Pyrosequencing as a tool for better understanding of human microbiomes

Abstract

Next-generation sequencing technologies have revolutionized the analysis of microbial communities in diverse environments, including the human body. This article reviews several aspects of one of these technologies, the pyrosequencing technique, including its principles, applications, and significant contribution to the study of the human microbiome, with especial emphasis on the oral microbiome. The results brought about by pyrosequencing studies have significantly contributed to refining and augmenting the knowledge of the community membership and structure in and on the human body in healthy and diseased conditions. Because most oral infectious diseases are currently regarded as biofilm-related polymicrobial infections, high-throughput sequencing technologies have the potential to disclose specific patterns related to health or disease. Further advances in technology hold the perspective to have important implications in terms of accurate diagnosis and more effective preventive and therapeutic measures for common oral diseases.

Keywords: human microbiome; next-generation DNA sequencing; oral microbiome; pyrosequencing.

Fig. 1

The 454 pyrosequencing approach.

Fig. 2

Variability within the 16S rRNA gene. From prealigned sequenced >1,200-bp downloaded from RDP, the variability, measured as Shannon information entropy, was calculated at each sequence position, using only positions without a gap in E. coli. The graph shows the Shannon entropy (Y-axis) averaged over 50-bp windows, centered at each position in the gene (X-axis). Shannon entropy at position x was calculated as – Σ p(xi) log2 p(xi), where p(xi) denotes the frequency of nucleotide i. Figure reproduced with permission from Andersson et al. (61).

Fig. 3

Shared abundant phylotypes in three oral microbiomes and their relative abundance. Relative abundance of shared phylotypes within an individual microbiome. Only abundant phylotypes that contributed to at least 0.1% of the individual microbiome are shown. The most abundant phylotypes (≥0.5% of the microbiome) are grouped separately in the upper panel. Phylotypes were defined as OTUs clustering sequences at a 3% genetic difference. The highest taxon (in most cases, genus) at which the OTU was identified is shown together with the cluster identification number. Different colors indicate three different microbiomes, S1, S2, and S3, respectively. Figure reproduced with permission from Zaura et al. (49).