Evaluating the latest high-throughput molecular techniques for the exploration of microbial gut communities

The human gut microbiota has become the subject of an increasing amount of attention, due to an emerging understanding of its role in maintaining health throughout our lives. Since only a small proportion of the gut bacteria can be quantified using traditional plate culturing methods, culture-independent approaches are required for determining the structure of complex microbial communities. To avoid cloning bias and low phylotype coverage that affects amplicon cloning and sequencing strategies, high-throughput methods such as phylogenetic arrays and massively parallel sequencing are now being used to find more than just the most abundant taxa, at significantly lower costs and higher speeds. The target for these methods is the 16S ribosomal RNA gene that is present in all prokaryotes. Since the gene is too long to be sequenced using high-throughput methods, regions of high variability (from V1-V9) are selected for amplification and either direct sequencing, or hybridization against phylogenetic microarrays. In our recent study,1 we compared sequencing of amplified V4 and V6 regions using 454 FLX Pyrosequencing(2) with the HITChip, an oligonucleotide microarray for taxonomic profiling of human intestinal tract communities based on concatenations of known V1 and V6 regions.(3) We found good correlations between the phylogenetic classifications stemming from the two technologies, especially at lowerorder ranks (phylum, class, order, and to a lesser extent, family), which indicates high robustness of both approaches. However, the V6 regions proved to be much less suitable for taxonomic classification than the V4 region, probably due to this region simply being too variable. Although this study was, to our knowledge, the deepest sequencing of single gastrointestinal samples reported to date, the microbial richness levels had still not leveled out, with up to 1,800 unique phylotypes detected in one community. Encouragingly for studies with lower sequencing coverage per sample, we also noticed that a fifth of the sequencing depth (40,000 as opposed to 200,000 reads) was sufficient for capturing a majority of microbial diversity within a sample.