，本文详述了针对45个单核苷酸多态性（single nucleotide polymorphisms, SNPs）的高效多重预扩增聚合酶链式反应（Polymerase Chain Reaction, PCR），如何在后续对红大麻哈鱼（Oncorhynchus nerka）历史鳞片样本的SNP基因分型反应中，生成高准确率的基因型，且失败率与错误率均处于较低水平。其次，本文展示了如何对该方法进行改造，以扩增微卫星位点，进而检测预先存在的DNA污染。本研究共对760份历史鳞片样本与182份当代鳍剪样本开展了基因分型与污染筛查工作。基因分型失败率与错误率极低，且历史样本与当代样本的该类比率相近。通过对扩增后的微卫星位点进行筛查，成功鉴定出21%的历史样本存在预先DNA污染。该方案具备自动化潜力，同时兼具低失败率与低错误率的优势，且可同时对预扩增与后续基因分型反应进行多重扩增，使其非常适合高效对大量潜在受污染的低质量DNA来源样本进行基因分型。