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16S rRNA Gene Sequencing Service

Introduction The 16S rRNA gene is a highly conserved component of the transcriptional machinery of all DNA-based life forms and thus is highly suited as a target gene for sequencing DNA in samples containing up to thousands of different species. 16S rRNA gene sequencing is commonly used for identification, classification and quantitation of microbes in complex biological mixtures such as environmental samples like water, soil or air, and microbiome samples such as milk or feces. 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. Conveniently, the 16S ribosomal RNA gene consists of both highly conserved and variable regions. 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. While the conserved region makes universal amplification possible, subsequent sequencing of the variable regions allows discrimination between specific microorganisms such as bacteria, archaea and microbial eukarya. LC Sciences offers a comprehensive 16S ribosomal RNA gene sequencing service for identification and classification of species in microbial samples. LC Sciences has developed a dual zone amplification strategy, targeting the V3 and V4 variable regions. Sample bar-coding enables multiplexing of hundreds of different samples in a single sequencing run, keeping per sample costs low. Sequencing is performed on the industry leading Illumina MiSeq platform and extensive data analysis is provided including: sequencing data output statistics, sequence clustering into operational taxonomic units or OTUs, diversity analysis, classification and abundance analysis. An important distinction of LC Sciences’ service is that we perform the classification analysis on 7 taxonomy levels, all the way down to species level. Species level ID is enabled through the curation of several genomic databases and advanced bioinformatics. OTU Analysis An operational taxonomic unit or OTU is a measure of microbial diversity commonly used when performing 16S sequencing analysis. Sequence data is clustered into OTUs based on sequence similarity and each OTU generally represents a distinct species. OTU Venn diagrams can be used to show the unique OTUs in different subsets of sample groups. Species Accumulation Curve Statistical analysis of OTU data also generates a species accumulation curve which is a method to estimate the number of additional OTUs that may be discovered through further effort and can be an indication of adequacy or deficiency in number of samples analyzed, similar to power analysis. Diversity Analysis LC Sciences’ comprehensive service includes both Alpha and Beta diversity analysis. Alpha diversity is a measurement of the variety of organisms that inhabit a defined region or habitat. LC Sciences calculates diversity based on the Shannon, Simpson, Chao1 and observed species indices. Rarefaction Curve & Rank Abundance Curve The diversity among samples can be observed from the rarefaction curves of each sample and the rank abundance curve indicates species abundance and uniformity. Beta Diversity Analysis Beta diversity represents the explicit comparison of microbial (or other) communities based on their composition. Beta diversity metrics thus assess the differences between microbial communities. Principal Coordinates Analysis Clustering analysis of beta diversity data by the UPGMA algorithm is performed to plot a phylogenetic tree and Principal Coordinates Analysis is performed to visualize and explore similarities or dissimilarities of data. Classification of Results by Taxonomic Level The taxonomic analysis is performed by mapping OTU representative tags to the RDP, Greengenes, and NCBI 16S microbial databases. Taxonomy results are displayed in the form of area charts, heat map, and phylogenetic trees. Note the heatmaps are plotted on 7 taxonomic levels, all the way down to species level. The phylogenetic trees show the phylogenesis among species. The tree is composed of nodes that represent taxonomic units (from phylum through genus), and branches that denote the phylogenetic relation between nodes. LC Sciences comprehensive data report is presented in a user friendly HTML format for easy visualization and interpretation of your experimental results.