16s Rrna Gene Sequencing

[Narkis Eatman]

16Sand Internal Transcribed Spacer (ITS) ribosomal RNA (rRNA) sequencing are common amplicon sequencing methods used to identify and compare bacteria or fungi present within a given sample. Next-generation sequencing (NGS)-based ITS and 16S rRNA gene sequencing are well-established methods for comparing sample phylogeny and taxonomy from complex microbiomes or environments that are difficult or impossible to study.

The prokaryotic 16S rRNA gene is approximately 1500 bp long, with nine variable regions interspersed between conserved regions. Variable regions of the 16S rRNA gene are frequently used for phylogenetic classification of genus or species in diverse microbial populations.1 The ITS1 region of the rRNA cistron is a commonly used DNA marker for identifying fungal species in metagenomic samples.2

A key benefit of 16S and ITS ribosomal RNA NGS methods is that they provide a cost-effective technique to identify strains that may not be found using traditional methods. Unlike capillary sequencing or PCR-based approaches, next-generation sequencing is a culture-free method that enables analysis of the entire microbial community within a sample.

16S rRNA NGS allows microbiologists to achieve genus-level sensitivity for metagenomic surveys of bacterial populations. ITS analysis with NGS enables rapid fungal identification to help advance our understanding of the mycobiome. Furthermore, NGS offers the ability to combine multiple samples in a sequencing run.

Illumina offers products to support NGS-based 16S and ITS rRNA analysis studies, from library preparation to data analysis and interpretation. Our user-friendly workflow can help take the guesswork out of your experiments.

This method involves comprehensively sampling all genes in all organisms present in a given complex sample. It allows microbiologists to evaluate bacterial diversity and detect the abundance of microbes in various environments.

eDNA sequencing is an emerging method for studying biodiversity and monitoring ecosystem changes. For some sample types, using a combination of 16S or ITS sequencing with other approaches can help uncover the full breadth of diversity in an ecological sample.

16s rRNA sequencing is a culture-free method to identify and compare bacterial diversity from complex microbiomes or environments that are difficult to study. It is commonly used to identify bacteria present within a given sample down to the genus and/or species level. Specifically, it is an amplicon-based sequencing method that targets the 16s rRNA bacteria-specific genetic marker using a single amplicon focused on a single gene.

Because the 16s rRNA sequence is ubiquitous in bacteria and archaea, it can be used to identify a wide diversity of microbes within a single sample and single workflow. Through 16s rRNA sequencing, one can identify taxa present in a sample. This leads to a greater understanding of our microbial communities and their interactions with us.

Both the ribosome and its subunits are characterized by their sedimentation coefficients, expressed in Svedberg units (symbol: S). In this case, 16s means it takes 16 Svedberg units of time for the ribosome to sediment in a solution.

16S DNA refers to the gene in the bacterial genome that codes for the 16S rRNA. 16S rRNA is the rRNA that is transcribed from the 16S DNA gene. The Illumina 16S Metagenomic Sequencing Library Preparation protocol uses DNA as input, and the PCR primers target the variable regions V3 and V4 of the 16S DNA gene for the amplicon PCR.

The 16S Demonstrated Protocol provides an option for creating Illumina compatible libraries from the target of your choice. Fungi and other organisms do not have 16s rRNA genes, however, they have other conserved regions such as 18S and ITS regions. Any amplicon can be used to do similar diversity analysis studies.

At Illumina, our goal is to apply innovative technologies to the analysis of genetic variation and function, making studies possible that were not even imaginable just a few years ago. It is mission critical for us to deliver innovative, flexible, and scalable solutions to meet the needs of our customers. As a global company that places high value on collaborative interactions, rapid delivery of solutions, and providing the highest level of quality, we strive to meet this challenge. Illumina innovative sequencing and array technologies are fueling groundbreaking advancements in life science research, translational and consumer genomics, and molecular diagnostics.

The genes coding for it are referred to as 16S rRNA genes and are used in reconstructing phylogenies, due to the slow rates of evolution of this region of the gene.[2] Carl Woese and George E. Fox were two of the people who pioneered the use of 16S rRNA in phylogenetics in 1977.[3] Multiple sequences of the 16S rRNA gene can exist within a single bacterium.[4]

The 16S rRNA gene is used for phylogenetic studies[7] as it is highly conserved between different species of bacteria and archaea.[8] Carl Woese pioneered this use of 16S rRNA in 1977.[2] It is suggested that 16S rRNA gene can be used as a reliable molecular clock because 16S rRNA sequences from distantly related bacterial lineages are shown to have similar functionalities.[9] Some thermophilic archaea (e.g. order Thermoproteales) contain 16S rRNA gene introns that are located in highly conserved regions and can impact the annealing of "universal" primers.[10] Mitochondrial and chloroplastic rRNA are also amplified.[11]

The most common primer pair was devised by Weisburg et al. (1991)[7] and is currently referred to as 27F and 1492R; however, for some applications shorter amplicons may be necessary, for example for 454 sequencing with titanium chemistry the primer pair 27F-534R covering V1 to V3.[12]Often 8F is used rather than 27F. The two primers are almost identical, but 27F has an M instead of a C. AGAGTTTGATCMTGGCTCAG compared with 8F.[13]

In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for identification of bacteria.[21][22]As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid and cheap alternative to phenotypic methods of bacterial identification.[23] Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new species,[24] or even genera.[7][25]It has also been used to describe new species that have never been successfully cultured.[26][27]With third-generation sequencing coming to many labs, simultaneous identification of thousands of 16S rRNA sequences is possible within hours, allowing metagenomic studies, for example of gut flora.[28] In samples collected from patients with confirmed infections, 16S rRNA next-generation sequencing (NGS) demonstrated enhanced detection in 40% of cases compared to traditional culture methods; moreover, pre-sampling antibiotic consumption did not significantly affect the sensitivity of 16S NGS.[29]

While 16S hypervariable region analysis is a powerful tool for bacterial taxonomic studies, it struggles to differentiate between closely related species.[35] In the families Enterobacteriaceae, Clostridiaceae, and Peptostreptococcaceae, species can share up to 99% sequence similarity across the full 16S gene.[37] As a result, the V4 sequences can differ by only a few nucleotides, leaving reference databases unable to reliably classify these bacteria at lower taxonomic levels.[37] By limiting 16S analysis to select hypervariable regions, these studies can fail to observe differences in closely related taxa and group them into single taxonomic units, therefore underestimating the total diversity of the sample.[35] Furthermore, bacterial genomes can house multiple 16S genes, with the V1, V2, and V6 regions containing the greatest intraspecies diversity.[8] While not the most precise method of classifying bacterial species, analysis of the hypervariable regions remains one of the most useful tools available to bacterial community studies.[37]

Under the assumption that evolution is driven by vertical transmission, 16S rRNA genes have long been believed to be species-specific, and infallible as genetic markers inferring phylogenetic relationships among prokaryotes. However, a growing number of observations suggest the occurrence of horizontal transfer of these genes. In addition to observations of natural occurrence, transferability of these genes is supported experimentally using a specialized Escherichia coli genetic system. Using a null mutant of E. coli as host, growth of the mutant strain was shown to be complemented by foreign 16S rRNA genes that were phylogenetically distinct from E. coli at the phylum level.[38][39] Such functional compatibility was also seen in Thermus thermophilus.[40] Furthermore, in T. thermophilus, both complete and partial gene transfer was observed. Partial transfer resulted in spontaneous generation of apparently random chimera between host and foreign bacterial genes. Thus, 16S rRNA genes may have evolved through multiple mechanisms, including vertical inheritance and horizontal gene transfer; the frequency of the latter may be much higher than previously thought.[41]

The 16S rRNA gene is used as the standard for classification and identification of microbes, because it is present in most microbes and shows proper changes.[42] Type strains of 16S rRNA gene sequences for most bacteria and archaea are available on public databases, such as NCBI. However, the quality of the sequences found on these databases is often not validated. Therefore, secondary databases that collect only 16S rRNA sequences are widely used.

MIMt is a compact non-redundant 16S database for a rapid metagenomic samples identification. It is composed of 39.940 full 16S sequences belonging to 17,625 well classified bacteria and archaea species. All sequences were obtained from complete genomes deposited in NCBI and for each of the sequences full taxonomic hierarchy is provided. It contains no redundancy, so only one representative for each species was considered avoiding same sequences from differente strains, isolates or patovars resulting in a very fast tool for microorganisms identification, compatible with any classification software (QIIME, Mothur, DADA, etc).[43]

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