High-throughput DNA Sequencing Analysis Research Articles

Use of commercial or indigenous yeast impacts the S. cerevisiae transcriptome during wine fermentation.

Understanding microbial functions during wine fermentation, particularly the role of Saccharomyces cerevisiae, is crucial for enhancing wine quality. While commercially available S. cerevisiae strains are commonly used, indigenous strains can offer unique flavors, potentially reflecting vineyard terroir. By leveraging high-throughput DNA sequencing and transcriptomic analysis, we explored the impact of vine age on the grape mycobiome and characterized microbial functions during grape fermentation. Our findings revealed that older vines harbor higher fungal loads, but fungal diversity remains similar across vine ages. Additionally, uninoculated fermentations exhibited diverse fungal dynamics, including the beneficial wine yeasts Metschnikowia and Pichia. Transcriptomic analysis uncovered significant differences in S. cerevisiae gene expression between inoculated and uninoculated fermentations, highlighting the potential of indigenous yeast to enhance wine diversity and inform winemaking practices.

High-throughput DNA sequence analysis elucidates novel insight into the genetic basis of adaptation in local sheep.

Understanding how evolutionary factors related to climate adaptation and human selection have influenced the genetic architecture of domesticated animals is of great interest in biology. In the current study, by using 304 whole genomes from different geographical regions (including Europe, north Africa, Southwest Asia, east Asia, west Africa, south Asia, east Africa, Australia and Turkey), We evaluate global sheep population dynamics in terms of genetic variation and population structure. We further conducted comparative population analysis to study the genetic underpinnings of climate adaption to local environments and also morphological traits. In order to identify genomic signals under selection, we applied fixation index (FST) and also nucleotide diversity (θπ) statistical measurements. Our results revealed several candidate genes on different chromosomes under selection for local climate adaptation (e.g. HOXC12, HOXC13, IRF1, FGD2 and GNAQ), body size (PDGFA, HMGA2, PDE3A) and also morphological related traits (RXFP2). The discovered candidate genes may offer newel insights into genetic underpinning of regional adaptation and commercially significant features in local sheep.

Efficient minimizer orders for large values of k using minimum decycling sets.

Minimizers are ubiquitously used in data structures and algorithms for efficient searching, mapping, and indexing of high-throughput DNA sequencing data. Minimizer schemes select a minimum k-mer in every L-long subsequence of the target sequence, where minimality is with respect to a predefined k-mer order. Commonly used minimizer orders select more k-mers than necessary and therefore provide limited improvement in runtime and memory usage of downstream analysis tasks. The recently introduced universal k-mer hitting sets produce minimizer orders with fewer selected k-mers. Generating compact universal k-mer hitting sets is currently infeasible for k > 13, and thus, they cannot help in the many applications that require minimizer orders for larger k Here, we close the gap of efficient minimizer orders for large values of k by introducing decycling-set-based minimizer orders: new minimizer orders based on minimum decycling sets. We show that in practice these new minimizer orders select a number of k-mers comparable to that of minimizer orders based on universal k-mer hitting sets and can also scale to a larger k Furthermore, we developed a method that computes the minimizers in a sequence on the fly without keeping the k-mers of a decycling set in memory. This enables the use of these minimizer orders for any value of k We expect the new orders to improve the runtime and memory usage of algorithms and data structures in high-throughput DNA sequencing analysis.

Analysis of differences in tobacco leaf microbial communities after redrying in Chinese provinces and from abroad

Microorganisms play an important role in the tobacco aging process. Before the aging process, raw tobacco leaves must be threshed and redried. In order to explore the differences of microbial community structure of threshed and redried tobacco leaves from different origins at home and abroad, 14 groups of tobacco leaves from 8 different countries were tested by high-throughput DNA sequencing and microbiology analysis. Then, through amplicon sequence variants (ASV) cluster analysis, Venn diagram and species labeling and other microbial diversity analysis, the dominant bacteria and fungi on the surface of threshed and redried tobacco leaves were obtained. The results showed that there were significant differences in the composition of tobacco bacteria and fungi after threshing and redrying from different geographical areas. The relative abundance of Microbacterium and Sphingomonas in domestic tobacco leaves was significantly higher than that of foreign tobacco leaves. The relative abundance of Pseudomonas in foreign tobacco bacterial colonies was significantly higher than that of domestic tobacco leaves. In terms of fungi, the relative abundance of Aspergillus and Alternaria in domestic tobacco leaves was significantly higher than that of foreign tobacco leaves. Septoria, Sampaiozyma, Cladosporium and Phoma account for significantly higher proportions of foreign tobacco leaves. These microorganisms may be indispensable in aging process to form different flavors of tobacco leaves. It provides an important theoretical basis for the further use of microorganisms to promote tobacco leaf aging.

Interactions between the transcription factors FfmA and AtrR are required to properly regulate gene expression in the fungus Aspergillus fumigatus.

Transcriptional regulation of azole resistance in the filamentous fungus Aspergillus fumigatus is a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility. Null alleles of ffmA exhibit a strongly compromised growth rate even in the absence of any external stress. Here, we employ an acutely repressible doxycycline-off form of ffmA to rapidly deplete FfmA protein from the cell. Using this approach, we carried out RNA-seq analyses to probe the transcriptome cells acutely deprived of FfmA. A total of 2,000 genes were differentially expressed upon acute depletion of FfmA, illustrating the broad transcriptomic effect of this factor. Interestingly, the transcriptome changes observed upon this acute depletion of FfmA expression only shared limited overlap with those found in an ffmAΔ null strain analyzed by others. Chromatin immunoprecipitation coupled with high throughput DNA sequencing analysis (ChIP-seq) identified 530 genes that were bound by FfmA. More than 300 of these genes were also bound by AtrR, a transcription factor important in azole drug resistance, demonstrating striking regulatory overlap with FfmA. However, while AtrR is an upstream activation protein with known specificity, our data suggest that FfmA is a chromatin-associated factor that binds DNA in a manner dependent on other factors. We provide evidence that AtrR and FfmA interact in the cell and show reciprocal expression modulation. Interaction of AtrR and FfmA is required for normal gene expression in A. fumigatus.

Understanding protein functions in the biological context.

Proteins are essential biomacromolecules in all living systems because they are the prominent ultimate executives of the genetic information stored in DNA. Thus, studying protein is one of the central tasks in biological sciences. The complexity, diversity, and dynamics of a protein's structure, function, and structure-function relationship, the inherent structural fragility and thus the requirements on handling proteins to maintain protein's structural and functional orderliness make it a rather tricky task to work with protein. The approach to understanding the functions of a protein has been progressing steadily. In this paper, we reviewed the progress on the approach to the functional study of proteins that tremendously contributed to understanding their biological significance. Emphasis was put on the advances in the age in which high-throughput DNA sequencing and bioinformatics analysis are revolutionizing biological study.

Structure and Functional Properties of Bacterial Communities in Surface Sediments of the Recently Declared Nutrient-Saturated Lake Villarrica in Southern Chile.

Lake Villarrica, one of Chile's main freshwater water bodies, was recently declared a nutrient-saturated lake due to increased phosphorus (P) and nitrogen (N) levels. Although a decontamination plan based on environmental parameters is being established, it does not consider microbial parameters. Here, we conducted high-throughput DNA sequencing and quantitative polymerase chain reaction (qPCR) analyses to reveal the structure and functional properties of bacterial communities in surface sediments collected from sites with contrasting anthropogenic pressures in Lake Villarrica. Alpha diversity revealed an elevated bacterial richness and diversity in the more anthropogenized sediments. The phylum Proteobacteria, Bacteroidetes, Acidobacteria, and Actinobacteria dominated the community. The principal coordinate analysis (PCoA) and redundancy analysis (RDA) showed significant differences in bacterial communities of sampling sites. Predicted functional analysis showed that N cycling functions (e.g., nitrification and denitrification) were significant. The microbial co-occurrence networks analysis suggested Chitinophagaceae, Caldilineaceae, Planctomycetaceae, and Phycisphaerae families as keystone taxa. Bacterial functional genes related to P (phoC, phoD, and phoX) and N (nifH and nosZ) cycling were detected in all samples by qPCR. In addition, an RDA related to N and P cycling revealed that physicochemical properties and functional genes were positively correlated with several nitrite-oxidizing, ammonia-oxidizing, and N-fixing bacterial genera. Finally, denitrifying gene (nosZ) was the most significant factor influencing the topological characteristics of co-occurrence networks and bacterial interactions. Our results represent one of a few approaches to elucidate the structure and role of bacterial communities in Chilean lake sediments, which might be helpful in conservation and decontamination plans.

High-throughput sequencing and marker pigment analysis of freshwater phytoplankton: A direct comparison with microscopic count data in the tropical crater lakes of Western Uganda

High-throughput DNA sequencing (HTS) and photosynthetic marker-pigment analysis can provide cost-and time-efficient alternatives for morphology-based microscopic identification and counting of lacustrine phytoplankton. However, application of particularly HTS in tropical lakes is still uncommon. We analyzed the community composition of cyanobacterial and eukaryotic phytoplankton in relation to trophic status and other environmental factors in 15 tropical crater lakes in western Uganda, using both high-performance liquid chromatography (HPLC) of photosynthetic marker pigments and HTS of 16 S and 18 S small subunit rRNA genes. We then compared the results with phytoplankton composition data based on microscopic identification and cell counting to evaluate whether the two former methods can be alternative or complementary approaches to analyze these lakes’ phytoplankton communities. HTS recorded 105 operational taxonomic units (OTUs) of cyanobacteria, and 197 OTUs of autotrophic eukaryotes mainly from dinoflagellates, cryptophytes and green algae. Ochrophyta (chrysophytes and diatoms) were present in low abundances only, and the few remaining sequences belonged to Streptophyta and Haptophyta. Marker pigment distribution among samples reflected the widespread and often dominant presence of cyanobacteria (primarily zeaxanthin, myxoxanthophyll and echinenone), followed by green algae (chlorophyll b, lutein, antheraxanthin, neoxanthin) and diatoms (fucoxanthin, diato-diadinoxanthin), and with cryptophyta (alloxanthin) important in some of the lakes. Direct and indirect ordinations of the OTUs and marker pigments revealed that, similar to patterns observed in cell counts, phytoplankton community composition differed between deep oligotrophic lakes and shallower more productive lakes, even though the distribution of cyanobacterial OTUs was more related to lake water conductivity and season than to trophic status. Procrustes analyses of the composition of green algae and Ochrophyta, the two dominant eukaryotic groups, based on HTS data showed patterns significantly similar to composition data based on cell counts, but no significant correlations were found between the complete eukaryotic or cyanobacterial HTS data and cell count data. While the latter discrepancies can be attributed to method-related biases in both HTS and microscopy, in contrast to the microscopic approach HTS failed to identify trophic indicator species and did not assign all lakes to the correct trophic category. Composition data based on marker pigments clustered the 15 lakes according to their trophic level only when pigment abundances were expressed as absolute concentrations, not when expressed as fractional abundances. We attribute this lower discriminating power partly to the wide distribution of cyanobacteria in these tropical crater lakes, besides the pigments’ low taxonomic resolution. Nevertheless, as the compositional patterns in relation to environmental variation observed in marker pigments and OTUs of the dominant eukaryotic phytoplankton groups are similar to those in cell count data, both methods hold potential for ecosystem-level monitoring of tropical crater lakes. However, the high-resolution HTS approach is handicapped by non-uniform amplification of gene sequences, while the low-resolution marker-pigment approach is handicapped by the widespread dominance of cyanobacteria in these systems irrespective of their trophic status.

ChIP-Seq Strategy to Identify Z-DNA-Forming Sequences in the Human Genome.

Different from the canonical right-handed B-DNA, a left-handed Z-DNA forms an alternating syn- and anti-base conformations along the double-stranded helix under physiological conditions. Z-DNA structure plays a role in transcriptional regulation, chromatin remodeling, and genome stability. To understand the biological function of Z-DNA and map the genome-wide Z-DNA-forming sites (ZFSs), a ChIP-Seq strategy is applied, which is a combination of chromatin immunoprecipitation (ChIP) and high-throughput DNA sequencing analysis. Cross-linked chromatin is sheared and its fragments associated with Z-DNA-binding proteins are mapped onto the reference genome sequence. The global information of ZFSs positioning can provide a useful resource for better understanding of DNA structure-dependent biological mechanism.

High-Throughput DNA Analysis Platform Based on an Optofluidic Ring Resonator Laser

We demonstrate an optofluidic laser using DNA microdroplets, an intercalating dye, and a glass capillary ring resonator. Only the target DNA emits the laser signal, while the non-target DNA, including those with single-base mismatches, exhibits zero optical signals. Subsequently, a single laser emission detected within a few milliseconds can distinguish the target DNA from others, allowing for truly digital DNA sequence analysis from an optical point of view. In addition, the microdroplets could be individually investigated using our optofluidic system. These advances in laser-based DNA analysis make our proposed scheme a promising, rapid, and high-throughput DNA sequence analysis platform.

Berberine suppresses colorectal cancer by regulation of Hedgehog signaling pathway activity and gut microbiota

BackgroundA growing body of evidence reveals that dysregulation of Hedgehog signaling pathway and dysbiosis of gut microbiota are associated with the pathogenesis of colorectal cancer (CRC). Berberine, a botanical benzylisoquinoline alkaloid, possesses powerful activities against various malignancies including CRC, with the underlying mechanisms to be illuminated. PurposeThe present study investigated the potencies of berberine on CRC and deciphered the action mechanisms in the context of Hedgehog signaling cascade and gut microbiota. MethodsThe effects of berberine on the malignant phenotype, apoptosis, cell cycle and Hedgehog signaling of CRC cells were examined in vitro. In azoxymethane/dextran sulfate sodium-caused mouse CRC, the efficacies of berberine on the carcinogenesis, pathological profile, apoptosis, cell cycle and Hedgehog signaling were determined in vivo. Also, the influences of berberine on gut microbiota in CRC mice were assessed by high-throughput DNA sequencing analysis of 16S ribosomal RNA of fecal microbiome in CRC mice. ResultsIn the present study, berberine was found to dampen the proliferation, migration, invasion and colony formation of CRC cells, without toxicity to normal colonic cells. Additionally, berberine induced apoptosis and arrested cell cycle at G0/G1 phase in CRC cells, accompanied by reduced Hedgehog signaling pathway activity in vitro. In mouse CRC, berberine suppressed tumor growth, ameliorated pathological manifestations, and potentially induced the apoptosis and cell cycle arrest of CRC, with lowered Hedgehog signaling cascade in vivo. Additionally, berberine decreased β-diversity of gut microbiota in CRC mice, without influence on α-diversity. Berberine also enriched probiotic microbes and depleted pathogenic microbes, and modulated the functionality of gut microbiota in CRC mice. ConclusionsOverall, berberine may suppress colorectal cancer, orchestrated by down-regulation of Hedgehog signaling pathway activity and modulation of gut microbiota.

Production of Universal Anti-Bcma CAR-T Cells with Reduced Alloreactivity, but Potent Effector Function for the Treatment of Multiple Myeloma

Chimeric antigen receptor (CAR)-T therapy has recently shown great promise against multiple myeloma (MM) by targeting the B cell maturation antigen (BCMA). However, the production of autologous CAR-T cells on an individualized basis has significant challenges such as manufacture time, reproducibility, consistency and cost. In addition, viral-vector-produced CAR-T cells tend to be comprised of differentiated T cell subsets, which correlate with poor in vivo durability. Using the high-fidelity NextGEN™ (NG) CRISPR gene editing system in combination with the non-viral piggyBac™ (PB) DNA transposition technology, which produces CAR-T cells predominantly in a highly desirable stem cell memory T cell subset, we have developed P-BCMA-ALLO1, an off-the-shelf allogeneic CAR-T cell product that overcomes these limitations and can be given to any MM patient.The NG system was used to disrupt both TCR and MHCI expression in P-BCMA-ALLO1 to reduce possible alloreactivities. The DNA cleavage specificity is guaranteed by a truly dimeric system requiring the simultaneous binding of two obligate homodimers. High-throughput DNA sequencing analysis was performed on human T cells with gene disruption of TCRα, β2M or PD-1. Indel frequencies of amplified genomic on-target and off-target sites were evaluated from T cells that had been edited with either single or dual guide RNAs (gRNA)s. Herein, an average of 100,000 sequences were analyzed per site and non-edited T cells were included as a background reference. For these targets, the NG system demonstrated high-efficiency gene editing (>84% for TCRα, >64% for β2M, and >50% for PD-1) that was performed in resting T cells. No indel events were detected at any off-target site, proving the exquisite specificity of this system.P-BCMA-ALLO1 was incapable of mediating allo-responses such as graft-vs-host disease (GvHD) and graft rejection. Using a clinically-scalable purification method, P-BCMA-ALLO1 cells were enriched to be >99.5% TCR/MHCI double-negative and alloreactivities were evaluated by standard mixed lymphocyte reaction for either cell proliferation or IFNγ production in two separate assays: a 7-12 day CFSE-based cell proliferation assay and a 20-hour IFNγ-ELISPOT assay. Compared to non-edited CAR-T cells, P-BCMA-ALLO1 did not exhibit any detectable response to allogeneic PBMC stimulation, nor did it trigger any allogeneic T cell proliferative response above background level of cell proliferation or IFNγ production.Pre-clinical evaluation of P-BCMA-ALLO1 demonstrated optimal T cell phenotype and potent in vitro and in vivo efficacy. More than 95% of the P-BCMA-ALLO1 cells were CAR+ and greater than 50% of the product were CD45RA+CD62L+ stem cell memory T cells (TSCM), a highly desirable early memory T cell subset that exhibits the greatest capacity for self-renewal and the multipotent capacity to the entire spectrum of memory and effector T cell subsets. In addition, the CAR-T cells did not express the exhaustion markers PD-1, TIM-3 and LAG-3, nor did they exhibit any evidence of tonic signaling as IFNγ production in the absence of target antigen. In a short-term 24-hour in vitro co-culture assay with BCMA+ MM cell lines, P-BCMA-ALLO1 secreted high levels of IFNγ and mediated robust (70-90%) killing of cell targets. In addition, more than 80% of the cells proliferated in a longer 72-hour CFSE-based proliferation assay. P-BCMA-ALLO1 also showed strong potency in a NSG mouse model engrafted with MM.1S cells, an aggressive human MM-derived tumor model. While all control animals showed rapid tumor growth by bioluminescent imaging (BLI) and succumbed to disease within five weeks, all treated mice survived for the duration of the study. After P-BCMA-ALLO1 administration, tumor burden was rapidly reduced to the limit of detection by BLI within 7-10 days. Lastly, several occurrences of tumor relapse were observed in some of these animals, but were subsequently controlled without re-administration of the product. Altogether, these data demonstrate that P-BCMA-ALLO1 exhibited significantly reduced alloreactivity yet retained potent effector function, suggesting its safe and effective use for treating any MM patient. DisclosuresWang:Poseida Therapuetics: Employment. Barnett:Poseida Therapeutics, Inc.: Employment. Martin:Poseida Therapeutics, Inc.: Employment. Hermanson:Poseida Therapeutics: Employment. Li:Poseida Therapuetics: Employment. Smith:Poseida Therapeutics, Inc.: Employment. Wu:Poseida Therapuetics: Employment. Cranert:Poseida Therapuetics: Employment. Rengarajan:Poseida Therapeutics, Inc.: Employment. Tan:Poseida Therapeutics, Inc.: Employment. Tong:Poseida Therapuetics: Employment. Codde:Poseida Therapeutics, Inc.: Employment. Down:Poseida Therapeutics, Inc.: Employment. Ostertag:Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock:Poseida Therapeutics, Inc.: Employment.

Influence of polycyclic aromatic hydrocarbon pollution on the diversity and function of bacterial communities in urban wetlands.

Human disturbance has become the primary driving factor behind declining urban wetland ecological health due to rapid urbanization. Sediment microbial communities are critical for wetland ecosystem functioning but experience a range of natural and anthropogenic stressors due to rapid urbanization and land use changes, especially in developing countries. Polycyclic aromatic hydrocarbons (PAHs) released into the environment primarily come from anthropogenic sources like industrial activities and traffic emissions. Environmental PAH contamination is accelerating due to rapid urbanization, which also increases potential PAH-related dangers to human health. However, PAHs are widely distributed and not easy to centrally control. Microorganisms are the primary mediators of wetland purification, with most PAH-degrading microorganisms being bacteria. To better understand the influence of PAH contamination on urban wetland microbial communities, bacterial community compositions within sediments of urban wetlands in three land use types were investigated using high-throughput DNA sequencing and bioinformatics analyses. Statistical analyses revealed significant differences in overall microbial compositions among the three land use types, although γ-proteobacteria was the dominant phyla across all samples. Among the potential PAH-degrading bacterial taxa in sediments, Sphingomonas was the most prevalent. The distributions of PAH-degrading taxa were primarily affected by variance in organic compound abundances in addition to various physico-chemical variables, among which high-ring PAH content was a key parameter associated with bacterial distributions, except in the riverine wetlands. Functional inference via phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) indicated that 30 of the 43 genes related to PAH metabolism were predicted to be present within the genomes of bacteria among the three land use type. In particular, dioxygenase and dehydrogenase genes involved in PAH degradation were inferred to be prevalent, indicating that the host urban wetlands exhibited strong potential for organic pollutant degradation.

Mycobiome of Fraxinus excelsior With Different Phenotypic Susceptibility to Ash Dieback

For the last two decades, large-scale population decline of European ash (Fraxinus excelsior) has occurred in Europe because of the introduction of the alien fungal pathogen, Hymenoscyphus fraxineus, from East Asia. Since European ash is a keystone species having critical importance for biodiversity, and only a small percentage of the ash population appears to show some tolerance against the pathogen, the loss of ash trees means that other associated organisms, especially those with high or obligate associations to ash, are at risk of further species declines. In this study, we used high throughput DNA sequencing and multivariate analysis to characterize: (i) the mycobiome in aerial tissues (i.e., leaf, bark, and xylem) of ash trees showing different phenotypic response to ash dieback, (ii) the temporal variation in fungal communities across the growing season, and (iii) the similarity in fungal community structure between ash and other common trees species that may serve as an ecological niche substitute for ash microfungi. Results showed that fungal communities differed among the three tissue types, susceptibility classes, in time and between sites. Trophic analysis of functional groups using the FUNGuild tool indicated a higher presence of pathotrophic fungi in leaves than in bark and xylem. The share of pathotrophic fungi increased along a gradient of low to high disease susceptibility in both bark and xylem tissue, while the proportion of symbiotrophic fungi correspondingly decreased in both tissue types. Neighboring, alternative host trees did not share all the fungal species found in ash, however, most microfungi uniquely associated to ash in this study are generalists and not strictly host specific. The progressive disappearance of ash trees on the landscape imposes a high risk for extinction of Red-listed macrofungal species, and breeding for resistance against ash dieback should help sustain important biodiversity associated to ash. Microfungal diversity though may be less prone to such demise since most ash-associated endophytes appear to occur on a broad range of host species.

Single-nucleotide resolution of N 6-adenine methylation sites in DNA and RNA by nitrite sequencing.

A single-nucleotide resolution sequencing method of N6-adenine methylation sites in DNA and RNA is described. Using sodium nitrite under acidic conditions, chemoselective deamination of unmethylated adenines readily occurs, without competing deamination of N6-adenine sites. The deamination of adenines results in the formation of hypoxanthine bases, which are read by polymerases and reverse transcriptases as guanine; the methylated adenine sites resist deamination and are read as adenine. The approach, when coupled with high-throughput DNA sequencing and mutational analysis, enables the identification of N6-adenine sites in RNA and DNA within various sequence contexts.

Cell-Free Exploration of the Natural Product Chemical Space.

Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.

MSphere of Influence: Translating Gut Microbiome Studies To Benefit Human Health.

Anna M. Seekatz works in the field of the gut microbiome as it related to infectious diseases. In this "mSphere of Influence" article, she reflects on how two studies, "The impact of a consortium of fermented milk strains on the gut microbiome of gnotobiotic mice and monozygotic twins" (N. P. McNulty, T. Yatsunenko, A. Hsiao, et al., Sci Transl Med 3:106ra106, 2011) and "High-throughput DNA sequence analysis reveals stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria" (M. J. Hamilton, A. R. Weingarden, T. Unno, A. Khoruts, and M. J. Sadowsky, Gut Microbes 4:125-135, 2013), shaped how she approaches interpreting microbiome studies.

Tasting the differences: Microbiota analysis of different insect-based novel food

Traceability, quality and safety of edible insects are important both for the producers and the consumers. Today, alongside the burst of edible insects in western countries, we are facing a gap of knowledge of insect microbiota associated with the microbial ecosystems of insect-based products. In this context, High-Throughput DNA Sequencing (HTS) techniques can give insight into the carryover of insect microbiota into final food products. In this study, we investigated the microbiota composition of insect-based commercial food products, applying HTS techniques coupled with bioinformatic analysis. The work aimed to analyse the microbiota variability of different categories of some insect-based commercial food products made of A. domesticus (house cricket), T. molitor (mealworm beetle), and A. diaperinus (lesser mealworm or litter beetle), including commercial raw materials and processed food items, purchased via e-commerce from different companies. Our data revealed that samples cluster per insect species based on microbiota profile and preliminary results suggested that a small number of prevalent bacteria formed a “core microbiota” characterizing the products depending on the insect. This microbial signature can be recognized despite the different food processing levels, rearing conditions and selling companies. Furthermore, differences between raw and processed food made of the same insect or similar product produced by different companies was found.These results support the application of HTS analysis for studying the composition of insect-based commercial food products in a wider perspective, for food traceability and food quality control.

A rapid high-throughput sequencing-based approach for the identification of unknown bacterial pathogens in whole blood.

High-throughput DNA sequencing (HTS) of pathogens in whole blood samples is hampered by the high host/pathogen nucleic acids ratio. We describe a novel and rapid bacterial enrichment procedure whose implementation is exemplified in simulated bacteremic human blood samples. The procedure involves depletion of the host DNA, rapid HTS and bioinformatic analyses. Following this procedure, Y. pestis, F. tularensis and B. anthracis spiked-in samples displayed an improved host/pathogen DNA ratio of 2.5–5.9 orders of magnitude, in samples with bacteria spiked-in at 103–105 CFU/ml. The procedure described in this study enables rapid and detailed metagenomic profiling of pathogens within 8–9 h, circumventing the challenges imposed by the high background present in the bacteremic blood and by the unknown nature of the sample.

Effects of Hydraulic Retention Time and Influent Nitrate-N Concentration on Nitrogen Removal and the Microbial Community of an Aerobic Denitrification Reactor Treating Recirculating Marine Aquaculture System Effluent

The effects of hydraulic retention time (HRT) and influent nitrate-N concentration on nitrogen removal and the microbial community composition of an aerobic denitrification reactor treating recirculating marine aquaculture system effluent were evaluated. Results showed that over 98% of nitrogen was removed and ammonia-N and nitrite-N levels were below 1 mg/L when influent nitrate-N was below 150 mg/L and HRT over 5 h. The maximum nitrogen removal efficiency and nitrogen removal rate were observed at HRT of 6 or 7 h when influent nitrate-N was 150 mg/L. High-throughput DNA sequencing analysis revealed that the microbial phyla Proteobacteria and Bacteroidetes were predominant in the reactor, with an average relative total abundance above 70%. The relative abundance of denitrifying bacteria of genera Halomonas and Denitratisoma within the reactor decreased with increasing influent nitrate-N concentrations. Our results show the presence of an aerobically denitrifying microbial consortium with both expected and unexpected members, many of them relatively new to science. Our findings provide insights into the biological workings and inform the design and operation of denitrifying reactors for marine aquaculture systems.