Zhang Han-Bo, Xu Chan-Wen, Wang Miao-Miao, Li Tao, Zhao Zhi-Wei
Laboratory of Conservation and Utilization for Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, PR China.
Can J Microbiol. 2008 Jun;54(6):479-82. doi: 10.1139/w08-031.
We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36-53 OTUs using the DOTUR program when sequence similarities of 95%-100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon-Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.
我们基于16S rRNA基因的限制性片段长度多态性（RFLP）模式对克隆分配的误差进行了定量评估。从一个16S rRNA基因文库中随机选取了80个克隆，并根据三种四聚体限制性内切酶RsaI、BsuRI和HinfI无法区分的酶切模式将它们归类为35个操作分类单元（OTU）。然后对所有这些克隆进行测序，并使用DOTUR程序在序列相似度为95%-100%时将它们重新归类为36-53个OTU。分配相同的克隆数量在53到61之间，百分比在66.3%到76.3%之间。通过RFLP分析观察到的细菌群落的香农-韦弗指数为2.75，与DOTUR在97%序列相似度时估计的指数相等。与在97%序列相似度下用DOTUR程序分配的克隆相比，RFLP分析仅正确分配了61个克隆（76.3%）。6个克隆（7.5%）在门水平上被错误分配，13个克隆（16.2%）在较低分类等级上的系统发育位置不同。
