Abstract

Despite the revolutionary advancements in DNA sequencing technology and cultivation techniques, few studies have been done to directly compare these methods. In this study, a 16S rRNA gene-based, integrative approach combining culture-independent techniques with culture-dependent methods was taken to investigate the bacterial community structure of coastal seawater collected from the Yellow Sea, Korea. For culture-independent studies, we used the latest model pyrosequencer, Roche/454 Genome Sequencer FLX Titanium. Pyrosequencing captured a total of 52 phyla including 27 candidate divisions from the water column, whereas the traditional cloning approach captured only 15 phyla including 2 candidate divisions. In addition, of 878 genera retrieved, 92.1% of the sequences were unique to pyrosequencing. For culture-dependent analysis, plate culturing, plate washing, enrichment, and high-throughput culturing (HTC) methods were applied. Phylogenetic analysis showed that the plate-washing clones formed a cluster devoid of any previously cultured representatives within the family Rhodobacteraceae. One HTC isolate (SF293) fell into the OM182 clade, which was not recovered by other culturing methods described here. By directly comparing the sequences obtained from cultures with those from culture-independent work, we found that only 33% of the culture sequences were identical to those from clone libraries and pyrosequences. This study presents a detailed comparison of common molecular and cultivation techniques available in microbial ecology. As different methods yielded different coverage, we suggest choosing the approach after carefully examining the scientific questions being asked.

• Keywords: diversity; pyrosequencing; Roche/454 GS-FLX titanium; cloning; high-throughput culturing (HTC); DGGE; plate-washing PCR (PWPCR)