环境样本中微型和微微型浮游植物高通量测序的引物优化

Optimization of high-throughput sequencing primers for nanophytoplankton and picophytoplankton in environmental samples

分别以18Sr DNA的V4区和V9区为目标基因,采用高通量测序平台和生物信息学方法,分析海水样品中微型和微微型浮游植物多样性。利用在线分析软件对V4(F/R)、V9(F/R)和C4(F/R)3对引物的敏感性、特异性进行了评估和比较,发现自行设计的引物V4(F/R)对真核藻类的扩增特异性高于V9(F/R)和C4(F/R)。高通量测序结果显示,检测样品平均获得68834条原始序列,高质量数据占99%以上,获得基因注释的序列数达94%以上。3对引物V4(F/R)、V9(F/R)、C4(F/R)鉴定的平均微型/微微型浮游植物OTUs数分别为78、42、58,引物V4(F/R)鉴定效率相对较高,同时对细小微胞藻(Micromonas pusilla)、(金牛微球藻Ostreococcus tauri)、密球藻(Pycnococcus provasolii)、抑食金球藻(Aureococcus anophagefferens)、赤潮异弯藻(Heterosigma akashiwo)等优势种检出频率高于引物V9(F/R)。相对已发表的2对引物,设计的引物V4(F/R)对海洋微型和微微型藻类多样性检测更为高效。

In this study, nanophytoplankton and picophytoplankton diversity in seawater samples was analyzed using a high- throughput sequencing platform and a series of bioinformatics tools, based on the V4 and V9 region of 18S rDNA as the target gene. High-throughput sequencing, which is considered as one of the most important tools in genomies research, is widely applied in the field of marine nanophytoplankton and picophytoplankton diversity studies. We successfully obtained a pair of nanophytoplankton and picophytoplankton PCR primers V4（F/R） by analyzing the nucleic acid database and using a series of bioinformatics tools. Two pairs of universal primers were also selected for comparative analysis, which amplified variable region V4 and V9 of the small subunit nuclear ribosomal DNA （ SSU nrDNA） .The sensitivity and specificity of PCR primers V4（ F/R）, V9（ F/R）, and C4（F/R） were also evaluated and compared using online bioinformatics software. The results showed that the amplification specificity of primer pair V4（F/R） was better than that of V9（F/R） and C4（F/R） in eukaryotie algae. High-throughput sequencing results showed that 68834 raw tags were amplified by the primers, 99% of which were effective tags. Sequences of more than 94% of the effective tags were identified by Ribosomal Database ProjectClassifier, among which 308 operational taxonomic units （OTUs） of one sample were used for further analysis. The average numbers of nanophytoplankton and picophytoplankton OTUs amplified by V4 （F/R）, V9 （F/R）, and C4 （F/R）, were 78, 42, 58, respectively. The primer pair V4（F/R） was found to have higher sensitivity and specificity for amplifying nanophytoplankton and picophytoplankton, including Micromonas pusilla, Ostreococcus tauri, Pycnococcus provasolii, Aureococcus anophagefferens,and Heterosigma akashiwo. The V4 region from the environmental eukaryotic 18S rDNA gene could be suitable for high-throughput sequencing technology, and it was also a good target gene formarine nanophytoplankton and picophytoplankton identification. This study demonstrates the use of a simple, rapid, high sensitivity, and low-cost technology to explore marine nanophytoplankton and picophytoplankton diversity. Moreover, it also provides a reference for the early warning and control of brown tide disasters.