Which Microbial Communities Are Present? Sequence-Based Metagenomics

Abstract

Analysis of the 16S rRNA gene sequences isolated from environmental samples has demonstrated the extraordinary microbial diversity missed by traditional culture-based methodologies (Tringe and Rubin, 2005). Figure 8.1 summarises several metagenomic sequencing approaches. Although there are many culture-independent methodologies capable of assess- ing microbial diversity discussed in this volume, none of these tools can identify all themicroorganisms or functions in a high-throughput way. PCR, FISH and microar- rays can only identify organisms or genes for which probes have been prepared and always miss unknown genes. When using universal primers for the 16S rRNA gene, DGGE and other post-PCR techniques use DNA amplified from nearly all the microorganisms in the sample, but this technique is limited by band resolu- tion and species identity is only determined for bands that have been individually excised and sequenced. Advances in sequencing technology over the past decade have increased sequencing throughput over 50-fold when compared to Sanger-based sequencing, making it possible to produce gigabases of sequencing information in just days (MacLean et al., 2009). The corresponding decreases in sequencing costs have made it feasible to employ enough sequencing runs to sequence the total DNA extracted from environmental samples, circumventing many of the limitations of other