Metagenomic analysis of the bacterial microbiome, resistome and virulome distinguishes Portuguese Serra da Estrela PDO cheeses from similar non-PDO cheeses: An exploratory approach.

This study aimed to evaluate the microbiome, resistome and virulome of two types of Portuguese cheese using high throughput sequencing (HTS). Culture-dependent chromogenic methods were also used for certain groups/microorganisms. Eight samples of raw ewe's milk cheese were obtained from four producers: two producers with cheeses with a PDO (Protected Designation of Origin) label and the other two producers with cheeses without a PDO label. Agar-based culture methods were used to quantify total mesophiles, Enterobacteriaceae, Escherichia coli, Staphylococcus, Enterococcus and lactic acid bacteria. The presence of Listeria monocytogenes and Salmonella was also investigated. The selected isolates were identified by 16S rRNA gene sequencing and evaluated to determine antibiotic resistance and the presence of virulence genes. The eight cheese samples analyzed broadly complied with EC regulations in terms of the microbiological safety criteria. The HTS results demonstrated that Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus plantarum, Lacticaseibacillus rhamnosus, Enterococcus durans and Lactobacillus coryniformis were the most prevalent bacterial species in cheeses. The composition of the bacterial community varied, not only between PDO and non-PDO cheeses, but also between producers, particularly between the two non-PDO cheeses. Alpha-diversity analyses showed that PDO cheeses had greater bacterial diversity than non-PDO cheeses, demonstrating that the diversity of spontaneously fermented foods is significantly higher in cheeses produced without the addition of food preservatives and dairy ferments. Despite complying with microbiological regulations, both PDO and non-PDO cheeses harbored potential virulence genes as well as antibiotic resistance genes. However, PDO cheeses exhibited fewer of these virulence and antibiotic resistance genes compared to non-PDO cheeses. Therefore, the combination of conventional microbiological methods and the metagenomic approach could contribute to improving the attribution of the PDO label to this type of cheese.

Genomic characterisation of bioaerosols within livestock facilities: A systematic review.

Livestock facilities are widely regarded as reservoirs of infectious disease, owing to their abundance in particulate matter (PM) and microbial bioaerosols. Over the past decade, bioaerosol studies have increasingly utilised high throughput sequencing (HTS) to achieve superior throughput, taxonomic resolution, and the detection of unculturable organisms. However, the prevailing focus on amplicon sequencing has limited the identification of viruses and microbial taxa at the species-level. Herein, a literature search was conducted to identify methods capable of overcoming the aforementioned limitations. Screening 1531 international publications resulted in 29 eligible for review. Metagenomics capable of providing rich insights were identified in only three instances. Notably, long-read sequencing was not utilised for metagenomics. This review also identified that sample collection methods lack a uniform approach, highlighted by the differences in sampling equipment, flow rates and durations. Further heterogeneity was introduced by the unique sampling conditions, which makes it challenging to ground new findings within the established literature. For instance, winter was associated with increased microbial abundance and antimicrobial resistance, yet less alpha diversity. Researchers implementing metagenomics into the livestock environment should consider season, the microclimate, and livestock growth stage as influential upon their findings. Considering the increasing accessibility of long-read sequencing, future research should explore its viability within a novel uniform testing protocol for bioaerosol emissions.

Effects of curing concentration and drying time on flavor and microorganisms in dry salted Spanish mackerel.

This study investigated the quality changes of dry salted mackerel during curing and drying process and the relationship between flavor substances and microorganisms. The results showed that the thiobarbituric acid reactive substances (TBARS) values increased gradually with the increase of salt concentration and treatment time. The total volatile base nitrogen (TVB-N) values and total viable counts (TVC) values showed the same trend. Under 3% condition, the TVB-N values exceeded the standard and was not suitable for consumption. A total of 61 volatile flavor substances were identified by Gas chromatography-ion mobility spectrometry (GC-IMS), among which aldehydes contributed the most. Staphylococcus and Cobetia were the most abundant by High-throughput sequencing (HTS). There was significant correlation between TOP15 microorganisms and TOP20 flavor substances. Staphylococcus and Cobetia were positively correlated with 13 volatile flavor substances, which contributed to the formation of flavor in naturally fermented Spanish mackerel.

From Metagenomics to Ecogenomics: NGS-Based Approaches for Discovery of New Circular DNA Single-Stranded Viral Species.

Viruses comprise the most abundant genetic material in the biosphere; however, global viral genomic population (virome) has been largely underestimated. Recently, high-throughput sequencing (HTS) has provided a powerful tool for the detection of known viruses and the discovery of novel viral species from environmental and individual samples using metagenomics and ecogenomics approaches, respectively. Viruses with circular DNA single-stranded (ssDNA) genomes belonging to the begomovirus genera (family Geminiviridae) constitute the largest group of emerging plant viruses worldwide. The knowledge of begomoviruses viromes is mostly restricted to crop plant systems; nevertheless, it has been described that noncultivated plants specifically at the interface between wild and cultivated plants are important reservoirs leading to viral evolution and the emergence of new diseases. Here we present a protocol that allows the identification and isolation of known and novel begomoviruses species infecting cultivated and noncultivated plant species. The method consists of circular viral molecules enrichment by rolling circle amplification (RCA) from begomovirus-positive total plant DNA, followed by NGS-based metagenomic sequencing. Subsequently, metagenomic reads are processed for taxonomic classification using Viromescan software and a customized Geminiviridae family database, and begomovirus-related reads are used for contigs assembly and annotation using Spades software and Blastn algorithm, respectively. Then, the obtained begomovirus-related signatures are used as templates for specific primers design and implemented for PCR-based ecogenomic identification of individual samples harboring the corresponding viral species. Lastly, full-length begomovirus genomes are obtained by RCA-based amplification from total plant DNA of selected individual samples, cloning, and viral molecular identity corroborated by Sanger sequencing. Conclusively, the identification and isolation of a novel monopartite begomovirus species native to the New World (NW) named Gallium leaf deformation virus (GLDV) is shown.

Viromes of Plants Determined by High-Throughput Sequencing of Virus-Derived siRNAs.

Plants growing in open airfields can be infected by several viruses even as a multiple infection. Virus infection in crops can lead to a serious damage to the harvest. In addition, virus presence in grapevine, fruit trees, and tuberous vegetables, propagated vegetatively affects the phytosanitary status of the propagation material (both the rootstock and the variety) having profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Small interfering (si) RNAs produced by the RNA silencing-based host immune system during viral infection can be sequenced by high-throughput techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample and therefore opening new avenues in virus diagnostics. This method is based on Illumina sequencing and bioinformatics analysis of virus-derived siRNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, to ensure success for every user.

The Oxford Nanopore MinION as a Versatile Technology for the Diagnosis and Characterization of Emerging Plant Viruses.

The emergence of novel viral epidemics that could affect major crops represents a serious threat to global food security. The early and accurate identification of the causative viral agent is the most important step for a rapid and effective response to disease outbreaks. Over the last years, the Oxford Nanopore Technologies (ONT) MinION sequencer has been proposed as an effective diagnostic tool for the early detection and identification of emerging viruses in plants, providing many advantages compared with different high-throughput sequencing (HTS) technologies. Here, we provide a step-by-step protocol that we optimized to obtain the virome of "Lamon bean" plants (Phaseolus vulgaris L.), an agricultural product with Protected Geographical Indication (PGI) in North-East of Italy, which is frequently subjected to multiple infections caused by different RNA viruses. The conversion of viral RNA in ds-cDNA enabled the use of Genomic DNA Ligation Sequencing Kit and Native Barcoding DNA Kit, which have been originally developed for DNA sequencing. This allowed the simultaneous diagnosis of both DNA- and RNA-based pathogens, providing a more versatile alternative to the use of direct RNA and/or direct cDNA sequencing kits.

Application of Nanopore-Based Sequencing to Identify Virus Infections in Woody Plants.

Plant viruses threaten the yield and quality of crops. Efficient and affordable pathogen diagnosis is crucial to regulate the trade of plant materials and for disease management and control. Sequencing technology based on Illumina platform is a powerful tool for the identification of plant viruses, but it requires long and expensive protocols, cumbersome equipment, and significant cost per library. Nanopore sequencing technology, developed by Oxford Nanopore Technologies (ONT), is a recent sequencing system very easy to use, suitable for onsite-field detection, and associated with low costs. Coupled with its portability, nanopore technology has great application prospects in the field of quick detection of plant viruses. In this protocol, we expose in detail the application of cDNA-PCR nanopore-based sequencing for the detection of plant viruses.

Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication.

We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio's single-molecule real-time sequencing and Oxford's Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X+ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767-8523 in the X+ virus. Thus, the X+ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X+ variant virus. Furthermore, the Sf-rhabdovirus X+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.

Application of VirCapSeq-VERT and BacCapSeq in the diagnosis of presumed and definitive neuroinfectious diseases.

Unbiased high-throughput sequencing (HTS) has enabled new insights into the diversity of agents implicated in central nervous system (CNS) infections. The addition of positive selection capture methods to HTS has enhanced the sensitivity while reducing sequencing costs and the complexity of bioinformatic analysis. Here we report the use of virus capture-based sequencing for vertebrate viruses (VirCapSeq-VERT) and bacterial capture sequencing (BacCapSeq) in investigating CNS infections. Thirty-four samples were categorized: (1) patients with definitive CNS infection by routine testing; (2) patients meeting clinically the Brighton criteria (BC) for meningoencephalitis; (3) patients with presumptive infectious etiology highest on the differential. RNA extracts from cerebrospinal fluid (CSF) were used for VirCapSeq-VERT, and DNA extracts were used for BacCapSeq analysis. Among 8 samples from known CNS infections in group 1, VirCapSeq and BacCapSeq confirmed 3 expected diagnoses (42.8%), were negative in 2 (25%), yielded an alternative result in 1 (11.1%), and did not detect 2 expected negative pathogens. The confirmed cases identified HHV-6, HSV-2, and VZV while the negative samples included JCV and HSV-2. In groups 2 and 3, 11/26 samples (42%) were positive for at least one pathogen; however, 27% of the total samples (7/26) were positive for commensal organisms. No microbial nucleic acids were detected in negative control samples. HTS showed limited promise for pathogen identification in presumed CNS infectious diseases in our small sample. Before conducting larger-scale prospective studies to assess the clinical value of this novel technique, clinicians should understand the benefits and limitations of using this modality.

Application of VirCapSeq-VERT and BacCapSeq In the Diagnosis of Presumed and Definitive Neuroinfectious Diseases.

Background: Unbiased high-throughput sequencing (HTS) has enabled new insights into the diversity of agents implicated in central nervous system (CNS) infections. The addition of positive selection capture methods to HTS has enhanced the sensitivity while reducing sequencing costs and complexity of bioinformatic analysis. Here we report the use of virus capture based sequencing for vertebrate viruses (VirCapSeq-VERT) and bacterial capture sequencing (BacCapSeq) in investigating CNS infections. Design/Methods: Thirty-four samples were categorized: (1) Patients with definitive CNS infection by routine testing; (2) Patients meeting clinically Brighton Criteria (BC) for meningoencephalitis (3) Patients with presumptive infectious etiology highest on the differential. RNA extracts from cerebrospinal fluid (CSF) were used for VirCapSeq-VERT and DNA extracts were used for BacCapSeq analysis. Results: Among 8 samples from known CNS infections in group 1, VirCapSeq and BacCapSeq confirmed 3 expected diagnoses (42.8%), were negative in 2 (25%), yielded an alternative result in 1 (11.1%), and did not detect 2 expected negative pathogens. The confirmed cases identified HHV-6, HSV-2, and VZV while the negative samples included JCV and HSV-2. In groups 2 and 3,11/26 samples (42%) were positive for at least one pathogen, however 27% of the total samples (7/26) were positive for commensal organisms. No microbial nucleic acids were detected in negative control samples. Conclusions: HTS showed limited promise for pathogen identification in presumed CNS infectious diseases in our small sample. Before conducting larger-scale prospective studies to assess clinical value of this novel technique, clinicians should understand benefits and limitations of using this modality.

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis.

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.

Characterization of Functional Microorganisms in Representative Traditional Fermented Dongcai from Different Regions of China.

Dongcai is loved for its delicious flavor and nutritional value. The microorganisms in Dongcai play a vital role in their flavor, quality, and safety, and the microbial communities of Dongcai vary greatly from region to region. However, it remains unknown what the predominant microorganisms are in different traditional Dongcai and how they affect its flavor. The objective of this study is to explore the microbial diversity of traditional fermented Dongcai in three representative Chinese regions (Tianjin, Sichuan, and Guangzhou) and further assess their microbial functions. The microbial diversity of fermented Dongcai in Guangdong has the lowest diversity compared to fermented Dongcai in Sichuan, which has the highest. The distribution of the main genera of fermented Dongcai varies from region to region, but Carnimonas, Staphylococcus, Pseudomonas, Sphingomonas, Burkholderia-Caballeronia-Paraburkholderia, and Rhodococcus are the dominant genera in common. In addition, halophilic bacteria (HAB, i.e., Halomonas Bacillus, Virgibacillus, etc.) and lactic acid bacteria (LAB, i.e., Weissella and Lactobacillus) are also highly abundant. Of these, Burkholderia-Caballeronia-Paraburkholderia, Rhodococcus, Sphingomonas, Ralstonia, and Chromohalobacter are dominant in the Sichuan samples. In the Tianjin samples, Lactobacillus, Weissella, Virgibacillus, Enterobacter, Klebsiella, and Pseudomonas are the most abundant. Predictions of microbial metabolic function reveal that carbohydrates, amino acids, polyketides, lipids, and other secondary metabolites are abundantly available for biosynthesis. In addition, the different flavors of the three types of Dongcai may be due to the fact that the abundance of HAB and LAB shows a significant positive correlation with the amounts of important metabolites (e.g., salt, acid, amino nitrogen, and sugar). These results contribute to our understanding of the link between the distinctive flavors of different types of Dongcai and the microorganisms they contain and will also provide a reference for the relationship between microbial communities and flavor substances in semi-fermented pickles.

Evolution of Cell-Type-Specific RNA Aptamers via Live Cell-Based SELEX.

Live cell-based SELEX (Systematic Evolution of Ligand EXponential enrichment) is a promising approach for identifying aptamers that can selectively bind to a cell-surface receptor or recognize a particular target cell population. In particular, it offers a facile selection strategy for some special cell-surface proteins that are originally glycosylated or heavily posttranslationally modified and are unavailable in their native/active conformation after in vitro expression and purification. In this chapter, we describe a generalized procedure for evolution of cell type-specific RNA aptamers targeting a cell membrane bound target by combining the live cell-based SELEX strategy with high-throughput sequencing (HTS) and bioinformatics analysis.

From Genomics to Metagenomics in the Era of Recent Sequencing Technologies.

Metagenomics, also known as environmental genomics, is the study of the genomic content of a sample of organisms obtained from a common habitat. Metagenomics and other "omics" disciplines have captured the attention of researchers for several decades. The effect of microbes in our body is a relevant concern for health studies. Through sampling the sequences of microbial genomes within a certain environment, metagenomics allows study of the functional metabolic capacity of a community as well as its structure based upon distribution and richness of species. Exponentially increasing number of microbiome literatures illustrate the importance of sequencing techniques which have allowed the expansion of microbial research into areas, including the human gut, antibiotics, enzymes, and more. This chapter illustrates how metagenomics field has evolved with the progress of sequencing technologies.Further, from this chapter, researchers will be able to learn about all current options for sequencing techniques and comparison of their cost and read statistics, which will be helpful for planning their own studies.

B cell M-CLL clones retain selection against replacement mutations in their immunoglobulin gene framework regions.

Introduction: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, accounting for 30-40% of all adult leukemias. The dynamics of B-lymphocyte CLL clones with mutated immunoglobulin heavy chain variable region (IgHV) genes in their tumor (M-CLL) can be studied using mutational lineage trees. Methods: Here, we used lineage tree-based analyses of somatic hypermutation (SHM) and selection in M-CLL clones, comparing the dominant (presumably malignant) clones of 15 CLL patients to their non-dominant (presumably normal) B cell clones, and to those of healthy control repertoires. This type of analysis, which was never previously published in CLL, yielded the following novel insights. Results: CLL dominant clones undergo - or retain - more replacement mutations that alter amino acid properties such as charge or hydropathy. Although, as expected, CLL dominant clones undergo weaker selection for replacement mutations in the complementarity determining regions (CDRs) and against replacement mutations in the framework regions (FWRs) than non-dominant clones in the same patients or normal B cell clones in healthy controls, they surprisingly retain some of the latter selection in their FWRs. Finally, using machine learning, we show that even the non-dominant clones in CLL patients differ from healthy control clones in various features, most notably their expression of higher fractions of transition mutations. Discussion: Overall, CLL seems to be characterized by significant loosening - but not a complete loss - of the selection forces operating on B cell clones, and possibly also by changes in SHM mechanisms.

Enhancing urinary tract infection diagnosis for negative culture patients with metagenomic next-generation sequencing (mNGS).

Background: Metagenomic next-generation sequencing (mNGS) is a promising technology that allows unbiased pathogen detection and is increasingly being used for clinical diagnoses. However, its application in urinary tract infection (UTI) is still scarce. Methods: The medical records of 33 patients with suspected UTI who were admitted to the Second Hospital of Tianjin Medical University from March 2021 to July 2022 and received urine mNGS were retrospectively analyzed. The performance of mNGS and conventional urine culture in diagnosing infection and identifying causative organisms was compared, and the treatment effects were evaluated in terms of changes in urinalyses and urinary symptoms. Results: In the detection of bacteria and fungi, mNGS detected at least one pathogen in 29 (87.9%) cases, including 19 (57.6%) with positive mNGS but negative culture results and 10 (30.3%) with both mNGS and culture positive results. The remaining 4 (12.1%) patients were negative by both tests. Overall, mNGS performed better than culture (87.9% vs. 30.3%, P < 0.001). Within the 10 double-positive patients, mNGS matched culture results exactly in 5 cases, partially in 4 cases, and not at all in 1 case. In addition, mNGS detected a broader pathogen spectrum, detecting 26 species compared to only 5 species found in culture. The most abundant bacteria detected by mNGS was Escherichia coli, detected in 9 (27.2%) patients. All anaerobic bacteria, Mycobacterium Tuberculosis and all mixed pathogens were detected by mNGS. The final clinical diagnosis of UTI was made in 25 cases, and the sensitivity of mNGS was significantly higher than culture (100.0% vs 40.0%; P < 0.001) when using the diagnosis as a reference standard; the positive predictive value, negative predictive value and specificity were 86.2%, 100% and 50.0%, respectively. Importantly, targeted antibiotic therapy based on mNGS resulted in significant improvement in urinalyses and urinary symptoms in patients. Conclusions: mNGS is a technology that has shown clear advantages over culture, particularly in the context of mixed infections and UTIs that are difficult to diagnose and treat. It helps to improve the detection of pathogens, guide changes in treatment strategies, and is an effective complement to urine culture.

Nanopore Technology Applied to Targeted Detection of Tomato Brown Rugose Fruit Virus Allows Sequencing of Related Viruses and the Diagnosis of Mixed Infections.

Tomato (Solanum lycopersicum) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.

The Presentation of Two Unrelated Clinical Cases from the Republic of North Ossetia-Alania with the Same Previously Undescribed Variant in the COL6A2 Gene.

Here, we described three affected boys from two unrelated families of Ossetian-Digor origin from the Republic of North Ossetia-Alania who were admitted to the Research Centre for Medical Genetics with unspecified muscular dystrophy. High-throughput sequencing was performed and revealed two novel frameshift variants in the COL6A2 gene (NM_001849.3) in a heterozygous state each in both cases: c.508_535delinsCTGTGG and c.1659_1660del (case 1) and c.1689del and c.1659_1660del (case 2). In two cases, the same nucleotide variant in the COL6A2 gene (c.1659_1660del) was observed. We have suggested that the variant c.1659_1660del may be common in the Ossetian-Digor population because two analyzed families have the same ancestry from the same subethnic group of Ossetians). The screening for an asymptomatic carriage of the nucleotide variant c.1659_1660del in 54 healthy donors from Ossetian-Digor population revealed that the estimated carrier frequency is 0.0093 (CI: 0.0002-0.0505), which is high for healthy carriers of the pathogenic variant. Molecular genetic, anamnestic data and clinical examination results allowed us to diagnose Ullrich muscular dystrophy in those affected boys. Genetic heterogeneity and phenotypic diversity of muscular dystrophies complicate diagnosis. It is important to make a differential diagnosis of such conditions and use HTS methods to determine the most accurate diagnosis.

DNA metabarcoding-based study on bacteria and fungi associated with house dust mites (Dermatophagoides spp.) in settled house dust.

House dust mites (HDMs) including Dermatophagoides spp. are an important cause of respiratory allergies. However, their relationship with microorganisms in house dust has not been fully elucidated. Here, we characterized bacteria and fungi associated with HDMs in house dust samples collected in 107 homes in Korea by using DNA barcode sequencing of bacterial 16S rRNA gene, fungal internal transcribed spacer 2 (ITS2) region, and arthropod cytochrome c oxidase I (COI) gene. Our inter-kingdom co-occurrence network analysis and/or indicator species analysis identified that HDMs were positively related with a xerophilic fungus Wallemia, mycoparasitic fungi such as Cystobasidium, and some human skin-related bacterial and fungal genera, and they were negatively related with the hygrophilous fungus Cephalotrichum. Overall, our study has succeeded in adding novel insights into HDM-related bacteria and fungi in the house dust ecosystem, and in confirming the historically recognized fact that HDMs are associated with xerophilic fungi such as Wallemia. Understanding the microbial ecology in house dust is thought to be important for elucidating the etiology of human diseases including allergies, and our study revealed baseline information of house dust ecology in relation to HDMs. The findings could be useful from a perspective of human health.