Animal Ecology, Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden.
Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Stockholm, Sweden.
Mol Ecol Resour. 2017 Nov;17(6):e111-e121. doi: 10.1111/1755-0998.12699. Epub 2017 Aug 3.
Population-scale molecular studies of endangered and cryptic species are often limited by access to high-quality samples. The use of noninvasively collected samples or museum-preserved specimens reduces the pressure on modern populations by removing the need to capture and handle live animals. However, endogenous DNA content in such samples is low, making shotgun sequencing a financially prohibitive approach. Here, we apply a target enrichment method to retrieve mitochondrial genomes from 65 museum specimens and 56 noninvasively collected faecal samples of two endangered great ape species, Grauer's gorilla and the eastern chimpanzee. We show that the applied method is suitable for a wide range of sample types that differ in endogenous DNA content, increasing the proportion of target reads to over 300-fold. By systematically evaluating biases introduced during target enrichment of pooled museum samples, we show that capture is less efficient for fragments shorter or longer than the baits, that the proportion of human contaminating reads increases postcapture although capture efficiency is lower for human compared to gorilla fragments with a gorilla-generated bait, and that the rate of jumping PCR is considerable, but can be controlled for with a double-barcoding approach. We succeed in capturing complete mitochondrial genomes from faecal samples, but observe reduced capture efficiency as sequence divergence increases between the bait and target species. As previously shown for museum specimens, we demonstrate here that mitochondrial genome capture from field-collected faecal samples is a robust and reliable approach for population-wide studies of nonmodel organisms.
对濒危和隐密物种进行大规模的分子研究，往往受到高质量样本获取的限制。通过使用非侵入性采集的样本或博物馆保存的标本，可以减少对现代种群的压力，无需捕捉和处理活体动物。然而，此类样本中的内源性 DNA 含量较低，使得 shotgun 测序在经济上不可行。在这里，我们应用靶向富集方法从 65 个博物馆标本和 56 个非侵入性采集的两种濒危大型类人猿物种（格雷尔氏大猩猩和东部黑猩猩）粪便样本中获取线粒体基因组。我们表明，所应用的方法适用于内源性 DNA 含量差异较大的各种样本类型，将目标读取的比例提高了 300 多倍。通过系统评估对混合博物馆样本进行靶向富集过程中引入的偏倚，我们表明，对于比诱饵短或长的片段，捕获效率较低，尽管与带有大猩猩生成的诱饵的大猩猩片段相比，人类污染读取的比例在捕获后增加，但跳跃 PCR 的速率相当大，但可以通过双条形码方法进行控制。我们成功地从粪便样本中捕获了完整的线粒体基因组，但观察到随着诱饵和目标物种之间的序列差异增加，捕获效率降低。正如之前对博物馆标本所展示的那样，我们在这里证明了从野外采集的粪便样本中捕获线粒体基因组是一种用于非模式生物全种群研究的稳健可靠的方法。
