Department of Integrative Biology and Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada N1G 2W1;
Department of Integrative Biology and Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada N1G 2W1;Department of Microbiology, Mansoura University, Mansoura, Egypt 35516;
Proc Natl Acad Sci U S A. 2014 Jun 3;111(22):8007-12. doi: 10.1073/pnas.1406468111. Epub 2014 May 7.
Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works.
传统的生态系统样本组成评估是基于个体的形态学或 DNA 条码鉴定。这两种方法都既昂贵又耗时，特别是当应用于生态调查中通常包含的大量样本和分类群时。下一代测序方法可以通过同时对大量混合物中的所有分类群的标本进行测序来克服个体标本分离和鉴定的瓶颈。在这里，我们应用多个平行扩增引物、多个 DNA 条码标记物、454 焦磷酸测序和 Illumina MiSeq 测序对同一样本进行测序，以最大限度地恢复节肢动物宏基因组和细菌及其他微生物微生物组的混合样本。我们用一个含有 1066 个形态上可区分的热带陆地生态系统节肢动物的复杂样本验证了这种方法，该样本具有高度的分类多样性。多扩增子下一代 DNA 条码能够从环境混合样本中恢复 91%的可区分个体的序列，以及许多作为不可区分组织存在的物种。与对每个个体标本进行常规 Sanger 测序相比，454 焦磷酸测序能够恢复 10 个更多的节肢动物科和 30 个更多的物种。使用其他基因座（16S 和 18S 核糖体 DNA 基因区域）还增加了检测与这些陆地节肢动物相关的微生物物种的能力。这种方法大大减少了对生态系统样本之间基于 DNA 的生物多样性进行比较所需的时间和金钱。这种方法为更经济实惠且更具容量的生态和进化研究打开了大门，适用于广泛的社会经济问题，以及对世界运作方式的基本理解。
