Metagenomic Methods Research Articles

Unveiling early-life microbial colonization profile through characterizing low-biomass maternal-infant microbiomes by 2bRAD-M

IntroductionThe microbial composition of human breast milk and infant meconium offers critical insights into the early microbial colonization profile, and it greatly contributes to the infant’s immune system and long-term health outcomes. However, analyzing these samples often faces technical challenges and limitations of low-resolution using conventional approaches due to their low microbial biomass.MethodsHere, we employed the type IIB restriction enzymes site-associated DNA sequencing for microbiome (2bRAD-M) as a reduced metagenomics method to address these issues and profile species-level microbial composition. We collected breast milk samples, maternal feces, and infant meconium, comparing the results from 2bRAD-M with those from both commonly used 16S rRNA amplicon sequencing and the gold-standard whole metagenomics sequencing (WMS).ResultsThe accuracy and robustness of 2bRAD-M were demonstrated through its consistently high correlation of microbial individual abundance and low whole-community-level distance with the paired WMS samples. Moreover, 2bRAD-M enabled us to identify clinical variables associated with infant microbiota variations and significant changes in microbial diversity across different lactation stages of breast milk.DiscussionThis study underscores the importance of employing 2bRAD-M in future large-scale and longitudinal studies on maternal and infant microbiomes, thereby enhancing our understanding of microbial colonization in early life stages and demonstrating further translational potential.

Assessing Bias and Reproducibility of Viral Metagenomics Methods for the Combined Detection of Faecal RNA and DNA Viruses

Whole transcriptome amplification (WTA2) and sequence-independent single primer amplification (SISPA) are two widely used methods for combined metagenomic sequencing of RNA and DNA viruses. However, information on the reproducibility and bias of these methods on diverse viruses in faecal samples is currently lacking. A mock community (MC) of diverse viruses was developed and used to spike faecal samples at different concentrations. Virus-like particles (VLPs) were extracted, nucleic acid isolated, reverse-transcribed, and PCR amplified using either WTA2 or SISPA and sequenced for metagenomic analysis. A bioinformatics pipeline measured the recovery of MC viruses in replicates of faecal samples from three human donors, analysing the consistency of viral abundance measures and taxonomy. Viruses had different recovery levels with VLP extraction introducing variability between replicates, while WTA2 and SISPA produced comparable results. In comparing WTA2- and SISPA-generated libraries, WTA2 gave more uniform coverage depth profiles and improved assembly quality and virus identification. SISPA produced more consistent abundance, with a 50% difference between replicates occurring in ~20% and ~10% of sequences for WTA2 and SISPA, respectively. In conclusion, a bioinformatics pipeline has been developed to assess the methodological variability and bias of WTA2 and SISPA, demonstrating higher sensitivity with WTA2 and higher consistency with SISPA.

Potential roles of cigarette smoking on gut microbiota profile among Chinese men

BackgroundCigarette smoking is posited as a potential factor in disrupting the balance of the human gut microbiota. However, existing studies with limited sample size have yielded inconclusive results.MethodsHere, we assessed the association between cigarette smoking and gut microbial profile among Chinese males from four independent studies (N total = 3308). Both 16S rRNA and shotgun metagenomic sequencing methods were employed, covering 206 genera and 237 species. Microbial diversity and abundance were compared among non-smokers, current smokers, and former smokers.ResultsActinomyces[g], Atopobium[g], Haemophilus[g], Turicibacter[g], and Lachnospira[g] were found to be associated with smoking status (current smokers vs. non-smokers). Metagenomic data provided a higher resolution at the species level, particularly for the Actinomyces[g] branch. Additionally, serum γ-glutamylcysteine (γ-Glu-Cys) was found to have a potential role in connecting smoking and Actinomyces[g]. Furthermore, we revealed putative mediation roles of the gut microbiome in the associations between smoking and common diseases including cholecystitis and type 2 diabetes.ConclusionsWe characterized the gut microbiota profile in male smokers and further revealed their potential involvement in mediating the impact of smoking on health outcomes. These findings advance our understanding of the intricate association between cigarette smoking and the gut microbiome.

Dissemination of genes associated with antibiotic resistance and bacterial virulence during ecosystem succession in two Tibetan glacier forefields.

The release of pathogens and DNA from the cryosphere (glacier, permafrost, and, sea ice) has become a new threat to society and environment. Due to enhanced glacier retreat, the size of glacier forefields has greatly expanded. Herein, we used a combination of metagenomic and metatranscriptomic methods and adopted a sequence-based approach to investigate the distribution and changing patterns of virulence factor genes (VFGs) and antibiotic resistance genes (ARGs) in two glacier forefields. The forefields are separated by approximately 400km located in the center and north of the Tibetan Plateau, which are used to demonstrate the gene dissemination capacity across short (10m) and long (730m) spatial transects. The results revealed a diverse range of actively transcribed VFGs and ARGs. The relative abundance of ARG reduced with ecosystem succession, while that of VFG was similar, suggesting that the ARG is under a stronger environmental selection pressure. VFGs and ARGs were dominated by those associated with adherence and vancomycin resistance, respectively. Notably, toxin production related genes were identified but a low abundance, indicating a low risk to health in glacier forefields. The dissemination risks were low for both VFGs and ARGs, which was strongly constrained by dispersal limitation. Additionally, the limited dissemination was mainly through vertical transmission, instead of horizontal transfer. In conclusion, the sequence-based approach revealed a low risk to health in recently deglaciated areas, with the risk of VFGs and ARGs being disseminated into downstream ecosystems remaining low.

Metagenomic exploration and computational prediction of novel enzymes for polyethylene terephthalatedegradation.

As a global environmental challenge, plastic pollution raises serious ecological and health concerns owing to the excessive accumulation of plastic waste, which disrupts ecosystems, harms wildlife, and threatens human health. Polyethylene terephthalate (PET), one of the most commonly used plastics, has contributed significantly to this growing crisis. This study offers a solution for plastic pollution by identifying novel PET-degrading enzymes. Using a combined approach of computational analysis and metagenomic workflow, we identified a diverse array of genes and enzymes linked to plastic degradation. Our study identified 1305,282 unmapped genes, 36,000 CAZymes, and 317 plastizymes in the soil samples were heavily contaminated with plastic. We extended our approach by training machine learning models to discover candidate PET-degrading enzymes. To overcome the scarcity of known PET-degrading enzymes, we used a Generative Adversarial Network (GAN) model for dataset augmentation and a pretrained deep Evolutionary Scale Language Model (ESM) to generate sequence embeddings for classification. Finally, 21 novel PET-degrading enzymes were identified. These enzymes were further validated through active site analysis, amino acid composition analysis, and 3D structure comparison. Additionally, we isolated bacterial strains from contaminated soils and extracted plastizymes to demonstrate their potential for environmental remediation. This study highlights the importance of biotechnological solutions for plastic pollution, emphasizing scalable, cost-effective processes and the integration of computational and metagenomic methods.

Impacts of aquaculture on nitrogen cycling and microbial community dynamics in coastal tidal flats.

The expansion of aquaculture areas has encroached upon vast areas of coastal wetlands and introduced excessive nitrogen inputs, disrupting microbial communities and contributing to various environmental issues. However, investigations on how aquaculture affects microbial communities and nitrogen metabolism mechanisms in coastal tidal flats remain scarce. Hence, we explored the composition, diversity, and assembly processes of nitrogen-cycling (N-cycling) microbial communities in tidal flats in Jiangsu using metagenomic assembly methods. Our study further delved into the seasonal variations of these microbial characteristics to better explore the effects of seasonal changes in aquaculture areas on microbial community. Nitrogen metabolism-related processes and functional genes were identified through the KEGG and NCyc databases. The results revealed significant seasonal variation in the relative abundance and composition of microbial communities. Higher diversity was observed in winter, while the co-occurrence network of microbial communities was more complex in summer. Pseudomonadota emerged as the most abundant phylum in the N-cycling community. Furthermore, pH and NO3-N were identified as the primary factors influencing bacterial community composition, whereas NO2-N was more strongly associated with the N-cycling community. Regarding the nitrogen metabolism processes, nitrogen mineralization and nitrification were predominant in the tidal flat regions. NO2-N and NO3-N exhibited significant effects on several N-cycling functional genes (e.g., nirB, hao, and narG). Finally, neutral and null modeling analyses indicated that bacterial communities were predominantly shaped by stochastic processes, whereas N-cycling communities were largely driven by deterministic processes. These findings highlighted the significant role that aquaculture pollution plays in shaping the N-cycling communities in tidal flats. This underscored the importance of understanding microbial community dynamics and nitrogen metabolism in tidal flats to improve environmental management in coastal aquaculture areas.

Isolation and Metagenomic Analysis of Halophilic Microorganisms from Jintan Salt Mine in China

The Jintan Salt Mine in China has a long history and substantial reserves, with a salinity level reaching 85.9%. In order to gain a deeper understanding of the microbial community structure and its functional properties in the Jintan Salt Mine, and to assess its potential impact on the salt cavern hydrogen storage project, further investigation is required. The diversity of microbial community structure and function in the brine of the Jintan Salt Mine, which contains up to 30% salinity, was assessed using culture and macrogenomic approaches. A total of 71 bacterial strains and 5 archaeal strains were isolated and purified from brine using different media. After 16S rRNA molecular characterization, the isolated 71 strains were identified as members of Marinobacter, Halomonas, Halovibrio, Sutcliffiella, Bacillus, Halobacillus, and other genera. Furthermore, the metagenomics method revealed that the dominant phyla in brine were Euryarchaea, Proteobacteria, Balneolaeota, and Bacteroides. Metabolism was also found tobe the main functional activity of microorganisms in the brines. The metabolic processes of sulfur, methane, and carbon fixation in prokaryotes represent significant forms of energy metabolism. These processes are attributed to the presence of methanogenic and sulfate-reducing bacteria in brine samples. The findings of this study will considerably enhance the extant knowledge of microorganisms in salt mines.

Meadow Degradation Affects Microbial Community Structure and Vegetation Characteristics by Increasing Soil pH

ABSTRACTSoil is the natural habitat for microorganisms, providing water and nutrients for vegetation, but the influence of meadow degradation on the “vegetation‐soil‐microorganism” system is still controversial, and the drivers of soil microbial and vegetation characteristics change are still unclear. This study explores in‐depth the vegetation and soil changes in four different degradation levels, that is, non‐degraded (ND), lightly degraded (LD), moderately degraded (MD), and severely degraded (SD), in the northeastern region of the Qinghai‐Tibetan Plateau. Soil metagenomic sequencing methods were employed to understand the soil microbial and functional diversity and the factors that influence them. The findings indicated that alpine meadow degradation decreased the vegetation height, coverage, and above‐ground biomass (AGB), but increased the vegetation diversity index. In addition, meadow degradation reduces soil properties (organic carbon, total nitrogen and phosphorus, available nitrogen and phosphorus) and enzyme activities (sucrase, cellulase, urease, and catalase) but increases soil pH. The copiotrophic groups (Actinobacteria, Ascomycota) decreased while oligotrophic groups (Acidobacteria, Glomeromycota) increased with meadow degradation. In particular, changes in vegetation characteristics and microbial community structure in alpine meadows are strongly influenced by changes in soil pH. In conclusion, the degradation of alpine meadows leads to the growth of some barren‐tolerant species and deviation of soil pH from neutrality, which increases vegetation diversity but surface exposure reduces the vegetation richness, affects the type and amount of root secretions and changes in soil microbial communities, which reduce the fixation and release of soil nutrients. This study provides the theoretical basis for further analyzing the inner mechanism of alpine meadow degradation.

Gut Microbiome modulation in patients treated with an enriched nutraceutical composition: a multi target strategy

Introduction: The contribution of gut microbiota to health and disease is now well accepted and under continuous investigation. It is the most representative in the body (about 70% of the total) with over 400 different species of microorganisms living there. It is exclusive to each individual and therefore represents a real biological imprint, capable of distinguishing humans from one another. Gut microbiota works synergically with the host, promoting health by performing protective, metabolic and structural functions. Thus, when a modification of the personal normal gut microbiota composition occurs, a predisposition to develop different diseases may increase. Probiotics are functional food that may modulate gut microbiota stimulating protective bacterial properties and inhibiting detrimental activity. We have previously reported a beneficial effect from the administration of an enriched nutraceutical composition containing probiotics in reducing irritable bowel syndrome-related symptoms and improving metabolic profile by lowering triglycerides. However, if these effects are attributed to gut microbiota remain unknown. Objective: This analysis aims at evaluating microbiota modulation in subjects before and after Triobiotix® administration using a metagenomic approach Methods: Triobiotix® is an enriched nutraceutical composition containing maltodextrin, hyaluroic acid and mineralized seaweed extract Lithothamnion Calcareum plus a mix of five different lactic bacterial species. A total of 46 subjects with availability of sample test before and after treatment were included in the present analysis selected from 265 subjects. A fecal sample was collected before and up to 90 days after the administration of Triobiotix®. Feces were analyzed by Wellmicro® gut test that uses the next-generation sequencing of the 16S rRNA gene on Illumina MiSeq. Additionally, for 171 subjects a 90-day clinical evaluation was available. Results: Treatment with this enriched nutraceutical for up to 3 months resulted in a positive modulation of the main indices of improved gut microbiota, such as biodiversity and dysbiosis. Improvement on metabolic functions, such as glycemic and lipid metabolisms, as well as on immunological and anti-inflammatory activities have been described, thus indicating that the observed clinical benefits associated with the administration of this nutraceutical formulation might be also attributed to a gut microbiota modulation. Improvement in main IBS-related symptoms and bloating was maintained up to 90 days as well as microbiota diversity. Conclusions: Our analysis suggests that the gut microbiota of subjects taking Triobiotix® rapidly returns to a high diversity with reduced dysbiosis after treatment with associated improvement of main physiological functions linked to brain, heart and liver. Keywords: gut microbiota; microbiome, metagenomic; Nutraceuticals

Comparison of Plasma Metagenomic Next-generation Sequencing and PCR Methods for Epstein-Barr Virus Viral Load Monitoring in Nasopharyngeal Carcinoma.

Plasma Epstein-Barr virus (EBV) viral load measurement is prognostic in nasopharyngeal carcinoma (NPC) disease monitoring; however, a consensus measurement approach does not exist. This study characterized the clinical performance of metagenomic next-generation sequencing (mNGS), an unbiased sequencing-based assay distinct from polymerase chain reaction (PCR) or targeted sequencing approaches, in 73 peripheral blood specimens from 32 patients diagnosed with NPC. Samples were analyzed for plasma EBV viral load either by mNGS profiling or PCR-based assays (either LMP2 or BAMHI-W PCR) and compared to tumor presence by clinical assessment. Plasma mNGS-based EBV detection was quantified as reads per million (RPM). Plasma mNGS displayed similar overall performance (100% sensitivity, 86% specificity, 92% accuracy) to BAMHI-W PCR (100% sensitivity, 86% specificity, 94% accuracy) and superior performance to the LMP2 PCR assay (36% sensitivity, 56% specificity, 45% accuracy). In a subset of 13 patients who underwent longitudinal analysis, plasma mNGS EBV RPM correlated with cancer recurrence (95%CI Pre-CRT=232.10±214; 95%CI Post-CRT=0.34±0.32; 95%CI difference=-231.70±214; *p=0.03, paired t-test), suggesting plasma mNGS exhibits potential for monitoring recurrence. Plasma mNGS is a distinct method for EBV titer measurement in NPC patients and more broadly, is a promising method for non-invasive monitoring of disease status for infection-associated malignancies.

Metagenomics reveals the profiles and drivers of antibiotic resistance genes and virulence factors in Glebionis coronaria L. planting soil

Due to the application of contaminated water irrigation and bio-amendments, the emergence and spread of antibiotic resistance genes (ARGs) and virulence factors (VFs) in soil have become a global problem, thus having potential threats to human health and ecology. However, limited knowledge exists regarding how variations in the soils of Glebionis coronaria L. (previously called Chrysanthemum coronarium L., or the crown daisy) planting sites influence the profiles of ARGs and VFs. In this study, ARGs, VFs, mobile genetic elements (MGEs), and microbial communities were comprehensively characterized using metagenomic methods in the soils from five different Glebionis coronaria L. planting sites in Shanghai. The dominant microbial populations in Glebionis coronaria L. planting soil were not affected by different planting sites, but there were changes in their relative abundance. The abundance and diversity of ARGs, VFs, and MGEs were different among the soils of five planting sites. The overall distribution characteristics of ARGs and VFs in the soils of each planting site remained consistent. Potential hosts shared by ARGs and VFs were present at each site, raising the risk of soil antibiotic resistance. Fe and T were the primary driving factors for the changes in ARGs and VFs, contributing to 11.32 % and 10.74 % of the overall variation in ARGs and VFs, respectively. Taken together, the identification of biological and non-biological factors that affect ARGs and VFs in the soils from different planting sites could provide valuable perspectives for controlling the dissemination of these genes. This study underscored the necessity for accurate and coordinated strategies to tackle the worldwide antibiotic resistance challenge.

Metagenome-Assembled Genomes of Pig Fecal Samples in Nine European Countries: Insights into Antibiotic Resistance Genes and Viruses.

Gut microbiota plays a crucial role in the health and productivity of pigs. However, the spread of antibiotic resistance genes (ARGs) and viruses within the pig intestinal microbiota poses significant threats to animal and public health. This study utilized 181 pig samples from nine European countries and employed metagenomic assembly methods to investigate the dynamics and distribution of ARGs and viruses within the pig intestinal microbiota, aiming to observing their associations with potential bacterial hosts. We identified 4605 metagenome-assembled genomes (MAGs), corresponding to 19 bacterial phyla, 97 families, 309 genera, and a total of 449 species. Additionally, 44 MAGs were classified as archaea. Analysis of ARGs revealed 276 ARG types across 21 ARG classes, with Glycopeptide being the most abundant ARG class, followed by the class of Multidrug. Treponema D sp016293915 was identified as a primary potential bacterial host for Glycopeptide. Aligning nucleotide sequences with a viral database, we identified 1044 viruses. Among the viral genome families, Peduoviridae and Intestiviridae were the most prevalent, with CAG-914 sp000437895 being the most common potential host species for both. These findings highlight the importance of MAGs in enhancing our understanding of the gut microbiome, revealing microbial diversity, antibiotic resistance, and virus-bacteria interactions. The data analysis for the article was based on the public dataset PRJEB22062 in the European Nucleotide Archive.

Evaluating metagenomic analyses for undercharacterized environments: what's needed to light up the microbial dark matter?

Non-human-associated microbial communities play important biological roles, but they remain less understood than human-associated communities. Here, we assess the impact of key environmental sample properties on a variety of state-of-the-art metagenomic analysis methods. In simulated datasets, all methods performed similarly at high taxonomic ranks, but newer marker-based methods incorporating metagenomic assembled genomes outperformed others at lower taxonomic levels. In real environmental data, taxonomic profiles assigned to the same sample by different methods showed little agreement at lower taxonomic levels, but the methods agreed better on community diversity estimates and estimates of the relationships between environmental parameters and microbial profiles.

Cross-comparison of gut metagenomic profiling strategies

The rapid advancements in sequencing technologies and bioinformatics have enabled metagenomic research of complex microbial systems, but reliable results depend on consistent laboratory and bioinformatics approaches. Current efforts to identify best practices often focus on optimizing specific steps, making it challenging to understand the influence of each stage on microbial population analysis and compare data across studies. This study evaluated DNA extraction, library construction methodologies, sequencing platforms, and computational approaches using a dog stool sample, two synthetic microbial community mixtures, and various sequencing data sources. Our work, the most comprehensive evaluation of metagenomic methods to date. We developed a software tool, termed minitax, which provides consistent results across the range of platforms and methodologies. Our findings showed that the Zymo Research Quick-DNA HMW MagBead Kit, Illumina DNA Prep library preparation method, and the minitax bioinformatics tool were the most effective for high-quality microbial diversity analysis. However, the effectiveness of pipelines or method combinations is sample-specific, making it difficult to identify a universally optimal approach. Therefore, employing multiple approaches is crucial for obtaining reliable outcomes in microbial systems.

Empirical assessment of the enrichment-based metagenomic methods in identifying diverse respiratory pathogens.

Probe-based nucleic acid enrichment represents an effective route to enhance the detection capacity of next-generation sequencing (NGS) in a set of clinically diverse and relevant microbial species. In this study, we assessed the effect of the enrichment-based sequencing on identifying respiratory infections using tiling RNA probes targeting 76 respiratory pathogens and sequenced using both Illumina and Oxford Nanopore platforms. Forty respiratory swab samples pre-tested for a panel of respiratory pathogens by qPCR were used to benchmark the sequencing data. We observed a general improvement in sensitivity after enrichment. The overall detection rate increased from 73 to 85% after probe capture detected by Illumina. Moreover, enrichment with probe sets boosted the frequency of unique pathogen reads by 34.6 and 37.8-fold for Illumina DNA and cDNA sequencing, respectively. This also resulted in significant improvements on genome coverage especially in viruses. Despite these advantages, we found that library pooling may cause reads mis-assignment, probably due to crosstalk issues arise from post-capture PCR and from pooled sequencing, thus increasing the risk of bleed-through signal. Taken together, an overall improvement in the breadth and depth of pathogen coverage is achieved using enrichment-based sequencing method. For future applications, automated library processing and pooling-free sequencing could enhance the precision and timeliness of probe enrichment-based clinical metagenomics.

New perspectives on metagenomic analysis for pathogen monitoring in sustainable freshwater aquaculture production: a systematic review

The freshwater and saltwater aquatic food sector has experienced the most significant growth in recent years and is increasingly recognized as a sustainable alternative for fostering prosperous societies self-sufficiently and ecologically. One primary economic and health risk factor in aquaculture production is health control, with potentially more severe impacts observed in tropical and developing countries. While metagenomics holds great promise for application in agro-industrial fields like aquaculture, its adoption remains limited. Consequently, this study aimed to assess the prospects for developing and applying metagenomics in identifying pathogens in freshwater aquaculture. The WIPO database was used to search for patents developed using metagenomics to monitoring pathogens in freshwater aquaculture. Metagenomics methods have been extensively employed in different fields, such as, medicine, veterinary, biotechnology, agriculture, particularly in studies focusing on microbial communities in different ecosystems. In aquaculture, the utilization of metagenomics has predominantly revolved around investigating antibiotic resistance genes, primarily in saltwater farms. Despite this, freshwater aquaculture, particularly in fish and crustacean farming, aligns closely with sustainable development goals, notably (SDGs) 2, 3, 6, and 13. Countries such as the United States of America, South Korea, and Canada stand at the forefront of utilizing metagenomics for disease monitoring in freshwater aquaculture, evidenced by their active patent developments. The metagenomic analysis, coupled with bioinformatics tools and databases, represents a rapid, secure, and non-invasive approach to environmental monitoring for preventive purposes.Systematic review registrationhttps://osf.io/srpyz/, identifier 10.17605/OSF.IO/SRPYZ.

Identification and characterization of multiple novel viruses in fecal samples of ruddy shelducks using viral metagenomics methods

The viral metagenomics approach is an effective technique for investigating and analysing both existing and emerging viruses in humans and diverse animal samples. The ruddy shelduck, a nationally protected secondary key species of wild animals, has become the predominant species among overwintering waterbirds in Qinghai Lake. Viruses carried by ruddy shelducks can potentially infect humans or other animals; however, limited research on the faecal virome of ruddy shelducks is currently available. In the present study, faecal samples of ruddy shelducks collected from Saga County, Shigatse City, Tibet, China, were subjected to viral metagenomic analysis. The predominant viral families identified in ruddy shelduck samples were Picornaviridae, Parvoviridae, Microviridae, Vilyaviridae, Astroviridae, and Caliciviridae. Among these, two picornavirus genomes have been identified as new strains of the genus Megrivirus in the family Parvoviridae. In addition, viruses that infect parasites and bacteria have been identified and characterised. The present study enhances our comprehension of the composition of the viral community in ruddy shelducks faeces and highlights the dynamic nature of viral evolution and the significance of continuous monitoring to assess potential risks to wildlife and public health.

Binning Metagenomic Contigs Using Contig Embedding and Decomposed Tetranucleotide Frequency.

Metagenomic binning is a crucial step in metagenomic research. It can aggregate the genome sequences belonging to the same microbial species into independent bins. Most existing methods ignore the semantic information of contigs and lack effective processing of tetranucleotide frequency, resulting in insufficient and complex feature information extracted for binning and poor binning results. To address the above problems, we propose CedtBin, a metagenomic binning method based on contig embedding and decomposed tetranucleotide frequency. First, the improved BERT model is used to learn the contigs to obtain their embedding representation. Secondly, the tetranucleotide frequencies are decomposed using a non-negative matrix factorization (NMF) algorithm. After that, the two features are spliced and input into the clustering algorithm for binning. Considering the sensitivity of the DBSCAN clustering algorithm to input parameters, in order to solve the drawbacks of manual parameter input, we also propose an Annoy-DBSCAN algorithm that can adaptively determine the parameters of the DBSCAN algorithm. This algorithm uses Approximate Nearest Neighbors Oh Yeah (Annoy) and combines it with a grid search strategy to find the optimal parameters of the DBSCAN algorithm. On simulated and real datasets, CedtBin achieves better binning results than mainstream methods and can reconstruct more genomes, indicating that the proposed method is effective.

Transfer and persistence of microbiota markers from the human hand to the knife: A preliminary study

New scientific techniques and methods are always needed to link the perpetrators to the incident or the crime scene. Recent microbiota studies based on NGS (Next-generation sequencing) show that various biological samples from crime scenes have the potential to be used in forensic investigations. Especially when DNA traces belonging to more than one person are insufficient to fully determine the genetic profile, a secret sample, such as a microbiota sample created by the suspect's touch, can be used. In this preliminary study, a fictionalized experimental model was designed to investigate the transfer and persistence of the hand microbiome on the knife handle, which has a high potential to be used in criminal incidents, by metagenomic analysis methods. In addition, it was aimed to determine the transfer of specific bacterial species identified only to the person among the five participants onto the knife handle and their persistence over time. In the first stage of the research, samples were collected from the hands of 5 volunteer participants using the swap method, including their palms. Then, after each participant held a different knife, samples were collected from the knife handles via swabs from different angles of the knives at 4 and 24 h and analyzed by metagenomic methods. The findings of this preliminary study showed that the heatmap graphs generated after UniFrac distance analysis were not successful in establishing any similarity between the hand samples and the post-transfer knife handle samples. Nonetheless, it was observed that the transfer of bacterial species detected in the hand samples to knives differed according to the individuals and some bacterial species were transferred to the knife samples held by the participants. The number of bacterial species detected that are specific to each participant's hand sample was 302 in total, and it was determined that a total of 8.28 % of these bacterial species were transferred to the knife handle samples of the 4th hour and 6.95 % to the knife samples of the 24th hour. In the presented study, considering the transfer of some bacterial species in the hand microbiome, which are effective in the variation between individuals, onto the knife; It has been evaluated that some rare bacterial species can be important potential markers to associate the object with the perpetrator.

Genome-resolved metagenomics revealed novel microbial taxa with ancient metabolism from macroscopic microbial mat structures inhabiting anoxic deep reefs of a Maldivian Blue Hole.

Blue holes are vertical water-filled openings in carbonate rock that exhibit complex morphology, ecology, and water chemistry. In this study, macroscopic microbial mat structures found in complete anoxic conditions in the Faanu Mudugau Blue Hole (Maldives) were studied by metagenomic methods. Such communities have likely been evolutionary isolated from the surrounding marine environment for more than 10,000 years since the Blue Hole formation during the last Ice Age. A total of 48 high-quality metagenome-assembled genomes (MAGs) were recovered, predominantly composed of the phyla Chloroflexota, Proteobacteria and Desulfobacterota. None of these MAGs have been classified to species level (<95% ANI), suggesting the discovery of several new microbial taxa. In particular, MAGs belonging to novel bacterial genera within the order Dehalococcoidales accounted for 20% of the macroscopic mat community. Genome-resolved metabolic analysis of this dominant microbial fraction revealed a mixotrophic lifestyle based on energy conservation via fermentation, hydrogen metabolism and anaerobic CO2 fixation through the Wood-Ljungdahl pathway. Interestingly, these bacteria showed a high proportion of ancestral genes in their genomes providing intriguing perspectives on mechanisms driving microbial evolution in this peculiar environment. Overall, our results provide new knowledge for understanding microbial life under extreme conditions in blue hole environments.