Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences

Abstract

A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.

FIG. 1

DGGE analysis of individual clones and mixed PCR products. Vm, Gm, and Ws designations indicate PCR products derived from the original seven clones used to create the template mix (21). Lanes labeled a to g give examples of recovered clones whose DGGE mobilities match those of one of the original clones. The numbers between parentheses indicate the number of recovered clones showing the given DGGE mobility. pMix indicates that the original plasmid mix was used as a template in an amplification reaction using the 357f-GC and 518 primers, and the nMix sample used products of the βAMOf/βAMOr PCR as a template. The arrow indicates E. coli contamination. The poor amplification of clone Vm11 is due to a single mismatch with the 518r primer. The marker lanes (M) contained PCR-amplified 16S rDNA fragments of Lactococcus lactis and E. coli according to the method of Zwart et al. (27). The bands labeled A and B in the nMix sample were excised for DNA elution and reamplification using the 357f-GC and 518 primers. The DGGE analysis of these products is shown in the right panel, with lanes labeled according to the original excised band.

FIG. 2

Compressed alignment and distance matrix of original and novel clones. The names of clones derived in this study are shown in boldface type. The alignment was compressed by deleting constant characters (except positions adjacent to gaps). Dots denote no difference from the consensus sequence. Base numbering is according to E. coli convention (1) and is given vertically above the consensus sequence. Bases given in lowercase indicate characters that are not present in any of the original clones used in the template mixture. Boldface type, including heavy dots, designates positions for a potential chimera or heteroduplex correction. The grey shading represents the region of the mutational hot spot and the introduction of a foreign 3-bp sequence (CAG). Clones were ordered according to the relationship between novel clones and the original clones to which they are most closely related, and these levels of sequence divergence are shaded in grey and labeled with the appropriate original clone designation in the distance matrix. Both the alignment and the distance matrix were constructed with the SeqPup program (D. B. Gilbert, SeqPup, a biosequence editor and analysis application [ftp://iubio.bio.indiana.edu/molbio/seqpup/], 1996).