Preferential ligation during TA-cloning of multitemplate PCR products--a factor causing bias in microbial community structure analysis.

The description of microbial community structure is always biased by the selectivity of the methods applied. Although TA cloning of PCR amplified community DNA is one of the most widely used techniques in bacterial community analysis, no thorough comparative testing has been carried out on different TA cloning systems. In this study, we measured and compared the selectivity of two widely used TA-cloning kits in experimental setups where the length heterogeneity of the inserts modeled the natural length variation of the 16S rRNA gene and the 16S-23S intergenic spacer region. Both TOPO TA (Invitrogen, CA USA) and pGem-T vector system (Promega, WI USA) cloning kits showed significant and reproducible insert size related selectivity. The effect of ligation time and temperature was also studied in case of the pGem-T vector system. We compared the performance of the two cloning kits on an environmental sample, along with a semiquantitative community fingerprinting method to gain reference data free of cloning bias. The two clone libraries showed significantly different compositions, and were also differing from the community structure revealed by length heterogeneity PCR.