Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study.
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.
Zhang
Han-Bo
HB
Laboratory of Conservation and Utilization for Bio-resources, and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, PR China. zhhb@ynu.edu.cn
Xu
Chan-Wen
CW
Wang
Miao-Miao
MM
Li
Tao
T
Zhao
Zhi-Wei
ZW
D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000191:economics / Q000379:methods* / Q000592:standards; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D004262:DNA Restriction Enzymes