近期，中科院武汉病毒研究所周宁一研究员课题组在基因组内16S rRNA基因的差异性而导致的原核生物多样性高估研究方面取得重要进展。相关结果发表在10月的微生物学刊物Applied and Environmental Microbiology上，并被本期杂志选为亮点文章（Spotlight: Articles of Significant Interest Selected from This Issue by the Editors）。
自从Carl Woese应用16S rRNA基因来确定原核生物的亲缘关系，这个方法已成为原核生物系统发育和分子生态学研究中公认的经典标准。然而，该基因在原核生物内往往同时存在多个拷贝，而且拷贝之间的基因序列并不完全一致。因此，在原核生物生态学研究中，基于16S rRNA基因的菌群多样性分析会引起一定程度的高估。该组从目前2013个测序的原核生物基因组中，对16S rRNA基因的拷贝数及基因组内部异质性做了详细的分析研究，发现细菌基因组中包含1-15个拷贝，古菌基因组中包含1-4个拷贝。在952个基因组（隶属于585个种）内发现16S rRNA基因存在异质性。并发现16S rRNA基因不同区域存在不同程度的异质性，因而利用不同区域的序列进行基于焦磷酸测序的分子生态学研究会造成不同程度的多样性高估。
本研究指出，对于经常用来进行焦磷酸测序的区域中，V4-V5区域显示了最低的高估程度（约为3.0%），而V6区域的高估程度最高（约为13%）。因此在原核生物分子生态学研究中，16S rRNA基因的V4-V5区域更适合作为焦磷酸测序的目的片段。这是迄今为止最全面的关于16S rRNA基因的基因组内差异的研究，本研究不但提供了在针对不同的16S rRNA基因区段时不同丰度的高估程度，而且提出了可利用V4-V5区来降低这一高估（生物谷Bioon.com）。
生物谷推荐的英文摘要
Applied and Environmental Microbiology doi: 10.1128/AEM.01282-13
Intragenomic Heterogeneity of 16S rRNA Genes Causes Overestimation of Prokaryotic Diversity
Dong-Lei Suna， Xuan Jianga， Qinglong L. Wub and Ning-Yi Zhoua
Ever since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes， this method has been regarded as the “gold standard” in both microbial phylogeny and ecology studies. However， intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study， 2，013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species)， with 87.5% of the divergence detected being below the 1% level. In particular， some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that， at the unique level， full-length 16S rRNA genes can produce an overestimation of as much as 123.7%， while at the 3% level， an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions， with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods， due to its intragenomic heterogeneity， but also recommends that， for bacteria， this overestimation be minimized using primers targeting the V4 and V5 regions.
