Metagenomic pyrosequencing and microbial identification

Abstract

Background: The Human Microbiome Project has ushered in a new era for human metagenomics and high-throughput next-generation sequencing strategies.

Content: This review describes evolving strategies in metagenomics, with a special emphasis on the core technology of DNA pyrosequencing. The challenges of microbial identification in the context of microbial populations are discussed. The development of next-generation pyrosequencing strategies and the technical hurdles confronting these methodologies are addressed. Bioinformatics-related topics include taxonomic systems, sequence databases, sequence-alignment tools, and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is summarized with respect to potential pyrosequencing applications.

Summary: Both the approach of 16S rDNA amplicon sequencing and the whole-genome sequencing approach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to tackle such vast amounts of microbiological sequence diversity. Metagenomics, or genetic studies of microbial communities, may ultimately contribute to a more comprehensive understanding of human health, disease susceptibilities, and the pathophysiology of infectious and immune-mediated diseases.

Figure 1. Pyrosequencing chemistry

This figure shows the biochemical reactions and enzymes involved in the generation of light signals by DNA pyrosequencing (18). Each peak in the pyrograms represents a pulse of light detected in the instrument. ATP, adenosine triphosphate; ADP, adenosine diphosphate; dNDP, deoxy-nucleotidyl diphosphate; dNMP, deoxy-nucleotidyl monophosphate; PPi, pyrophosphate. Adapted from www.biotagebio.com

Figure 2. Conserved and hypervariable regions in the 16S rRNA gene

The interspersed conserved regions (C1–C9) are shown in gray, and the hypervariable regions (V1–V9) are depicted in different colors. An example of primer selection for DNA amplification and sequencing-based microbial identification is provided in the figure (V4 subregion with pink circles and arrows representing primer binding sites).

Figure 3. Deployment of 454 sequencing technology for metagenomics

A proposed pipeline is shown for high throughput 454 sequencing and associated bio-informatics strategies in metagenomics. Each box represents a discrete step in the process using either whole genome sequencing (WGS) or 16S rDNA amplicon sequencing. Note that WGS may be performed with prior whole genome amplification (WGA) or without prior amplification.