高通量测序和DGGE分析土壤微生物群落的技术评价

Comparative analysis of soil microbial communities by pyrosequencing and DGGE

摘要:

摘要:【目的】比较新一代高通量测序与传统的变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis，DGGE)指纹图谱技术，评价两种技术研究土壤微生物群落结构的优缺点。【方法】针对新西兰典型草地和森林土壤，以16S rRNA基因为标靶，通过高通量测序和DGGE技术分析土壤微生物群落的组成、丰度和多样性，比较两种方法在土壤微生物研究中的适用性。【结果】在不同的微生物分类水平，高通量测序草地土壤检测到22门，54纲，60目，131科，350属;而DGGE仅检测到6门，9纲，8目，10科，10属，表明DGGE显著低估了土壤微生物的群落组成。森林土壤也得到了类似规律，高通量测序的检测灵敏度是DGGE的3.8、6.7、6.4、19.2及39.4倍。进一步分析土壤中主要微生物类群的相对丰度，发现分类水平越低，高通量测序与DGGE的结果差异越大，尤其在科和属的水平上差异最大。以高通量测序结果为标准，DGGE明显高估了土壤中大多数微生物类群的相对丰度，最高可达2000倍。两种方法都表明草地土壤的多样性指数高于森林土壤，但DGGE多样性指数的绝对值远低于高通量测序结果。【结论】高通量测序能够较为全面和准确的反映土壤微生物群落结构，而DGGE仅能够反映有限的优势微生物类群，在很大程度上极可能低估土壤微生物的物种组成并高估其丰度。

Abstract:

Abstract:［Objective］ We aimed to assess the advantage and disadvantage of next-generation pyrosequencing and traditional Denaturing Gradient Gel Electrophoresis (DGGE) in fingerprinting analysis of soil microbial communities．［Methods］We analyzed microbial compositions，abundance and diversity of typical grassland and forest soils by 16S rRNA gene-based pyrosequencing and DGGE to compare the accuracy and reproducibility of the two techniques on soil microbial communities.［Results］ For grassland soils，pyrosequencing technique revealed 22 phyla，54 classes，60 orders，131 families and 350 genera; DGGE only detected 6 phyla，9 classes，8 orders，10 families and 10 genera. The results show that DGGE greatly underestimated soil community compositions．Similar results were obtained for forest soils，and the detection sensitivity of pyrosequencing of forest soils was 3.8，6.7，6.4，19.2 and 39.4 times higher than that of DGGE at the taxonomic levels of phylum， class， order，family and genera respectively． Furthermore， DGGE overwhelmingly overestimated the relative abundance of dominant microorganisms represented by the high-intensity bands，leading up to a 2000-fold difference． Both DGGE and pyrosequencing showed consistent results of microbial diversity changing patterns，although the DGGE-based diversity index was much lower than pyrosequencing.［Conclusion］ Pyrosequencing thus provides more comprehensive and accurate fingerprints of soil microbial community structure than DGGE. DGGE only can represent a few numerically dominant phylotypes with apparent overestimation of their relative abundance in soil microbial communities.