.Characterizing microbial communities in specimens is one of the main goals of microbiome research. The 16S ribosomal RNA (rRNA) gene is approximately 1.5 kb in length and contains several conserved and highly variable regions (V1-V9) that vary between bacteria. These highly variable sequences can be used to identify and characterize microbial diversity. Therefore, the 16S rRNA gene commonly apply to marker for characterizing microbial communities in various specimens.

Overview

The microbiota is the total microbial complex containing a wide range of bacterial species. It rang ubiquitously from humans (e.g., the microbiota inhabiting the animal gut) to the natural environment. In recent years, research on the human gut flora has been conducted worldwide. Besides, the human gut flora has been closely associated with the development of various diseases. Therefore, characterizing the diversity and composition of microbial communities in specimens is one of the main goals of current microbiological research.

With the rapid development of next-generation sequencing (NGS) technologies, metagenomic sequencing is emerging as a powerful approach to understand the complex microbial communities in the human gut.  Pacific Biosciences (PacBio) and Oxford Nanopore sequencing platforms overcome these technical limitations and are widely used, particularly those affecting read length. CD Genomics offers specialized full-length 16S/18S/ITS amplicon sequencing services to sequence PCR products from specific regions of 16SrDNA.

Full-length 16S Sequencing Technologies

Short-read Sequencing Platforms

Second-generation sequencing technologies analyze microbial diversity by reading the V1, V2, V3, V4, V5, and V6 regions of the 16S rRNA gene. High-throughput short-read sequencing of 16S rRNA gene amplicons based on the Illumina MiSeq 2 × 300 bp platform specifically targets the highly variable V3-V4 region of the nine variable regions. By reducing the high-cost burden of NGS, it is widely used in various metagenomic studies. However, this short-read amplicon-based platform is not only susceptible to identification bias from potential chimeric sequences generated during PCR amplification for library construction but also limited to genus-level microbial classification based on commonly used 16S rRNA genes based on microbial taxonomic databases. Therefore, the amplicon approach for partially variable regions (V3-V4) is limited to strains with a high degree of similarity at the species level.

Long-read Sequencing Platforms

In microbiome research, this long-read sequencing technology has led to changes in the analysis of complex microbial communities. It addresses the problem of identification accuracy that occurs when reading partially highly variable regions of 16S rRNA genes.

This device, although compact in size, has brought about a profound shift in our approach to sequencing the full-length 16S rRNA gene, which is the cornerstone of bacterial identification and classification studies. The advantages of the MinION^TMare primarily in its comprehensive targeting mechanism, which captures the entire spectrum of the 16S rRNA gene, thus avoiding the pitfalls of fragment sequencing. In this way, it improves the fineness of identification of bacterial species and subspecies, thus enhancing our diagnostic and research capabilities to an unprecedented level. In addition, the specificity of the MinION^TM method ensures a dramatic increase in sensitivity and accuracy, both of which are critical in microbiology research, where even small differences can represent very different bacterial taxa.

16S rRNA gene sequence analysis using the MinION™ nanopore sequencer. (Matsuo et al., 2021)

Pacific Bioscience SMRT sequencing technology enables accurate sequencing of the entire 16S rRNA gene, especially when delving into the implications of covering all variable regions. Typically, the read length provided by a sequencing platform is known to affect either accuracy or sequence length. However, PacBio introduces an innovative balance between these two parameters. Their high fidelity (HiFi) reads average approximately 1.5 kb in length, ensuring that the majority of genomic information is retained and accurately represented. A key factor driving PacBio’s sequencing proficiency is the utilization of the Cyclic Coherent Sequencing (CCS) mode.  As a result, PacBio HiFi reads offer a dual advantage – superior length and unrivaled accuracy. This combination raises the detection of microbial taxonomic units to previously unattained levels of granularity.

Two software packages, PacBio SMRT Link and the DADA2 R package(Callahan et al., 2019)

Synthetic Long Read Length Technology

Long-read sequencing platforms generate reads with lower nucleotide accuracy than Illumina platforms (~15% versus ~0.1% for Illumina) due to random base detection errors that occur during multiple sequencing of the same region. Loop Genomics has introduced a new 16S full-length-based synthetic long-read (sFL16S) technology. The method represents a hybrid approach . And it combines the reliability of short-read long sequencing with the depth and detail of long-read sequencing. The technology reads the entire variable region of the 16S rRNA gene (V1-V9) to elucidate microbial communities in metagenomic studies.

Technical introduction and analysis workflow for a new 16S full-length-based synthetic long-read (sFL16S) technology.  (Jeong et al., 2021)

Applications of Full-length 16S rRNA Sequencing

Characterization of microbial communities

The ability to sequence full-length 16S rRNA genes provides a panoramic view of microbial taxa in a sample, paving the way for detailed community characterization.

Disease surveillance

Changes in microbial communities that can serve as indicators of the onset. Besides, it also serve as progression, or resolution of certain diseases, particularly human intestinal diseases.

Drug development

Analyzing microbial communities at high resolution can help develop therapies. And the   target specific microbial pathogens or modulate microbial communities for beneficial effects.