Short-read full-length 16S rRNA amplicon sequencing for characterisation of the respiratory bacteriome of captive and free-ranging African elephants (Loxodonta africana).

Hypervariable region sequencing of the 16S ribosomal RNA (rRNA) gene plays a critical role in microbial ecology by offering insights into bacterial communities within specific niches. While providing valuable genus-level information, its reliance on data from targeted genetic regions limits its overall utility. Recent advances in sequencing technologies have enabled characterisation of the full-length 16S rRNA gene, enhancing species-level classification. Although current short-read platforms are cost-effective and precise, they lack full-length 16S rRNA amplicon sequencing capability. This study aimed to evaluate the feasibility of a modified 150 bp paired-end full-length 16S rRNA amplicon short-read sequencing technique on the Illumina iSeq 100 and 16S rRNA amplicon assembly workflow by utilising a standard mock microbial community and subsequently performing exploratory characterisation of captive (zoo) and free-ranging African elephant (Loxodonta africana) respiratory microbiota. Our findings demonstrate that, despite generating assembled amplicons averaging 869 bp in length, this sequencing technique provides taxonomic assignments consistent with the theoretical composition of the mock community and respiratory microbiota of other mammals. Tentative bacterial signatures, potentially representing distinct respiratory tract compartments (trunk and lower respiratory tract) were visually identified, necessitating further investigation to gain deeper insights into their implication for elephant physiology and health.

Martin LC ，O'Hare MA ，Ghielmetti G ，Twesigomwe D ，Kerr TJ ，Gumbo R ，Buss PE ，Kitchin N ，Hemmings SMJ ，Miller MA ，Goosen WJ ... -  《Scientific Reports》

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes.

Earl JP ，Adappa ND ，Krol J ，Bhat AS ，Balashov S ，Ehrlich RL ，Palmer JN ，Workman AD ，Blasetti M ，Sen B ，Hammond J ，Cohen NA ，Ehrlich GD ，Mell JC ... -  《Microbiome》

EnsembleSeq: a workflow towards real-time, rapid, and simultaneous multi-kingdom-amplicon sequencing for holistic and resource-effective microbiome research at scale.

Nagpal S ，Mande SS ，Hooda H ，Dutta U ，Taneja B ... -  《Microbiology Spectrum》

Evaluation of 16S rRNA amplicon sequencing using two next-generation sequencing technologies for phylogenetic analysis of the rumen bacterial community in steers.

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long "circular consensus" reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good's coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia (P < 0.05). Taxonomic classification accuracy was also greater in the near full-length read. UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing.

Myer PR ，Kim M ，Freetly HC ，Smith TPL ... -  《-》

Comparison of the full-length sequence and sub-regions of 16S rRNA gene for skin microbiome profiling.

Zhang H ，Wang X ，Chen A ，Li S ，Tao R ，Chen K ，Huang P ，Li L ，Huang J ，Li C ，Zhang S ... -  《mSystems》