Redirecting NK cells to the lymph nodes to augment their lymphoma-targeting capacity.

CAR-NK cells can induce remission in lymphoma patients. We speculate that the full potential of adoptive NK cell immunotherapy against lymphoma is restricted by their poor lymph node (LN) homing capacity. Here, we have utilized a clinically approved transfection method with the aim of redirecting NK cells to LNs. Electroporation of ex vivo expanded NK cells with mRNAs coding for CCR7, CXCR5, and CD62L resulted in increased in vitro migration towards chemokines and mouse LN-derived supernatant. Following infusion into SCID/Beige mice, modified NK cells showed enhanced LN homing. Importantly, lymphoma patient-derived NK cells were equally well expanded and engineered as healthy donor NK cells, highlighting their translational potential. Additionally, the introduction of high-affinity CD16, together with the homing molecules, also augmented their ADCC capacity against autologous lymphoma cells. Hence, genetic engineering can be utilized to enhance NK cell LN homing. The homing concept may synergize with CAR- or monoclonal/bi-/tri-specific antibody-based approaches.

Microbial hitchhikers on microplastics: The exchange of aquatic microbes across distinct aquatic habitats.

Microplastics (MPs) have the potential to modify aquatic microbial communities and distribute microorganisms, including pathogens. This poses a potential risk to aquatic life and human health. Despite this, the fate of 'hitchhiking' microbes on MPs that traverse different aquatic habitats remains largely unknown. To address this, we conducted a 50-day microcosm experiment, manipulating estuarine conditions to study the exchange of bacteria and microeukaryotes between river, sea and plastisphere using a long-read metabarcoding approach. Our findings revealed a significant increase in bacteria on the plastisphere, including Pseudomonas, Sphingomonas, Hyphomonas, Brevundimonas, Aquabacterium and Thalassolituus, all of which are known for their pollutant degradation capabilities, specifically polycyclic aromatic hydrocarbons. We also observed a strong association of plastic-degrading fungi (i.e., Cladosporium and Plectosphaerella) and early-diverging fungi (Cryptomycota, also known as Rozellomycota) with the plastisphere. Sea MPs were primarily colonised by fungi (70%), with a small proportion of river-transported microbes (1%-4%). The mere presence of MPs in seawater increased the relative abundance of planktonic fungi from 2% to 25%, suggesting significant exchanges between planktonic and plastisphere communities. Using microbial source tracking, we discovered that MPs only dispersed 3.5% and 5.5% of river bacterial and microeukaryotic communities into the sea, respectively. Hence, although MPs select and facilitate the dispersal of ecologically significant microorganisms, drastic compositional changes across distinct aquatic habitats are unlikely.

Endovascular transplantation of mRNA-enhanced mesenchymal stromal cells results in superior therapeutic protein expression in swine heart.

Heart failure has a poor prognosis and no curative treatment exists. Clinical trials are investigating gene- and cell-based therapies to improve cardiac function. The safe and efficient delivery of these therapies to solid organs is challenging. Herein, we demonstrate the feasibility of using an endovascular intramyocardial delivery approach to safely administer mRNA drug products and perform cell transplantation procedures in swine. Using a trans-vessel wall (TW) device, we delivered chemically modified mRNAs (modRNA) and mRNA-enhanced mesenchymal stromal cells expressing vascular endothelial growth factor A (VEGF-A) directly to the heart. We monitored and mapped the cellular distribution, protein expression, and safety tolerability of such an approach. The delivery of modRNA-enhanced cells via the TW device with different flow rates and cell concentrations marginally affect cell viability and protein expression in situ. Implanted cells were found within the myocardium for at least 3 days following administration, without the use of immunomodulation and minimal impact on tissue integrity. Finally, we could increase the protein expression of VEGF-A over 500-fold in the heart using a cell-mediated modRNA delivery system compared with modRNA delivered in saline solution. Ultimately, this method paves the way for future research to pioneer new treatments for cardiac disease.

Harnessing upregulated E-selectin while enhancing SDF-1α sensing redirects infused NK cells to the AML-perturbed bone marrow.

Increased bone marrow (BM) homing of NK cells is associated with positive outcome in patients with acute myeloid leukemia (AML) treated within adoptive NK cell transfer trials. While most efforts to further improve the efficacy focus on augmenting NK cell persistence and cytotoxicity, few address their ability to home to the tumor. Here, we decipher how AML growth alters the BM niche to impair NK cell infiltration and how insights can be utilized to resolve this issue. We show that AML development gradually impairs the BM homing capacity of infused NK cells, which was tightly linked to loss of SDF-1α in this environment. AML development also triggered up-regulation of E-selectin on BM endothelial cells. Given the poor E-selectin-binding capacity of NK cells, introduction of fucosyltransferase-7 (FUT7) to the NK cells per mRNA transfection resulted in potent E-selectin binding and stronger adhesion to E-selectin+ endothelial cells. Co-introduction of FUT7 and gain-of-function CXCR4 (CXCR4R334X) redirected NK cell homing to the BM of AML-bearing mice nearly to the levels in AML-free mice. This work shows how impaired NK cell homing caused by AML-induced microenvironmental changes can be overcome by genetic engineering. We speculate our insights can help further advance future NK cell immunotherapies.

A comprehensive dataset on spatiotemporal variation of microbial plankton communities in the Baltic Sea.

The Baltic Sea is one of the largest brackish water environments on earth and is characterised by pronounced physicochemical gradients and seasonal dynamics. Although the Baltic Sea has a long history of microscopy-based plankton monitoring, DNA-based metabarcoding has so far mainly been limited to individual transect cruises or time-series of single stations. Here we report a dataset covering spatiotemporal variation in prokaryotic and eukaryotic microbial communities and physicochemical parameters. Within 13-months between January 2019 and February 2020, 341 water samples were collected at 22 stations during monthly cruises along the salinity gradient. Both salinity and seasonality are strongly reflected in the data. Since the dataset was generated with both metabarcoding and microscopy-based methods, it provides unique opportunities for both technical and ecological analyses, and is a valuable biodiversity reference for future studies, in the prospect of climate change.

KIR2DS1 and KIR2DL1-C245 Dominantly Repress NK Cell Degranulation Triggered by Monoclonal or Bispecific Antibodies, whereas Education by Uptuning Inhibitory Killer Ig-related Receptors Exerts No Advantage in Ab-dependent Cellular Cytotoxicity.

NK cell responsiveness to target cells is tuned by interactions between inhibitory NK cell receptors and their cognate HLA class I ligands in a process termed "NK cell education." Previous studies addressing the role for NK cell education in Ab-dependent cellular cytotoxicity (ADCC) show ambiguous results and do not encompass full educational resolution. In this study, we systematically characterized human NK cell CD16-triggered degranulation toward defined human tumor cell lines in the presence of either the mAb rituximab or a recently developed CD34xCD16 bispecific killer engager. Despite positive correlation between killer Ig-related receptor (KIR)-mediated education and CD16 expression, NK cells educated by one or even two inhibitory KIRs did not perform better in terms of ADCC than uneducated NK cells in either missing-self or KIR-ligand matched settings at saturating Ab concentrations. Instead, NKG2A+ NK cells consistently showed more potent ADCC in the missing-self context despite lower levels of CD16 expression. KIR2DS1+ NK cells demonstrated dampened ADCC in both the missing-self and KIR-ligand matched settings, even in the presence of its ligand HLA C2. The lower response by KIR2DS1+ NK cells was also observed when stimulated with a bispecific killer engager. Surprisingly, repression of ADCC was also observed by NKG2A+ NK cells coexpressing the inhibitory KIR2DL1-C245 receptor that confers weak education. In conclusion, our study suggests that NK cell education by inhibitory KIRs does not augment ADCC per se, whereas expression of KIR2DS1 and KIR2DL1-C245 dominantly represses ADCC. These insights add to the fundamental understanding of NK cells and may have implications for their therapeutic use.

Glycemic variability and hypoglycemia before and after Roux-en-Y Gastric Bypass and Sleeve Gastrectomy - A cohort study of females without diabetes.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) lead to lower fasting glucose concentrations, but might cause higher glycemic variability (GV) and increased risk of hypoglycemia. However, it has been sparsely studied in patients without preoperative diabetes under normal living conditions. OBJECTIVES: To study 24-hour interstitial glucose (IG) concentrations, GV, the occurrence of hypoglycemia and dietary intake before and after laparoscopic RYGB and SG in females without diabetes. SETTING: Outpatient bariatric units at a community and a university hospital. METHODS: Continuous glucose monitoring and open-ended food recording over 4 days in 4 study periods: at baseline, during the preoperative low-energy diet (LED) regimen, and at 6 and 12 months postoperatively. RESULTS: Of 47 patients included at baseline, 83%, 81%, and 79% completed the remaining 3 study periods. The mean 24-hour IG concentration was similar during the preoperative LED regimen and after surgery and significantly lower compared to baseline in both surgical groups. GV was significantly increased 6 and 12 months after surgery compared to baseline. The self-reported carbohydrate intake was positively associated with GV after surgery. IG concentrations below 3.9 mmol/L were observed in 14/25 (56%) of RYGB- and 9/12 (75%) of SG-treated patients 12 months after surgery. About 70% of patients with low IG concentrations also reported hypoglycemic symptoms. CONCLUSIONS: The lower IG concentration in combination with the higher GV after surgery, might create a lower margin to hypoglycemia. This could help explain the increased occurrence of hypoglycemic episodes after RYGB and SG.

Pseudomonas ability to utilize different carbon substrates and adaptation influenced by protozoan grazing.

Bacteria are major utilizers of dissolved organic matter in aquatic systems. In coastal areas bacteria are supplied with a mixture of food sources, spanning from refractory terrestrial dissolved organic matter to labile marine autochthonous organic matter. Climate scenarios indicate that in northern coastal areas, the inflow of terrestrial organic matter will increase, and autochthonous production will decrease, thus bacteria will experience a change in the food source composition. How bacteria will cope with such changes is not known. Here, we tested the ability of an isolated bacterium from the northern Baltic Sea coast, Pseudomonas sp., to adapt to varying substrates. We performed a 7-months chemostat experiment, where three different substrates were provided: glucose, representing labile autochthonous organic carbon, sodium benzoate representing refractory organic matter, and acetate - a labile but low energy food source. Growth rate has been pointed out as a key factor for fast adaptation, and since protozoan grazers speed-up the growth rate we added a ciliate to half of the incubations. The results show that the isolated Pseudomonas is adapted to utilize both labile and ring-structured refractive substrates. The growth rate was the highest on the benzoate substrate, and the production increased over time indicating that adaptation did occur. Further, our findings indicate that predation can cause Pseudomonas to change their phenotype to resist and promote survival in various carbon substrates. Genome sequencing reveals different mutations in the genome of adapted populations compared to the native populations, suggesting the adaptation of Pseudomonas sp. to changing environment.

Retention of essential fatty acids in fish differs by species, habitat use and nutritional quality of prey.

Algae-produced long-chain polyunsaturated fatty acids (LC-PUFA; with ≥20 carbon atoms) are key biomolecules for consumer production and animal health. They are transferred to higher trophic levels and accumulated in food chains. However, LC-PUFA accumulation in consumers and their trophic transfer vary with the diet quality and the physiological demand for LC-PUFA of consumers. The goal of this study was to investigate spatial and taxonomic differences in LC-PUFA retention of coastal fish predators that potentially differ in their habitat use (benthic versus pelagic) and prey quality. We analyzed the fatty acid (FA) composition of common fish species, namely roach and European perch, as well as their potential prey from benthic and pelagic habitats in three bays of the northern Baltic Sea. We then assessed whether the fish LC-PUFA retention differed between species and among the study bays with different diet quality, that is, LC-PUFA availability. Our data indicated taxon-specific differences in the retention of LC-PUFA and their precursor FA in fish (i.e., short-chain PUFA with <20 carbon atoms). Perch did not show any spatial variation in the retention of all these FA, while roach showed spatial differences in the retention of docosahexaenoic acid (DHA) and their precursor FA, but not eicosapentaenoic acid (EPA). Data suggest that diet quality and trophic reliance on benthic prey underlay the DHA retention differences in roach. Although the PUFA supply might differ among sites, the low spatial variation in LC-PUFA content of perch and roach indicates that both fishes were able to selectively retain dietary LC-PUFA. Climate change together with other existing human-caused environmental stressors are expected to alter the algal assemblages and lower their LC-PUFA supply for aquatic food webs. Our findings imply that these stressors will pose heterogeneous impacts on different fish predators. We advocate further investigations on how environmental changes would affect the nutritional quality of the basal trophic level, and their subsequent impacts on LC-PUFA retention, trophic ecology, and performance of individual fish species.

Documented nutritional therapy in relation to nutritional guidelines post burn injury - a retrospective observational study.

BACKGROUND & AIMS: Intensive nutritional therapy is an essential component of burn care. Regarding post-minor burn injuries, the literature is lacking. The aim of this study was to evaluate documented nutritional therapy in relation to international guidelines after both minor and major burn injuries. The secondary aim of this study was to evaluate the adequacy of energy and protein intake compared to individual nutritional goals post-burn injury. METHODS: A retrospective observational single-centre study including patients admitted between 2017 and 2019 at a burn centre in Sweden was performed. The patients included in the study were ≥18 years old and in need of hospital care for ≥72 h post-burn injury. Information about patients' demographics, nutritional therapy, and clinical characteristics of burn injury was collected. The patients were divided according to total body surface area burnt (TBSA %) into minor burn injuries (TBSA <20%) and major burn injuries (TBSA ≥20%). Descriptive statistics were used to analyse data. Adherence to guidelines was established by comparing 24 nutritional therapy recommendations to documented treatment. If documented nutritional treatment were in accordance with guidelines, adherence was considered high (≥80%), moderate (60-79.9%) or low (<59.9%). RESULTS: One hundred thirty-four patients were included, 90 patients with minor burn injuries and 44 patients with major burn injuries. Documented adherence to the nutritional guideline was overall low. After minor burn injury, 8% (2/24) of nutritional therapy recommendations had a high adherence (fat intake <35% of total energy intake and enteral nutrition as prioritized feeding route), 17% (4/24) a moderate adherence, and 75% (18/24) a low adherence. In patients treated after a major burn injury, there were two recommendations with documented high adherence (Vitamin C and Zinc); 25% (6/24) had moderate adherence, and 67% (16/24) had low adherence. In addition, quite a large amount of missing data was found. Adequacy of documented nutritional intake, compared to the individual documented goal, was 78% (±23%) for energy and 66% (±22%) for protein after minor burn injury. After major burn injury, the adequacy was 89% (±21%) for energy and 78% (±19%) for protein, respectively. CONCLUSIONS: This study revealed low adherence to nutritional guidelines in patients treated for minor and major burn injuries. Compared to major burn injuries, lower documented adequacy for both energy and proteins was found in minor burn injuries. Given the disparity between guidelines and documented nutritional therapy, and the lack of specific guidelines for minor burn injuries, there could be a considerable risk of inadequate nutritional therapy post-burn injury.

Halomethoxybenzenes in air of the Nordic region.

Halomethoxybenzenes (HMBs) are a group of compounds with natural and anthropogenic origins. Here we extend a 2002-2015 survey of bromoanisoles (BAs) in the air and precipitation at Råö on the Swedish west coast and Pallas in Subarctic Finland. New BAs data are reported for 2018 and 2019 and chlorinated HMBs are included for these and some previous years: drosophilin A methyl ether (DAME: 1,2,4,5-tetrachloro-3,6-dimethoxybenzene), tetrachloroveratrole (TeCV: 1,2,3,4-tetrachloro-5,6-dimethoxybenzene), and pentachloroanisole (PeCA). The order of abundance of HMBs at Råö was ΣBAs > DAME > TeCV > PeCA, whereas at Pallas the order of abundance was DAME > ΣBAs > TeCA > PeCA. The lower abundance of BAs at Pallas reflects its inland location, away from direct marine influence. Clausius-Clapeyron (CC) plots of log partial pressure (Pair)/Pa versus 1/T suggested distant transport at both sites for PeCA and local exchange for DAME and TeCV. BAs were dominated by distant transport at Pallas and by both local and distant sources at Råö. Relationships between air and precipitation concentrations were examined by scavenging ratios, SR = (ng m-3)precip/(ng m-3)air. SRs were higher at Pallas than Råö due to greater Henry's law partitioning of gaseous compounds into precipitation at colder temperatures. DAME is produced by terrestrial fungi. We screened 19 fungal species from Swedish forests and found seven of them contained 0.01-3.8 mg DAME per kg fresh weight. We suggest that the volatilization of DAME from fungi and forest litter containing fungal mycelia may contribute to atmospheric levels at both sites.

Co-occurrences enhance our understanding of aquatic fungal metacommunity assembly and reveal potential host-parasite interactions.

Our knowledge of aquatic fungal communities, their assembly, distributions and ecological roles in marine ecosystems is scarce. Hence, we aimed to investigate fungal metacommunities of coastal habitats in a subarctic zone (northern Baltic Sea, Sweden). Using a novel joint species distribution model and network approach, we quantified the importance of biotic associations contributing to the assembly of mycoplankton, further, detected potential biotic interactions between fungi-algae pairs, respectively. Our long-read metabarcoding approach identified 493 fungal taxa, of which a dominant fraction (44.4%) was assigned as early-diverging fungi (i.e. Cryptomycota and Chytridiomycota). Alpha diversity of mycoplankton declined and community compositions changed along inlet-bay-offshore transects. The distributions of most fungi were rather influenced by environmental factors than by spatial drivers, and the influence of biotic associations was pronounced when environmental filtering was weak. We found great number of co-occurrences (120) among the dominant fungal groups, and the 25 associations between fungal and algal OTUs suggested potential host-parasite and/or saprotroph links, supporting a Cryptomycota-based mycoloop pathway. We emphasize that the contribution of biotic associations to mycoplankton assembly are important to consider in future studies as it helps to improve predictions of species distributions in aquatic ecosystems.

Inputs of Terrestrial Dissolved Organic Matter Enhance Bacterial Production and Methylmercury Formation in Oxic Coastal Water.

Methylmercury (MeHg) is a potent neurotoxin commonly found in aquatic environments and primarily formed by microbial methylation of inorganic divalent mercury (Hg(II)) under anoxic conditions. Recent evidence, however, points to the production of MeHg also in oxic pelagic waters, but the magnitude and the drivers for this process remain unclear. Here, we performed a controlled experiment testing the hypothesis that inputs of terrestrial dissolved organic matter (tDOM) to coastal waters enhance MeHg formation via increased bacterial activity. Natural brackish seawater from a coastal area of the Baltic Sea was exposed to environmentally relevant levels of Hg(II) and additions of tDOM according to climate change scenarios. MeHg formation was observed to be coupled to elevated bacterial production rates, which, in turn, was linked to input levels of tDOM. The increased MeHg formation was, however, not coupled to any specific change in bacterial taxonomic composition nor to an increased abundance of known Hg(II) methylation genes. Instead, we found that the abundance of genes for the overall bacterial carbon metabolism was higher under increased tDOM additions. The findings of this study may have important ecological implications in a changing global climate by pointing to the risk of increased exposure of MeHg to pelagic biota.

'Between foods and medicines': A qualitative interview study of patient experiences of the meaning and usage of oral nutritional supplements.

The aim of this study was to deepen the understanding of what oral nutritional supplements mean to patients and how this meaning connects to supplement usage, by exploring patient experiences of such supplements. Qualitative interviews were conducted in June 2019-March 2020 with ten patients with malnutrition or at nutritional risk, prescribed oral nutritional supplements by dietitians. Data were thematically analysed using systematic text condensation. Two final categories were identified: 'Oral nutritional supplements are a one-dimensional remedy' and 'Everyday oral nutritional supplement usage is regulated autonomously'. The patients described the meaning of oral nutritional supplements as nutrition. While the supplements could compensate for nutrients not eaten or be part of a helpful compensation strategy, they could not lessen the burden of altered eating. Supplement usage was described as dependent on the acceptance of taste and the priority given to nutrition in everyday life. Usage was greater when nutrients were perceived as needed, such as when striving for higher bodyweight or disease recovery. Usage was lower when a patient's own goals were not increased nutrient intake or bodyweight or when other activities were perceived as more important. Patient experiences indicated that oral nutritional supplements could serve as a remedy for malnutrition, but not for a situation of altered eating. Supplement usage was described as being regulated autonomously based on patient views on the importance of nutrition. Those views were diverse, highlighting the importance of supplement prescribers discussing treatment goals with each patient. A deeper understanding of the meaning of oral nutritional supplements and reasons for their usage from a patient perspective is crucial in order for healthcare to provide appropriate, effective nutrition therapy for malnutrition.

Microbial Interactions - Underexplored Links Between Public Health Relevant Bacteria and Protozoa in Coastal Environments.

The co-existence of bacteria and protozoa in aquatic environments has led to the evolution of predation defense mechanisms by the bacteria. Some of the predation-resistant bacteria (PRB) are also pathogenic to humans and other mammals. The links between PRB and protozoa in natural aquatic systems are poorly known, but they are important in predicting outbreaks and determining the long-term consequences of a contamination event. To elucidate co-occurrence patterns between PRB (16S rRNA) and bacterivorous protozoa (18S rRNA), we performed a field study in a coastal area in the northern Baltic Sea. Interactions between bacteria and protozoa were explored by using two complementary statistical tools. We found co-occurrence patterns between specific PRB and protozoa, such as Legionella and Ciliophora, and we also found that the interactions are genotype-specific as, for example, Rickettsia. The PRB sequence diversity was larger in bays and freshwater inlets compared to offshore sites, indicating local adaptions. Considering the PRB diversity in the freshwater in combination with the large spring floods in the area, freshwater influxes should be considered a potential source of PRB in the coastal northern Baltic Sea. These findings are relevant for the knowledge of survival and dispersal of potential pathogens in the environment.

Geochemical and Dietary Drivers of Mercury Bioaccumulation in Estuarine Benthic Invertebrates.

Sediments represent the main reservoir of mercury (Hg) in aquatic environments and may act as a source of Hg to aquatic food webs. Yet, accumulation routes of Hg from the sediment to benthic organisms are poorly constrained. We studied the bioaccumulation of inorganic and methylmercury (HgII and MeHg, respectively) from different geochemical pools of Hg into four groups of benthic invertebrates (amphipods, polychaetes, chironomids, and bivalves). The study was conducted using mesocosm experiments entailing the use of multiple isotopically enriched Hg tracers and simulation of estuarine systems with brackish water and sediment. We applied different loading regimes of nutrients and terrestrial organic matter and showed that the vertical localization and the chemical speciation of HgII and MeHg in the sediment, in combination with the diet composition of the invertebrates, consistently controlled the bioaccumulation of HgII and MeHg into the benthic organisms. Our results suggest a direct link between the concentration of MeHg in the pelagic planktonic food web and the concentration of MeHg in benthic amphipods and, to some extent, in bivalves. In contrast, the quantity of MeHg in benthic chironomids and polychaetes seems to be driven by MeHg accumulation via the benthic food web. Accounting for these geochemical and dietary drivers of Hg bioaccumulation in benthic invertebrates will be important to understand and predict Hg transfer between the benthic and the pelagic food web, under current and future environmental scenarios.

Dataset on seston and zooplankton fatty-acid compositions, zooplankton and phytoplankton biomass, and environmental conditions of coastal and offshore waters of the northern Baltic Sea.

We analyzed the taxonomic and fatty-acid (FA) compositions of phytoplankton and zooplankton, and the environmental conditions at three coastal and offshore stations of the northern Baltic Sea. Plankton samples for FA analyses were collected under the framework of sampling campaigns of the Swedish National Marine Monitoring program in September 2017. Monitoring data of phytoplankton and zooplankton biomass, and environmental variables at each station were extracted from the Swedish Meteorological and Hydrological Institute database (https://sharkweb.smhi.se/). Monthly phytoplankton biomass at each station in July-September 2017 was aggregated by class (i.e., chyrsophytes, cryptophytes, dinoflagellates, diatoms, euglenophytes, cyanobacteria, etc.). Zooplankton biomass in September 2017 was aggregated by major taxa (i.e., Acartia sp. [Calanoida], Eurytemora affinis [Calanoida], Cladocera, Limnocalanus macrurus and other copepods (i.e. excluding Eurytemora and Acartia)). Environmental variables monthly monitored in January-October 2017 included salinity, concentrations of dissolved organic carbon, humic substances, total nitrogen and total phosphorus. These variables were measured from 0 to 10 m depth below water surface, and the depth-integrated averages were used for data analyses. Seston and zooplankton (Eurytemora affinis, Acartia sp. and Cladocera) FA compositions were analyzed using gas chromatography and mass spectroscopy (GC-MS). Our dataset could provide new insights into how taxonomic composition and biochemical quality of the planktonic food chains change with the environmental conditions in subarctic marine ecosystems.

Short- and long-read metabarcoding of the eukaryotic rRNA operon: Evaluation of primers and comparison to shotgun metagenomics sequencing.

High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500 bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.