High‐throughput sequencing has become an accurate method for the identification of species present in soil, water, faeces, gut or stomach contents. However, information at the species level is limited due to the choice of short barcodes and based on the idea that DNA is too degraded to allow longer sequences to be amplified. We have therefore developed a long DNA metabarcoding method based on the sequencing of short reads followed by de novo assembly, which can precisely identify the taxonomic groups of organisms associated with complex diets, such as omnivorous individuals. The procedure includes 11 different primer pairs targeting the COI gene, the large subunit of the ribulose‐1,5‐bisphosphate carboxylase gene, the maturase K gene, the 28S rRNA and the trnL‐trnF chloroplastic region. We validated this approach using 32 faeces samples from an omnivorous reptile, the European pond turtle (Emys orbicularis, L. 1758). This metabarcoding approach was assessed using controlled experiments including mock communities and faecal samples from captive feeding trials. The method allowed us to accurately identify prey DNA present in the diet of the European pond turtles to the species level in most of the cases (82.4%), based on the amplicon lengths of multiple markers (168–1,379 bp, average 546 bp), and produced by de novo assembly. The proposed approach can be adapted to analyse various diets, in numerous conservation and ecological applications. It is consequently appropriate for detecting fine dietary variations among individuals, populations and species as well as for the identification of rare food items.

中文翻译：

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一种强大的长元条形码方法，用于从欧洲池塘龟的粪便分析中确定复杂的饮食 (Emys orbicularis, L. 1758)

高通量测序已成为鉴定土壤、水、粪便、肠道或胃内容物中存在的物种的准确方法。然而，由于选择了较短的条形码，并且基于 DNA 降解过度而无法扩增更长序列的想法，物种水平的信息是有限的。因此，我们开发了一种基于短读长测序和从头组装的长 DNA 元条形码方法，该方法可以精确识别与复杂饮食相关的生物分类群，例如杂食性个体。该程序包括针对 COI 基因、核酮糖-1,5-二磷酸羧化酶基因的大亚基、成熟酶 K 基因、28S rRNA 和trnL-trnF 的11 对不同引物对叶绿体区域。我们使用来自杂食性爬行动物欧洲池龟 ( Emys orbicularis) 的32 个粪便样本验证了这种方法, L. 1758)。这种元条形码方法使用受控实验进行评估，包括模拟社区和圈养喂养试验的粪便样本。该方法使我们能够根据多个标记的扩增子长度（168–1,379 bp，平均 546 bp），在大多数情况下（82.4%）将欧洲池龟饮食中存在的猎物 DNA 准确识别到物种水平，并由从头组装生产。所提出的方法可适用于在众多保护和生态应用中分析各种饮食。因此，它适用于检测个体、种群和物种之间的细微饮食差异以及稀有食品的识别。