16S rRNA Sequencing for Gut Microbiota Research

The human gut microbiome is known to influence the health of individuals and is closely related to the occurrence and recovery from diseases. Gut microbiota offers beneficial health effects and, conversely, the compositional disruption of gut microbiota observed in dysbiosis is correlated to various human diseases, including obesity, inflammatory bowel disease, type 2 diabetes, CNS related disorders, and cancer. In the gastrointestinal microbiome specifically, 16S rRNA profiling has also been widely applied. Creative Biolabs is one of the largest live biotherapeutic products (LBP) development services suppliers. We have expertise and capabilities in providing high-quality gut microbiota research solutions.

16S rRNA sequencing for Human Gut Microbiota

Many studies have employed 16S rRNA gene sequencing to profile the gut microbiota composition. The most important step in the study of the human gut microbiome is bacterial community profiling; the genomic region encoding the RNA for the ribosomal small subunit (SSU), known as 16S, is the most commonly analyzed. The 16S subunit ribosomal gene is highly conserved between bacteria and archaea and is therefore widely used as a marker gene for assessing microbial phylogeny. The 16S rRNA gene contains nine hypervariable regions (V1-V9) with bacterial species-specific variation, flanked by conserved regions. Thus, amplification in the hypervariable region of 16S rRNA can be used to detect microbial communities in samples, often down to the genus level, or for species-level assignment, if a full-length 16S sequence is retrieved. Due to its high resolution and cost-effective approach, 16S rRNA gene sequencing has become the commonest approach for microbial community profiling of the human gut.

Workflows of 16S rRNA Sequencing

  • Sample Collection and Storage

An important but rarely emphasized aspect of 16S rRNA gene sequencing research is sample integrity. For intestinal microbiota analysis studies, biopsy, surgical tissues, and stool samples are common biological samples collected for characterization. Sample contamination minimization ensures that each biological sample retains as much of its original microbiome as possible and contributes to accurate results of gut microbiome analysis. Short-term and long-term storage conditions after sample transportation are very important to minimize microbiome spectrum differences caused by storage conditions. Low-temperature transportation, such as on ice or dry ice, ethanol-stored and long-term storage in -80℃ are strongly recommended to maximize microbiome recovery within the sample prior to nucleic acid extraction.

  • Methods in Nucleic Acid Extraction

Sample nucleic acid extraction is a simple but critical step in microbial research. Most separation protocols involve three basic steps, including cell lysis, elimination of non-DNA macromolecules, and DNA separation and collection. Gram-positive organisms require stronger lysis conditions due to their thicker cell walls, unlike Gram-negative organisms which require only gentle lysis.

  • Selection of Universal 16S rRNA Gene Primers, Sequencing Technologies, and Databases

The 16S rRNA gene consists of nine hypervariable conserved regions (V1 to V9) separated by ten highly conserved regions. Most studies were done via sequencing of either one of the V3 or V4 regions, otherwise, a combination of two or more 16S rRNA gene hypervariable regions, whereby the most commonly used were the V3/V4 regions. Another important parameter in microbiome studies is bioinformatics analysis. As far as we know, only RDP and SILVA update frequently. The Greengenes database has not been updated since 2013. On the other hand, the National Center for Biotechnology Information (NCBI) recommended the RefSeq-targeted site as the "gold standard" for the analysis of 16S rRNA gene sequencing data. It is also important to select the appropriate computing tool for the analysis of the 16S rRNA gene sequencing dataset.

General overview of the workflow for 16S rRNA gene-based and metagenomics analysis of microbial communities.Fig.1 General overview of the workflow for 16S rRNA gene-based and metagenomics analysis of microbial communities. (Deo, 2019)

In the rapidly-evolving world of LBP, Creative Biolabs' comprehensive portfolio of research and services allows us to help our clients research, discover, and develop new LBP. The human gut microbiota holds promises as a reservoir of novel potential therapeutic targets as well as novel therapeutic agents, paving a new and exciting avenue in drug discovery. If you are interested in our 16S rRNA sequencing services for gut microbiota research, please contact us for more detail.


  1. Deo, P.N.; Deshmukh, R. Oral microbiome: Unveiling the fundamentals. Journal of oral and maxillofacial pathology. 2019, 23(1): 122.

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