[Comparative analysis of soil microbial communities by pyrosequencing and DGGE].

OBJECTIVE

We aimed to assess the advantage and disadvantage of next-generation pyrosequencing and traditional Denaturing Gradient Gel Electrophoresis (DGGE) in fingerprinting analysis of soil microbial communities.

METHODS

We analyzed microbial compositions, abundance and diversity of typical grassland and forest soils by 16S rRNA gene-based pyrosequencing and DGGE to compare the accuracy and reproducibility of the two techniques on soil microbial communities.

RESULTS

For grassland soils, pyrosequencing technique revealed 22 phyla, 54 classes, 60 orders, 131 families and 350 genera; DGGE only detected 6 phyla, 9 classes, 8.orders, 10 families and 10 genera. The results show that DGGE greatly underestimated soil community compositions. Similar results were obtained for forest soils, and the detection sensitivity of pyrosequencing of forest soils was 3.8, 6.7, 6.4, 19.2 and 39.4 times higher than that of DGGE at the taxonomic levels of phylum, class, order, family and genera respectively. Furthermore, DGGE overwhelmingly overestimated the relative abundance of dominant microorganisms represented by the high-intensity bands, leading up to a 2000-fold difference. Both DGGE and pyrosequencing showed consistent results of microbial diversity changing patterns, although the DGGE-based diversity index was much lower than pyrosequencing.

CONCLUSION

Pyrosequencing thus provides more comprehensive and accurate fingerprints of soil microbial community structure than DGGE. DGGE only can represent a few numerically dominant phylotypes with apparent overestimation of their relative abundance in soil microbial communities.