Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification.
Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification.
复制标题
DOI：
10.1186/2047-217x-2-4
复制
发表时间：
2013-03-27
期刊：
影响因子：
9.2
通讯作者：
Huang Q
中科院分区：
文献类型：
--
作者：
Zhou X;Li Y;Liu S;Yang Q;Su X;Zhou L;Tang M;Fu R;Li J;Huang Q
下一代测序(NGS)技术与经典的DNA条形码方法相结合，能够快速可靠地测量混合环境样本的生物多样性。然而，几乎所有现有的NGS协议中涉及的PCR扩增都不可避免地引入分类学偏见。在本研究中，我们开发了一种新的无PCR扩增的Illumina管道来分析陆地节肢动物群落。线粒体富集后，Illumina鸟枪测序，以超高的测序量，能够恢复细胞色素c氧化酶亚单位1(COI)条形码序列，这使得能够高保真地估计陆地昆虫群落的物种组成。对于15.5 GBP的Illumina数据，在37个输入的操作分类单元(OTU)中，无论是否使用参考条形码文库，分别检测到约97%和92%，而后者仅发现1个新的OTU。此外，大量样本中物种的测序量和总生物量之间的相关性相对较强，这表明了一种潜在的解决方案来揭示相对丰度。新的Illumina无聚合酶链式反应DNA代谢编码管道能够检测小型节肢动物样本，并倾向于避免大多数(如果不是全部)假阳性，这表明它在生物多样性相关监测方面具有巨大的潜力，例如在生物监测项目中。然而，为了将新的管道应用于分析具有较高多样性的节肢动物群落，线粒体浓缩方面可能需要进一步的改进。
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.