16S rRNA next-generation sequencing

This method is used to identify intestinal bacteria of interest (targeted identification: “Who is there? How many?”). The most common component of the human microbiome that is sequenced by next-generation sequencing is the small 30S ribosome subunit (SSU rRNA), which is unique to prokaryotes - the gene sequence of the Heat shock protein 60 (Hsp60) is preferentially used
(Picard et al. 2005) - and has the 16S ribosomal RNA (or 16S rRNA) as the RNA component that varies in nine hypervariable regions V1-V9 greatly between different species of bacteria
McQuade et al. 2019 Lancet Oncol 20: e77-91
Cahana & Iraqi 2020 Anim Models Exp Med. 3: 229-236

Amplicon sequence variants (ASVs) are considered to make marker-gene sequencing more precise and reproducible.
Zhu et al. 2021a Neurosci. Bull. 37(10): 1510-1522

The sequences of the bacterial 16S rRNA gene fragments are obtained from the extracted DNA. By using PCR amplification and DNA sequencing of various bacterial 16S rRNA genes present in a biofluid, it becomes possible to identify the different bacteria within the samples.
Mullish et al. 2021

In order to categorize them, they need to be assigned to operational taxonomic units (OTUs) using public databases like the National Center for Biotechnology Information (NCBI) or EzBioCloud, which collect 16S rRNA sequences. An OTU is essentially a sequence that typically identifies an organism at the genus or species level. OTUs of 97% similarity are assumed to characterize one species. This threshold was derived from an empirical study that showed that most strains had 97% of 16S rRNA sequence similarity
Cahana & Iraqi 2020 Anim Models Exp Med. 3: 229-236

The hypervariable regions are unique to each bacterial species and allow for classification or taxonomy.
Cahana & Iraqi 2020 Anim Models Exp Med. 3: 229-236

These DNA sequencing techniques produce shorter fragments than Sanger sequencing
Cahana & Iraqi 2020 Anim Models Exp Med. 3: 229-236

A common similarity threshold is 97%, which was derived from an empirical study that showed that most strains had 97% of 16S rRNA sequence similarity
Cahana & Iraqi 2020 Anim Models Exp Med. 3: 229-236

The early studies focused on the short 16S rRNA sequences, often conserved within a species and generally different between species.
The hypervariable regions are unique to each bacterial species and allow for classification or taxonomy
Cahana & Iraqi 2020 Anim Models Exp Med. 3: 229-236

Many 16S rRNA sequences have been found that do not belong to any known cultured species, suggesting numerous unisolated organisms.
Cahana & Iraqi 2020 Anim Models Exp Med. 3: 229-236

For 16S rRNA gene sequencing using Illumina Technology, fresh feces have to be collected, snap-frozen in liquid nitrogen and stored at −80 ◦ C. 16S rRNA gene amplification and sequencing has to be done using the Illumina MiSeq technology
Viennois et al. 2020 Cell Rep. 33: 108229.

The majority of gut microbiota cannot be cultured using standard techniques, so the development of culture-independent molecular methods based on sequencing of the phylogenetic marker - 16S /18S ribosomal RNA offers better insight into the gut microbiota structure.
Milosevic et al. 2019 Int. J. Mol. Sci. 20: 395

This technique uses the polymerase chain reaction to amplify genetic sequences present in the microbiota
Cryan et al. 2019 Physiol. Rev. 99: 1877-2013

These amplified sequences (amplicons), have been clustered into operational classification units (OTUs) according to their genomic relationships so that their relative abundance in samples can be calculated. The methods for defining OTUs include de novo methods (where reads are useless outside the reference database) and closed-reference methods (where reads outside the reference database cannot be captured).
Callahan et al. 2017 ISME J 11: 2639-2643

Recently, amplicons have been inferred as amplicon sequence variants (ASVs) with different de-noising algorithms, such as Deblur
Amir et al. 2017 mSystems 2: e00191-e00116) and DADA2 (Callahan et al. 2016 Nat Methods 13: 581-583).

These methods attempt to infer the biological sequences by obtaining single-nucleotide resolution from Illumina data. Compared to OTUs, ASVs can capture all biological variation without the restriction of a reference database, and ASVs can be replicated and compared across different datasets
Callahan et al. 2016 Nat Methods 13: 581-583).

Most microbiome studies characterize the presence of bacteria in a sample by sequencing a fragment of the gene encoding 16S rRNA.
Tamburini et al. 2016 Nat Med. 22(7): 713-22

Using the 16S rRNA gene to confidently identify strain-level variants of a given species requires a high degree of sequencing accuracy.
Ahern et al. 2014

The microbiome is characterized using 16S ribosomal RNA (rRNA) gene sequencing, shotgun, or high-throughput metagenome sequencing techniques
Goodrich et al. 2014 Cell 158(2): 250-262

Here, we focus primarily on 16S rRNA gene surveys because they are economical and scale to larger projects.
Goodrich et al. 2014 Cell 158(2): 250-262

16S rRNA gene sequence data provide a relatively unbiased characterization of bacterial and archaeal diversity
Goodrich et al. 2014 Cell 158(2): 250-262

For the nine hypervariable regions, some regions characterize bacteria better, and the choice of region, and therefore the appropriate primers to use, are an important step in study design
Ghyselinck et al. 2013 PLoS One 8(8): e71360

The establishment of culture-independent DNA sequencing technologies marked a significant advancement, alongside the development of bioinformatic tools. These innovations have made it possible to characterize bacterial and archaeal communities by sequencing their highly conserved 16S ribosomal RNA (16S rRNA) gene.
Woese & Fox 1977 Proc. Natl. Acad. Sci. USA. 74: 5088–5090
Eckburg et al. 2005

The elucidation of bacterial phylogeny based on the well-conserved small-subunit 16S ribosomal RNA (rRNA) sequence.
Woese & Fox 1977 Proc. Natl. Acad. Sci. USA. 74: 5088–5090
Woese 1987 Microbiol. Rev. 51: 221–271

The sequencing of the small subunit (SSU) 16S rRNA is a widely used method in research and analysis due to its cost-effectiveness. It is primarily employed to identify bacteria and assess bacterial diversity. Numerous companies and laboratories around the globe are engaged in analyzing gut microbiota using fecal samples. This technique allows for the identification of bacteria at the genus level and, in certain instances, at the species level. However, the use of short reads can complicate classification and does not offer insights into bacterial functions.