Application Note

pdf16S rRNA gene sequencing is commonly used for identification, classification and quantitation of microbes within complex biological mixtures such as environmental samples (ex marine water[1]) and gut samples (ex human gut microbiome[2]). The 16S rRNA gene is a highly conserved component of the transcriptional machinery of all DNA-based life forms[3] and thus is highly suited as a target gene for sequencing DNA in samples containing up to thousands of different species. Universal PCR primers can be designed to target the conserved regions of 16S making it possible to amplify the gene in a wide range of different microorganisms from a single sample. Conveniently, the 16S rRNA gene consists of both conserved and variable regions (Fig. 1). While the conserved region makes universal amplification possible, sequencing the variable regions allows discrimination between specific different microorganisms such as bacteria, archaea and microbial eukarya. Identification of viruses requires metagenomic sequencing (the direct sequencing of the total DNA extracted from a microbial community) due to their lack of the phylogenetic marker gene 16S.

 16S rRNA Sequencing

Fig 1 – Approximately 1.5 kb 16S rRNA gene of E.coli showing the nine variable regions that make it an ideal target as a phylogenetic marker gene[3].

Originally, studies of environmental samples required cultivation and isolation of species for identification, a laborious and time consuming process[4]. However, the coupling of 16S rRNA PCR with next-generation sequencing has enabled the study of many samples at low cost. Early 16S rRNA sequencing studies have already found many sequences which do not belong to any known cultured species, indicating that there are numerous non-isolated organisms and that cultivation based methods find only a small percentage of the bacterial and archaeal species in a sample[5]. Additionally, with the multiplexing of many samples and high depth of coverage afforded by today’s next-gen platforms, we can now analyze samples from comprehensive time series to quantify microbial community dynamics across many sites, or produce detailed 3D maps of microbial communities, as well as explore whether changes in rare or abundant species are associated with health and disease[6].

16S rRNA Gene Sequencing

Fig 2 – clustering of 5′ and 3′ reads from different environmental samples show that samples from a given environment type cluster together well[6].

Reads from next-gen sequencing can be BLASTED against curated databases such as The Ribosomal Database Project (RDP), GreenGenes, and SILVA for identification and classification. Related sequences are “clustered” and the number of representatives of each cluster counted. Clusters of similar sequences are referred to as “operational taxonomic units” (OTUs). OTU counts are summarized in a table of relative abundances for each organism in each sample[3]. To date, several analysis pipelines have been developed for analysis of 16S rRNA gene sequence data and two commonly used pipelines are QIIME and Mothur. QIIME takes users from their raw sequencing output through initial analyses such as OTU picking, taxonomic assignment, and construction of phylogenetic trees from representative sequences of OTUs, and through downstream statistical analysis, visualization, and production of publication-quality graphics.

LC Sciences offers a comprehensive 16SrRNA gene sequencing service for identification and classification of species in microbial samples. We use a dual zone (amplified zones V3 + V4) 16S rDNA fragment amplification strategy, sequence on the industry leading Illumina MiSeq platform and provide extensive data analysis including: sequencing data output statistics, sequence clustering into operational taxonomic units (OTU), diversity analysis, species classification and abundance analysis.

Recently, one of LC Sciences’ customers used 16S rRNA gene sequencing to study air microbial propagation in polluted areas where adverse weather conditions of cause a double pollution of dust and smog[7].

 


16S rRNA Gene Sequencing of Human Intestinal Microflora Low cost error removal from microarray synthesized DNA using immobilized MutS