Data from: A non-lethal sampling method to obtain, generate and assemble whole-blood transcriptomes from small, wild mammals

The acquisition of tissue samples from wild populations is a constant challenge in conservation biology, especially for endangered species and protected species where non-lethal sampling is the only option. Whole blood has been suggested as a non-lethal sample type that contains a high percentage of body-wide and genome-wide transcripts and therefore can be used to assess the transcriptional status of an individual, and to infer a high percentage of the genome. However, only limited quantities of blood can be non-lethally sampled from small species and it is not known if enough genetic material is contained in only a few drops of blood, which represents the upper limit of sample collection for some small species. In this study, we developed a non-lethal sampling method, the laboratory protocols and a bioinformatic pipeline to sequence and assemble the whole blood transcriptome, using Illumina RNA-Seq, from wild greater mouse-eared bats (Myotis myotis). For optimal results, both ribosomal and globin RNAs must be removed before library construction. Treatment of DNase is recommended but not required enabling the use of smaller amounts of starting RNA. A large proportion of protein-coding genes (61%) in the genome were expressed in the blood transcriptome, comparable to brain (65%), kidney (63%) and liver (58%) transcriptomes, and up to 99% of the mitogenome (excluding D-loop) was recovered in the RNA-Seq data. In conclusion, this non-lethal blood sampling method provides an opportunity for a genome-wide transcriptomic study of small, endangered or critically protected species, without sacrificing any individuals.

在保护生物学领域，从野生种群获取组织样本始终是一项长期存在的难题，对于仅能采用非致死性采样方式的濒危物种与保护物种而言尤为突出。全血作为一种非致死性样本类型，其所含的全身及全基因组转录本占比颇高，因此可用于评估个体的转录状态，并推断出大部分基因组信息。不过，小型物种的非致死性采血样本量往往有限，而仅数滴血液（这也是部分小型物种采样的上限）中是否含有足够的遗传物质，目前仍未明确。本研究以野生大鼠耳蝠（Myotis myotis）为研究对象，基于Illumina RNA测序（RNA-Seq）技术，开发了一套用于全血转录组测序与组装的非致死性采样方法、实验室流程及生物信息学分析管线。若要获得最佳实验结果，需在文库构建前去除核糖体RNA（ribosomal RNA）与珠蛋白RNA（globin RNA）。建议使用脱氧核糖核酸酶（DNase）进行处理，但并非强制要求，因此可使用起始量更少的RNA样本。该血液转录组中表达的蛋白质编码基因占基因组的61%，与脑组织（65%）、肾脏组织（63%）及肝脏组织（58%）的转录组表达占比相当；同时，RNA测序数据可覆盖高达99%的线粒体基因组（mitogenome，排除D-loop区域）。综上，该非致死性采血方法为小型濒危或高度保护物种开展全基因组转录组学研究提供了可行途径，且无需牺牲任何实验个体。