Illumina-based analysis of microbial community diversity

Abstract

Microbes commonly exist in milieus of varying complexity and diversity. Although cultivation-based techniques have been unable to accurately capture the true diversity within microbial communities, these deficiencies have been overcome by applying molecular approaches that target the universally conserved 16S ribosomal RNA gene. The recent application of 454 pyrosequencing to simultaneously sequence thousands of 16S rDNA sequences (pyrotags) has revolutionized the characterization of complex microbial communities. To date, studies based on 454 pyrotags have dominated the field, but sequencing platforms that generate many more sequence reads at much lower costs have been developed. Here, we use the Illumina sequencing platform to design a strategy for 16S amplicon analysis (iTags), and assess its generality, practicality and potential complications. We fabricated and sequenced paired-end libraries of amplified hyper-variable 16S rDNA fragments from sets of samples that varied in their contents, ranging from a single bacterium to highly complex communities. We adopted an approach that allowed us to evaluate several potential sources of errors, including sequencing artifacts, amplification biases, non-corresponding paired-end reads and mistakes in taxonomic classification. By considering each source of error, we delineate ways to make biologically relevant and robust conclusions from the millions of sequencing reads that can be readily generated by this technology.

Bacterial Diversity in Bentonites, Engineered Barrier for Deep Geological Disposal of Radioactive Wastes

“…The latter method provides longer reads, analyzing almost the full 16S rRNA gene, which makes allowance to go deeper into the taxonomy, up to genera and species. However, it is necessary to analyze a high amount of clones per sample to reach sufficient reads to completely characterize the most abundant representatives of the community [26]. The work presented in this study is focused on the bacterial diversity analysis of the already mentioned Spanish bentonites, by two different culture-independent molecular approaches based on the 16S ribosomal RNA gene analysis via (i) Illumina sequencing platform and (ii) cloning and sequencing, to get a better knowledge about what kind of microorganisms are present and how these microorganisms can potentially influence the performance of the nuclear waste repository.…”

Automated and accurate estimation of gene family abundance from shotgun metagenomes

“…High-throughput amplicon sequencing of taxonomically informative loci (e.g., small-subunit rRNA genes) has shed light on the tremendous diversity and distribution of microbial communities in nature [1] and revealed patterns and processes related to the assembly [2], diversification [3], and scaling [4] of these communities. Shotgun sequencing of total DNA from microbial communities (i.e., metagenomics) is gaining popularity, as it provides insight into the genomic composition of microbes as they exist in nature and enables inference of the community's biological functional potential [5].…”

Contemporary molecular tools in microbial ecology and their application to advancing biotechnology

“…Additionally, it is difficult to trace the taxonomic source of short reads from a complex community. The presence of unwanted host DNA or environmental contamination in metagenomic sequencing data is another important issue (Schmieder andEdwards 2011, Degnan andOchman 2012).…”