Full-length 16S Amplicon Sequencing Provides A New Idea for Microecological Research

Based on PacBio and Nanopore sequencing platform, the microbial diversity can be revealed by full-length amplification of all V1-V9 variable regions of 16S in prokaryotes or 18S highly variable regions or ITS regions in eukaryotes by using single-molecule real-time sequencing method, which can not only improve the resolution of species identification, but also improve the resolution of species identification. It can also improve the accuracy of microbial composition identification in samples, so as to reflect the microbial community structure more comprehensively.

Full-length 16S sequencing for more accurate species prediction

Recently, researchers used simulated bacterial communities, feces from 4 healthy people, and 381 isolated strains to re-evaluate the taxonomic potential of 16S gene at the species and strain level based on biogenic analysis and sequencing experiments, and found that only some variable regions could distinguish species. The results showed that such intragenomic 16S gene copy variants are highly prevalent in taxa isolated from the human gut microbiome, suggesting they may be used to improve discrimination between species and even strains in 16S gene-based microbiome studies. Moreover, different variable regions have different species classification abilities, while the full length of 16S can annotate all sequences to specific species, which is crucial for the discovery of new species.

In-silico comparison of 16S rRNA variable regions.

Full length 16S sequencing is revealing microbial community

Recent study discusses whether the maternal fecal and vaginal microbiota are transmitted vertically from mother to child or from laboratory reagents or DNA contamination acquired during delivery. The inability of conventional second-generation amplicon sequencing to accurately distinguish real signals beyond background contamination levels has hindered the accuracy of microbial community analysis in low-micro biomass samples (e. g. placenta, amniotic fluid, meconium). Researchers analyzed the high taxonomic resolution microbiota profiles of samples of 39 maternal-neonate pairs and aimed to explore the maternal origin of the microbiota of neonate meconium by using the PacBio single-molecule real-time circular consensus sequencing technology. The results indicated that the meconium micro-biota was seeded from multiple maternal body sites, and the amniotic fluid microbiota contributed most to the seeding of the meconium microbiota among the investigated maternal body sites. Moreover, long-read long-sequencing technology has potential advantages in reducing the pollution risk of low-micro biomass samples. Therefore, full-length 16S rRNA was used in this paper to analyze the microbial community of various samples, providing more accurate strain level data and more truly restoring the microbial community.

Dissimilarity-based multivariate analyses of microbiota communities of different sample types.

Full-length 16S sequencing helps to excavate more beneficial population resources

Due to the low phylogenetic resolution of traditional second-generation amplicon sequencing, to improve understanding of the complexity of the anaerobic digester microbial community, PacBio-Sequel was used to sequence the full-length 16S rRNA gene amplicon and to sequence sludge samples from 19 anaerobic digesters worldwide. Sixteen methanogenic archaea were identified at the species level, including those neglected by the second generation. The unique diversity in fermenters, syntrophs, and methanogens of anaerobic digesters associated with operating conditions was addressed. These results gleamed identify overlooked microbiomes and cross-link with digestion operations through the full-length 16S rRNA amplicon PacBio Sequel sequencing.

16S amplification sequencing is very useful as a complementary technique for genome wide sequencing, greatly simplifying the experimental process and analysis objectives. This technology has been proven to be a fast and effective technology, and has played a unique role in the next generation of high throughput sequencing. It has produced many exciting new discoveries and has been increasingly applied in a wide range of fields.


  1. He, Qiuwen et al. "The meconium microbiota shares more features with the amniotic fluid microbiota than the maternal fecal and vaginal microbiota." Gut microbes vol. 12,1 (2020): 1794266.
  2. Johnson, Jethro S et al. "Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis." Nature communications vol. 10,1 5029. 6 Nov. 2019
  3. Lam, Theo Y C et al. "Superior resolution characterisation of microbial diversity in anaerobic digesters using full-length 16S rRNA gene amplicon sequencing." Water research vol. 178 (2020): 115815.
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