Natural Systems and Sustainability Area, Universidad EAFIT, Medellín, Antioquia, Colombia.
Syndesis Health, Palm Beach Gardens, Florida, United States.
PeerJ. 2024 Sep 25;12:e17887. doi: 10.7717/peerj.17887. eCollection 2024.
The Neotropics harbors the largest species richness of the planet; however, even in well-studied groups, there are potentially hundreds of species that lack a formal description, and likewise, many already described taxa are difficult to identify using morphology. Specifically in small mammals, complex morphological diagnoses have been facilitated by the use of molecular data, particularly from mitochondrial sequences, to obtain accurate species identifications. Obtaining mitochondrial markers implies the use of PCR and specific primers, which are largely absent for non-model organisms. Oxford Nanopore Technologies (ONT) is a new alternative for sequencing the entire mitochondrial genome without the need for specific primers. Only a limited number of studies have employed exclusively ONT long-reads to assemble mitochondrial genomes, and few studies have yet evaluated the usefulness of such reads in multiple non-model organisms.
We implemented fieldwork to collect small mammals, including rodents, bats, and marsupials, in five localities in the northern extreme of the Cordillera Central of Colombia. DNA samples were sequenced using the MinION device and Flongle flow cells. Shotgun-sequenced data was used to reconstruct the mitochondrial genome of all the samples. In parallel, using a customized computational pipeline, species-level identifications were obtained based on sequencing raw reads (Whole Genome Sequencing). ONT-based identifications were corroborated using traditional morphological characters and phylogenetic analyses.
A total of 24 individuals from 18 species were collected, morphologically identified, and deposited in the biological collection of Universidad EAFIT. Our different computational pipelines were able to reconstruct mitochondrial genomes from exclusively ONT reads. We obtained three new mitochondrial genomes and eight new molecular mitochondrial sequences for six species. Our species identification pipeline was able to obtain accurate species identifications for up to 75% of the individuals in as little as 5 s. Finally, our phylogenetic analyses corroborated the identifications from our automated species identification pipeline and revealed important contributions to the knowledge of the diversity of Neotropical small mammals.
This study was able to evaluate different pipelines to reconstruct mitochondrial genomes from non-model organisms, using exclusively ONT reads, benchmarking these protocols on a multi-species dataset. The proposed methodology can be applied by non-expert taxonomists and has the potential to be implemented in real-time, without the need to euthanize the organisms and under field conditions. Therefore, it stands as a relevant tool to help increase the available data for non-model organisms, and the rate at which researchers can characterize life specially in highly biodiverse places as the Neotropics.
新热带地区拥有地球上最大的物种丰富度；然而，即使在研究充分的类群中，也可能有数百种尚未正式描述的物种，同样，许多已经描述的分类单元也很难仅通过形态学进行识别。特别是在小型哺乳动物中，复杂的形态学诊断已经通过使用分子数据（特别是线粒体序列）来获得准确的物种鉴定得到了促进。获得线粒体标记物意味着需要使用 PCR 和特定引物，而对于非模式生物，这些引物在很大程度上是不存在的。牛津纳米孔技术（ONT）是一种无需特定引物即可对整个线粒体基因组进行测序的新选择。只有少数研究完全使用 ONT 长读长来组装线粒体基因组，并且很少有研究评估此类读长在多种非模式生物中的有用性。
我们在哥伦比亚中央山脉北部的五个地点开展了实地工作，以收集小型哺乳动物，包括啮齿动物、蝙蝠和有袋动物。使用 MinION 设备和 Flongle 流池对 DNA 样本进行测序。使用 Shotgun 测序数据来重建所有样本的线粒体基因组。同时，使用定制的计算管道，基于测序原始读数（全基因组测序）获得种级别的鉴定。ONT 基鉴定结果通过传统形态学特征和系统发育分析得到证实。
共收集了 18 个物种的 24 只个体，进行了形态学鉴定，并保存在 EAFIT 大学的生物收藏中。我们的不同计算管道能够仅从 ONT 读长重建线粒体基因组。我们获得了三个新的线粒体基因组和六个物种的八个新的分子线粒体序列。我们的物种鉴定管道能够在短短 5 秒内获得多达 75%个体的准确物种鉴定结果。最后，我们的系统发育分析证实了我们自动化物种鉴定管道的鉴定结果，并揭示了对新热带小型哺乳动物多样性知识的重要贡献。
本研究能够评估使用仅 ONT 读长从非模式生物重建线粒体基因组的不同管道，并在多物种数据集上对这些方案进行基准测试。所提出的方法可以由非专家分类学家使用，并且有可能在没有需要处死生物体的情况下实时实施，并在野外条件下实施。因此，它是一个相关的工具，可以帮助增加非模式生物的可用数据，以及研究人员描述生命的速度，特别是在新热带等生物多样性极高的地方。
