鱼耳骨,被称为耳石,经常在渔业中收集以协助管理,是博物馆和国家档案馆中常见的样本类型。除了它们对衰老的效用之外,形态和微量元素分析,耳石是有价值的基因组信息库。先前的工作表明,可以从保留在耳石表面的痕量组织中提取DNA,尽管它们通常在室温下干燥储存。然而,大部分工作都在干净的实验室条件下使用了简化的代表性测序方法,为了获得足够的DNA产量,文库和最终的单核苷酸多态性(SNP)。这里,我们率先使用小规模(掺入)测序来筛选在常规分子生物学(与清洁相比)实验室制备的当代耳石样品的污染和质量水平,仅提交高于定义的内源性DNA阈值的样本进行全基因组重测序.尽管从耳石中提取的DNA质量和数量通常都很低,我们能够产生全基因组文库和最终过滤的集合,无联系的,甚至是推定的适应性SNP,有足够的数量用于人口的下游用途,气候和保护基因组学。通过与一组来自同一物种的组织样本进行比较,我们能够从DNA提取和文库制备中突出耳石样品的质量和功效,生物信息学预处理和SNP调用。我们提供详细的原理图,我们方法的协议和脚本,这样它就可以被社区广泛采用,改善耳石作为有价值的基因组数据来源的使用。
Fish ear bones, known as otoliths, are often collected in fisheries to assist in management, and are a common sample type in museum and national archives. Beyond their utility for ageing, morphological and trace element analysis, otoliths are a repository of valuable genomic information. Previous work has shown that DNA can be extracted from the trace quantities of tissue remaining on the surface of otoliths, despite the fact that they are often stored dry at room temperature. However, much of this work has used reduced representation sequencing methods in clean lab conditions, to achieve adequate yields of DNA, libraries and ultimately single-nucleotide polymorphisms (SNPs). Here, we pioneer the use of small-scale (spike-in) sequencing to screen contemporary
otolith samples prepared in regular molecular biology (in contrast to clean) laboratories for contamination and quality levels, submitting for whole-genome resequencing only samples above a defined endogenous DNA threshold. Despite the typically low quality and quantity of DNA extracted from otoliths, we are able to produce whole-genome libraries and ultimately sets of filtered, unlinked and even putatively adaptive SNPs of ample numbers for downstream uses in population, climate and conservation genomics. By comparing with a set of tissue samples from the same species, we are able to highlight the quality and efficacy of
otolith samples from DNA extraction and library preparation, to bioinformatic preprocessing and SNP calling. We provide detailed schematics, protocols and scripts of our approach, such that it can be adopted widely by the community, improving the use of otoliths as a source of valuable genomic data.