BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, Guangdong Province 518083, China.
Gigascience. 2013 Mar 27;2(1):4. doi: 10.1186/2047-217X-2-4.
Next-generation-sequencing (NGS) technologies combined with a classic DNA barcoding approach have enabled fast and credible measurement for biodiversity of mixed environmental samples. However, the PCR amplification involved in nearly all existing NGS protocols inevitably introduces taxonomic biases. In the present study, we developed new Illumina pipelines without PCR amplifications to analyze terrestrial arthropod communities.
Mitochondrial enrichment directly followed by Illumina shotgun sequencing, at an ultra-high sequence volume, enabled the recovery of Cytochrome c Oxidase subunit 1 (COI) barcode sequences, which allowed for the estimation of species composition at high fidelity for a terrestrial insect community. With 15.5 Gbp Illumina data, approximately 97% and 92% were detected out of the 37 input Operational Taxonomic Units (OTUs), whether the reference barcode library was used or not, respectively, while only 1 novel OTU was found for the latter. Additionally, relatively strong correlation between the sequencing volume and the total biomass was observed for species from the bulk sample, suggesting a potential solution to reveal relative abundance.
The ability of the new Illumina PCR-free pipeline for DNA metabarcoding to detect small arthropod specimens and its tendency to avoid most, if not all, false positives suggests its great potential in biodiversity-related surveillance, such as in biomonitoring programs. However, further improvement for mitochondrial enrichment is likely needed for the application of the new pipeline in analyzing arthropod communities at higher diversity.
下一代测序（NGS）技术与经典 DNA 条码方法相结合，实现了对混合环境样本生物多样性的快速和可靠测量。然而，几乎所有现有的 NGS 协议中涉及的 PCR 扩增不可避免地会引入分类偏差。在本研究中，我们开发了新的无需 PCR 扩增的 Illumina 管道，用于分析陆地节肢动物群落。
线粒体富集直接进行 Illumina 鸟枪法测序，超高测序量，能够恢复细胞色素 c 氧化酶亚基 1（COI）条码序列，从而以高保真度估计陆地昆虫群落的物种组成。使用 15.5 Gbp Illumina 数据，无论是否使用参考条码库，大约有 97%和 92%的输入操作分类单元（OTUs）被检测到，而后者只发现了 1 个新的 OTU。此外，对于大块样本中的物种，测序量与总生物量之间存在较强的相关性，这表明该方法可能是揭示相对丰度的一种潜在解决方案。
新的 Illumina 无 PCR 管道在 DNA 宏条形码中检测小型节肢动物标本的能力，以及它避免大多数（如果不是全部）假阳性的趋势，表明其在与生物多样性相关的监测（如生物监测计划）中具有巨大的潜力。然而，新管道在分析更高多样性的节肢动物群落时，可能需要进一步改进线粒体富集。
