Since the first discovery of 16s rRNA sequences in environmental samples in 1990 and their value in microbial community research, we have entered the booming era of microbial community research. Today, community-based research has entered the era of high-throughput sequencing, and the mainstream of current research is at the intersection of next-generation amplicon sequencing and long-read amplicon sequencing. .
Next Generation Amplicon Sequencing
The most common targets for next-generation amplicon sequencing are bacterial 16S ribosomal RNA (16S rRNA), 18S rRNA, and fungal internal transcribed spacer (ITS) with taxonomic information. However, NGS techniques provide limited information in the area of microbiome function prediction due to their short read lengths, with the limiting length of short-read 16s amplicon fragments being only 500–600 bp. . At most, only three consecutive variable regions can be analyzed. Selection of variable regions by next-generation amplicons is a daunting challenge and usually involves a trade-off with information loss. Therefore, NGS emphasizes microbial composition at the phylum or genus level, focusing on overall community diversity. It does not discriminate well at the species level and is unable to distinguish between highly related strains.
Long read Amplicon Sequencing
The long-read sequencing technology (Oxford Nanopore and PacBio SMRT sequencing) can easily cover a total of 9 variable regions of 16s with a total length of approximately 1500 bp, maximally preserving the possibility of species identification. Taking PacBio SMRT sequencing technology as an example, the extra-long read length and circular pattern allow the insertion of full-length 16S fragments to be repeatedly sequenced, and random errors can be reduced by cross-checking through to repeated sequencing to achieve high quality Consensus Sequencing (CCS). The feasibility of PacBio technology for comprehensive 16S rRNA gene sequencing analysis has proven to be applicable to several study topics such as gut microbiome, soil microbiome, and water microbiome. The number of microbial community species identified at the species level was 2.3 to 15 times higher than at the gate level, with detectable abundance as low as 0.05%.
Long-read amplicon sequencing goes further than short-read technology by focusing more on the association between other omics, not only species abundance at the phylum and genus level, but also being capable of exploring collaborative and competitive species at high performance resolution. Full 16s sequencing can provide a more comprehensive analysis at the strain level, which is of great importance for multi-omics association and subsequent topics. In the future, full 16S sequencing may gradually replace short-read sequencing as the main force in predicting microbiome function.
How Long to Read Multi-Omics 16S Sequencing Facilities?
Full 16S seq can be used to combine with metagenome analysis. The complete 16S seq can describe the composition of the microbial community, while metagenomics validates the results. Additionally, metagenomics provides access to genetic and functional annotation information, allowing functional-level resolution of differences between sample communities.
16s amplicon sequencing can also be combined with metabolomics for association analysis. By correlating metabolomics with the species abundance distribution obtained from 16s sequencing, the network of relationships can be constructed from different molecular levels using statistical models to explore potential key regulatory factors.
Sequencing of full-length, long-read 16s has great potential as a next-generation community-based microbial research tool and is a major tool needed for future strain-level research. Full-length amplicon sequencing can obtain sequence information of all variant regions, which can not only improve the resolution of species identification, but also improve the accuracy and completeness of microbial identification in samples, thus a more realistic restoration of the structure of the microbial community.