More Like this

1. Characterizing organisms from three domains of life with universal primers from throughout the global ocean

   https://doi.org/10.1038/s41597-025-05423-9  

   McNichol, Jesse; Williams, Nathan_L R; Raut, Yubin; Carlson, Craig; Halewood, Elisa R; Turk-Kubo, Kendra; Zehr, Jonathan P; Rees, Andrew P; Tarran, Glen; Gradoville, Mary R; et al (December 2025, Scientific Data)

   Abstract We introduce the Global rRNA Universal Metabarcoding Plankton database (GRUMP), which consists of 1194 samples that were collected from 2003–2020 and cover extensive latitudinal and longitudinal transects, as well as depth profiles in all major ocean basins. DNA from unfractionated (>0.2 µm) seawater samples was amplified using the 515Y/926 R universal three-domain rRNA gene primers, simultaneously quantifying the relative abundance of amplicon sequencing variants (ASVs) from bacteria, archaea, eukaryotic nuclear 18S, and eukaryotic plastid 16S. Thus, the ratio between taxa in one sample is directly comparable to the ratio in any other GRUMP sample, regardless of gene copy number differences. This obviates a problem in prior global studies that used size-fractionation and different rRNA gene primers for bacteria, archaea, and eukaryotes, precluding comparisons across size fractions or domains. On average, bacteria contributed 71%, eukaryotes 19%, and archaea 8% to rRNA gene abundance, though eukaryotes contributed 32% at latitudes >40°. GRUMP is publicly available on the Simons Collaborative Marine Atlas Project (CMAP), promoting the global comparison of marine microbial dynamics. 

   more » « less
2. NifH gene amplicon sequencing and metagenomic approaches are complementary in assessing diazotroph diversity

   https://doi.org/10.1093/ismeco/ycaf038  

   Cheung, Shunyan; Morando, Michael; Magasin, Jonathan; Cornejo-Castillo, Francisco M; Zehr, Jonathan P; Turk-Kubo, Kendra A (January 2025, ISME Communications)

   Abstract Exploring the diversity of diazotrophs is key to understanding their role in supplying fixed nitrogen that supports marine productivity. A nested PCR assay using the universal primer set nifH1-nifH4, which targets the nitrogenase (nifH) gene, is a widely used approach for studying marine diazotrophs by amplicon sequencing. Metagenomics, direct sequencing of DNA without PCR, has provided complementary views of the diversity of marine diazotrophs. A significant fraction of the metagenome-derived nifH sequences (e.g. Planctomycete- and Proteobacteria-affiliated) were reported to have nucleotide mismatches with the nifH1-nifH4 primers, leading to the suggestion that nifH amplicon sequencing does not detect specific diazotrophic taxa and underrepresents diazotroph diversity. Here, we report that these mismatches are mostly located in a single-base at the 5′-end of the nifH4 primer, which does not impact detection of the nifH genes. This is demonstrated by the presence of nifH genes that contain the nucleotide mismatches in a recent compilation of global ocean nifH amplicon datasets, with high relative abundances detected in a variety of samples. While the metagenome- and metatranscriptome-derived nifH genes accounted for 4.4% of the total amplicon sequence variants from the global ocean nifH amplicon database, the corresponding amplicon sequence variants can have high relative abundances (accounting for 47% of the reads in the database). These analyses underscore that nifH amplicon sequencing using the nifH1-nifH4 primers is an important tool for studying diversity of marine diazotrophs, particularly as a complement to metagenomics which can provide taxonomic and metabolic information for some dominant groups. 

   more » « less
3. A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

   https://doi.org/10.3390/agronomy11071274  

   Chen, Ko-Hsuan; Longley, Reid; Bonito, Gregory; Liao, Hui-Ling (July 2021, Agronomy)

   null (Ed.)

   High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest. 

   more » « less
4. Contrasting diversity patterns of prokaryotes and protists over time and depth at the San-Pedro Ocean Time series

   https://doi.org/10.1038/s43705-022-00121-8  

   Yeh, Yi-Chun; Fuhrman, Jed A. (December 2022, ISME Communications)

   Abstract Community dynamics are central in microbial ecology, yet we lack studies comparing diversity patterns among marine protists and prokaryotes over depth and multiple years. Here, we characterized microbes at the San-Pedro Ocean Time series (2005–2018), using SSU rRNA gene sequencing from two size fractions (0.2–1 and 1–80 μm), with a universal primer set that amplifies from both prokaryotes and eukaryotes, allowing direct comparisons of diversity patterns in a single set of analyses. The 16S + 18S rRNA gene composition in the small size fraction was mostly prokaryotic (>92%) as expected, but the large size fraction unexpectedly contained 46–93% prokaryotic 16S rRNA genes. Prokaryotes and protists showed opposite vertical diversity patterns; prokaryotic diversity peaked at mid-depth, protistan diversity at the surface. Temporal beta-diversity patterns indicated prokaryote communities were much more stable than protists. Although the prokaryotic communities changed monthly, the average community stayed remarkably steady over 14 years, showing high resilience. Additionally, particle-associated prokaryotes were more diverse than smaller free-living ones, especially at deeper depths, contributed unexpectedly by abundant and diverse SAR11 clade II. Eukaryotic diversity was strongly correlated with the diversity of particle-associated prokaryotes but not free-living ones, reflecting that physical associations result in the strongest interactions, including symbioses, parasitism, and decomposer relationships. 

   more » « less
5. Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians

   https://doi.org/10.1186/s12862-022-01994-y  

   Chan, Kin Onn; Hertwig, Stefan T.; Neokleous, Dario N.; Flury, Jana M.; Brown, Rafe M. (December 2022, BMC Ecology and Evolution)

   Abstract Background The 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence ( p -distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths. Results Using empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p -distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content. Conclusions Taken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost. 

   more » « less