Data from: Long-range PCR allows sequencing of mitochondrial genomes from environmental DNA

As environmental DNA (eDNA) from macro-organisms is often assumed to be highly degraded, current eDNA assays target small DNA fragments to estimate species richness by metabarcoding. A limitation of this approach is the inherent lack of unique species-specific single-nucleotide polymorphisms available for unequivocal species identification.
We designed a novel primer pair capable of amplifying whole mitochondrial genomes and evaluated it in silico for a wide range of ray-finned fishes (Class: Actinopterygii). We tested the primer pair using long-range PCR and Illumina sequencing in vitro on a mock community of fish species assembled from pooling genomic DNA extracted from tissues. In situ we utilized long-range PCR and Illumina sequencing to generate fragments between 16 and 17 kb from eDNA extracted from filtered water samples. Water samples were sourced from a mesocosm experiment and from a natural stream.
We validated our method in silico for 61 orders of Actinopterygii; we successfully sequenced mitogenomes in vitro from all six species in our mock community. In situ we recovered mitogenomes for all species present in our mesocosms. We additionally recovered mitogenomes from 10 of 12 species caught at the time of water sampling and two species previously only detected from eDNA metabarcoding of short DNA fragments from a natural stream.
Successful amplification of large fragments (>16 kb) from eDNA demonstrates that not all eDNA is highly degraded. Sequencing whole mitogenomes from filtered water samples will alleviate many problems associated with identification of species from short-fragment PCR amplicon-based methods.

由于大型生物来源的环境DNA（environmental DNA，eDNA）通常被认为降解程度极高，当前的eDNA检测技术多通过元条形码测序（metabarcoding）靶向小型DNA片段，以此估算物种丰富度。该方法的局限性在于，其本质上缺乏可用于明确物种鉴定的物种特异性单核苷酸多态性（single-nucleotide polymorphisms）。
本研究设计了一种可扩增完整线粒体基因组的新型引物对，并通过计算机模拟（in silico）对辐鳍鱼纲（Actinopterygii）的多个类群进行了评估。我们通过体外实验（in vitro），将从组织中提取的基因组DNA混合构建鱼类模拟群落，利用长距PCR（long-range PCR）与Illumina测序技术对该引物对进行了验证。在原位实验（in situ）中，我们同样采用长距PCR与Illumina测序技术，从过滤水样提取的eDNA中扩增得到了16~17 kb的片段。本研究的水样来源于中宇宙实验与一条天然溪流。
我们通过计算机模拟（in silico）验证了该方法对辐鳍鱼纲61个目物种的适用性；在体外实验中，我们成功对模拟群落中的全部6个物种完成了线粒体基因组（mitogenome）测序。原位实验中，我们成功获取了中宇宙实验体系内所有物种的线粒体基因组。此外，我们从水样采集时捕获的12个物种中的10个，以及此前仅通过天然溪流短DNA片段eDNA元条形码测序检测到的2个物种中，均成功获取了线粒体基因组。
从eDNA中成功扩增得到大片段（>16 kb）DNA的结果证明，并非所有的环境DNA都发生了高度降解。通过过滤水样获取完整线粒体基因组序列的方法，将有效缓解基于短片段PCR扩增子的物种鉴定方法所存在的诸多问题。