Several studies have documented the significant impact of methodological choices in microbiome analyses. The myriad of methodological options available complicate the replication of results and generally limit the comparability of findings between independent studies that use differing techniques and measurement pipelines. Here we describe the Mosaic Standards Challenge (MSC), an international interlaboratory study designed to assess the impact of methodological variables on the results. The MSC did not prescribe methods but rather asked participating labs to analyze 7 shared reference samples (5 × human stool samples and 2 × mock communities) using their standard laboratory methods. To capture the array of methodological variables, each participating lab completed a metadata reporting sheet that included 100 different questions regarding the details of their protocol. The goal of this study was to survey the methodological landscape for microbiome metagenomic sequencing (MGS) analyses and the impact of methodological decisions on metagenomic sequencing results. A total of 44 labs participated in the MSC by submitting results (16S or WGS) along with accompanying metadata; thirty 16S rRNA gene amplicon datasets and 14 WGS datasets were collected. The inclusion of two types of reference materials (human stool and mock communities) enabled analysis of both MGS measurement variability between different protocols using the biologically-relevant stool samples, and MGS bias with respect to ground truth values using the DNA mixtures. Owing to the compositional nature of MGS measurements, analyses were conducted on the ratio of Firmicutes: Bacteroidetes allowing us to directly apply common statistical methods. The resulting analysis demonstrated that protocol choices have significant effects, including both bias of the MGS measurement associated with a particular methodological choices, as well as effects on measurement robustness as observed through the spread of results between labs making similar methodological choices. In the analysis of the DNA mock communities, MGS measurement bias was observed even when there was general consensus among the participating laboratories. This study was the result of a collaborative effort that included academic, commercial, and government labs. In addition to highlighting the impact of different methodological decisions on MGS result comparability, this work also provides insights for consideration in future microbiome measurement study design.

Long-read metagenomic sequencing is a powerful approach for cataloging the microbial diversity present in complex microbiomes, including the human gut microbiome. We performed a deep-sequencing experiment using PacBio HiFi reads to obtain metagenome-assembled genomes (MAGs) from a pooled human gut microbiome. We performed long-read metagenome assembly using two methods (hifiasm-meta, metMDBG), used improved bioinformatic and proximity ligation binning strategies to cluster contigs and identify MAGs, and developed a novel framework to compare and consolidate MAGs (pb-MAG-mirror). We found proximity ligation binning yielded more MAGs than bioinformatic binning, but our novel comparison framework resulted in higher MAG yields than either binning strategy individually. In total, from 255 Gbp of total HiFi data we produced 595 total MAGs (including 175 high-quality MAGs) using hifiasm-meta, and 547 total MAGs (including 277 high-quality MAGs) with metaMDBG. Hifiasm-meta assembled almost twice as many strain-level MAGs as metaMDBG (246 vs. 156), but both assembly methods produced up to five strains for a species. Approximately 85% of the MAGs were assigned to known species, but we recovered >35 high-quality MAGs that represent uncultured diversity. Based on strict similarity scores, we found 125 MAGs were unequivocally shared across the assembly methods at the strain level, representing ∼22% of the total MAGs recovered per method. Finally, we detected more total viral sequences in the metaMDBG assembly versus the hifiasm-meta assembly (∼6,700 vs. ∼4,500). Overall, we find the use of HiFi sequencing, improved metagenome assembly methods, and complementary binning strategies is highly effective for rapidly cataloging microbial genomes in complex microbiomes.### Competing Interest StatementDMP, DJN, JEW, and SZ are employees of Pacific Biosciences of California, Inc. KL, ST, BF, and AD are employees of Zymo Research. MC and RC are the CEO and CSO of The BioCollective, respectively. IL is the CEO of Phase Genomics, and BA, HM, KWL, MI, JRG, and SJB are employees of Phase Genomics.

Colorectal cancer (CRC) is influenced by perturbations in the colonic microbiota, characterized by an imbalance favoring pathogenic bacteria over beneficial ones. This dysbiosis contributes to CRC initiation and progression through mechanisms such as carcinogenic metabolite production, inflammation induction, DNA damage, and oncogenic signaling activation. Understanding the role of external factors in shaping the colonic microbiota is crucial for mitigating CRC progression. This study aims to elucidate the gut microbiome’s role in CRC progression by analyzing paired tumor and mucosal tissue samples obtained from the colon walls of 17 patients. Through sequencing of the V3-V4 region of the 16S rRNA gene, we characterized the tumor microbiome and assessed its association with clinical variables. Our findings revealed a significant reduction in alpha diversity within tumor samples compared to paired colon biopsy samples, indicating a less diverse microbial environment within the tumor microenvironment. While both tissues exhibited dominance of similar bacterial phyla, their relative abundances varied, suggesting potential colon-specific effects. Fusobacteriota enrichment, notably in the right colon, may be linked to MLH1 deficiency. Taxonomy analysis identified diverse bacterial genera, with some primarily associated with the colon wall and others unique to this region. Conversely, several genera were exclusively expressed in tumor tissue. Functional biomarker analysis identified three key genes with differential abundance between tumor microenvironment and colon tissue, indicating distinct metabolic activities. Functional biomarker analysis revealed three key genes with differential abundance: K11076 (putrescine transport system) and K10535 (nitrification) were enriched in the tumor microenvironment, while K11329 (SasA-RpaAB circadian timing mediator) dominated colon tissue. Metabolic pathway analysis linked seven metabolic pathways to the microbiome. Collectively, these findings highlight significant gut microbiome alterations in CRC and strongly suggest that long-term dysbiosis profoundly impacts CRC progression.

Coprolites, or mummified feces, are valuable sources of information on ancient cultures as they contain ancient DNA (aDNA). In this study, we analyzed ancient plant DNA isolated from coprolites belonging to two pre-Columbian cultures (Huecoid and Saladoid) from Vieques, Puerto Rico, using shotgun metagenomic sequencing to reconstruct diet and lifestyles. We also analyzed DNA sequences of putative phytopathogenic fungi, likely ingested during food consumption, to further support dietary habits. Our findings show that pre-Columbian Caribbean cultures had a diverse diet consisting of maize (Zea mays), sweet potato (Ipomoea batatas), chili peppers (Capsicum annuum), peanuts (Arachis spp.), papaya (Carica papaya), tomato (Solanum lycopersicum) and, very surprisingly cotton (Gossypium barbadense) and tobacco (Nicotiana sylvestris). Modelling of putative phytopathogenic fungi and plant interactions confirmed the potential consumption of these plants as well as edible fungi, particularly Ustilago spp., which suggest the consumption of maize and huitlacoche. These findings suggest that a variety of dietary, medicinal, and hallucinogenic plants likely played an important role in ancient human subsistence and societal customs. We compared our results with coprolites found in Mexico and the United States, as well as present-day faeces from Mexico, Peru, and the United States. The results suggest that the diet of pre-Columbian cultures resembled that of present-day hunter-gatherers, while agriculturalists exhibited a transitional state in dietary lifestyles between the pre-Columbian cultures and larger scale farmers and United States individuals. Our study highlights differences in dietary patterns related to human lifestyles and provides insight into the flora present in the pre-Columbian Caribbean area. Importantly, data from ancient fecal specimens demonstrate the importance of ancient DNA studies to better understand pre-Columbian populations.

Dietzia natronolimnaea C79793-74 has emerged as a potential probiotic strain with implications for managing Crohn's disease. This study evaluates the safety profile of D. natronolimnaea C79793-74 as a probiotic sup-plement. Genotypic characterization involved a 16S rRNA gene and genomic sequencing and genome an-notation. The safety assessment included interrogation of the assembled genome for antibiotic resistance genes and virulence factors, utilizing the CARD and VFDB databases. Notably, the analysis revealed an ab-sence of antibiotic resistance or virulence factors in Dietzia natronolimnaea C79793-74. The safety and tol-erability of D. natronolimnaea C79793-74 were further investigated in an 8-week double-blind, place-bo-controlled clinical trial involving healthy adult participants. A daily dose of 5 x 109 CFU of the probiotic strain was administered. This clinical trial represents the first assessment of the safety of D. natronolimnaea C79793-74 in human subjects. Results demonstrated that participants in both the Dietzia and Placebo groups maintained clinical and hematologic markers within the normal range throughout the study. More-over, the probiotic strain was well-tolerated, with nearly all participants experiencing no severe or medium adverse events. Collectively, the comprehensive data obtained in this study support the inference that Dietzia natronolimnaea C79793-74 is safe and well-tolerated as a nutritional supplement for human con-sumption

Background: Inferior quality of biological material compromises data, slows discovery, and wastes research funds. The gut microbiome plays a critical role in human health and disease, yet little attention has been given to optimizing collection and processing methods of human stool. Methods: We collected the entire bowel movement from 2 healthy volunteers: one to examine stool sample heterogeneity and one to test stool sample handling parameters. Sequencing and bioinformatic analyses were used to examine the microbiome composition. Results: The microbiome profile varied depending on where the subsample was obtained from the stool. The exterior cortex of the stool was rich in specific phyla and deficient in others while the interior core of the stool revealed opposite microbiome profiles. Sample processing also resulted in varying microbiome profiles. Homogenization and stabilization at 4°C gave superior microbial diversity profiles compared to the fresh or frozen subsamples of the same stool sample. Bacterial proliferation continued in the fresh subsample when processed at ambient temperature. Bacteroidetes proliferated and Firmicutes diminished during the 30-minute processing of fresh sample. The frozen sample had good overall diversity but Proteobacteria diminished likely because of the freeze/thaw. Conclusion: The microbiome profile is specific to the section of the stool being sampled. Stool sample collection, homogenization and stabilization at 4°C for 24 hours provides a neat, high-quality sample of sufficient quantity that can be banked into aliquots with nearly identical microbial diversity profiles. This collection pipeline is essential to accelerate our understanding of the gut microbiome in health and disease.

Objective: The objective of this study is to assess the efficacy of BiotiQuest™ Sugar Shift, a probiotic formulated to transform monosaccharides, rehabilitated human gut microbiota, stabilize HbA1c and blood glucose, improve insulin resistance and reduce inflammation Methods: A double-blind, placebo-controlled study was carried out over 12 weeks with 64 Cubans, aged 30 to 65 with Type 2 Diabetes Mellitus diagnosed, 18 of whom being treated with insulin. Participants were randomly assigned to take either two capsules of the probiotic supplement or of placebo. Clinical measures were evaluated at 28-day intervals, included fasting and post-prandial glucose, HbA1c, lipid panel, insulin, creatinine, and serum lipopolysaccharide levels were assessed. Results: The treated group demonstrated a stabilization in their fasting blood glucose and postprandial glucose levels in treated group respect to the Placebo cohort. The HbA1c levels did not show significant changes in the treated group. The insulin levels decreased significantly in the treated group by day 84 compared to day 1 and to day 84 of the Placebo cohort (p=0.024 and p=0.015, respectively). Serum LPS levels also decreased significantly in the treated group (p=0.001). Conclusions: BiotiQuest™ Sugar Shift is suitable as adjunct therapy for the control of T2D. However, the 12-week trial period was not sufficient to detect significant reductions in all clinical parameters measured and a longer study is recommended.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by hyperglycemia, insulin resistance and chronic inflammation. Probiotics have been claimed effective in the management of obesity and type 2 diabetes mellitus. BiotiQuest™ Sugar Shift is a symbiotic formulation rationally designed for the endogenous conversion of glucose and fructose to support restoration of the human gut microbiota, modulation of intestinal glucose, and the production of anti-inflammatory metabolites.We report the results of a 12-week, double blind, placebo-controlled study designed to evaluate Sugar Shift in Cuban T2DM patients. Clinical parameters, including fasting and 2h post-prandial glucose, hemoglobin A1c, a lipid panel, insulin, creatinine, and serum lipopolysaccharide levels were assessed. Microbiome composition was assessed by 16S amplicon sequencing of the variable region V3-V4 of the 16S rRNA gene. Metabolic biomarkers were inferred from microbiome data by Kruskal-Wallis H test and LEfSe.Fasting glucose, Insulin, and serum LPS levels decreased significantly at day 84 as compared to day 1 in the treated group and to control group. Hb A1c remained stable in the treatment group as compared to the controls but not show significant improvement in the study period.Microbiome analysis showed significant increase in Chao1 alpha diversity in the treated group between day 1 and day 84. Taxonomic and functional biomarkers revealed significant differences between the Day 1 and Day 84 microbiome profiles in the treatment group, primarily associated with acetate, propionate, and butyrate production.Our results indicate that Sugar Shift can be a suitable adjunct therapy to standard of care therapy in the management of T2DM based upon the improvement in key inflammatory and insulin resistance markers. These results were interpreted as an indication of favorable microbiome changes during the course of the treatment for 12 weeks.### Competing Interest StatementThe authors have declared no competing interest.### Clinical TrialISRCTN48974890### Funding StatementThe author received no specific funding for this work.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This trial was evaluated and approved under the Hermanos Ameijeiras Ethic Committee for Clinical Investigation and The Hermanos Ameijeiras Scientific Council. The Sugar Shift Probiotic supplements were also evaluated and approved for their use by the Ethical Committee at the National Institute of Nutrition of Cuba and The Cuba Ministry of Health.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesThe data underlying the results presented in the study are available from Martha Carlin. Email martha.carlin@thebiocollective.com

We analyzed human coprolites from the Sacred City of Caral, the oldest civilization in America (3000- and 1800-years BC). Our objective was to know the microbial diversity of the Caral Civilization through the use of a mobile ancient laboratory. DNA extraction conducted in a mobile laboratory placed near the collection site to reduce exposure of samples to contaminants and favor a rapid molecular processing. Using 16S rRNA and ITS 1 amplicon sequencing, we have elaborated the first list of the microbiomes of Caral, based on the bacterial and fungal community fingerprints detected in the coprolites recovered in six sectors of that ancient urban center. Among the most abundant sequences were those associated with Firmicutes for bacteria, Ascomycota and Basidiomycota for fungi. Bacillus was the most abundant bacterial genera in all samples analyzed, compromising up to 24.81% of the total bacterial abundance; while Aspergillus (11.43%) was the most abundant genera among fungal communities.

We report the use of a mobile laboratory set up to extract ancient DNA (aDNA) from 34 human coprolites (fossilized faeces) samples. Our approach enabled the rapid genetic characterization of 5,000 years old archeological samples. It is useful for the on-site screening of museums and freshly excavated samples for DNA. This approach is accessible to other investigators as the mobile laboratory was set up using commercially available instruments.

ABSTRACT Genetic and microbiome studies of ancient Caral-Supe civilization have not yet been published. For this reason, the objective of this work is to identify the microorganisms and possible diseases that existed in this ancient civilization using coprolites samples. To do this, two coprolites samples were analyzing through high-throughput sequencing data of 16S rRNA gene and an intergenic region (ITS).

INTRODUCTION:To date, there has been little effort to develop standards for metabolome-based gut microbiome measurements despite the significant efforts toward standard development for DNA-based microbiome measurements.OBJECTIVES:The National Institute of Standards and Technology (NIST), The BioCollective (TBC), and the North America Branch of the International Life Sciences Institute (ILSI North America) are collaborating to extend NIST's efforts to develop a Human Whole Stool Reference Material for the purpose of method harmonization and eventual quality control.METHODS:The reference material will be rationally designed for adequate quality assurance and quality control (QA/QC) for underlying measurements in the study of the impact of diet and nutrition on functional aspects of the host gut microbiome and relationships of those functions to health. To identify which metabolites deserve priority in their value assignment, NIST, TBC, and ILSI North America jointly conducted a workshop on September 12, 2019 at the NIST campus in Gaithersburg, Maryland. The objective of the workshop was to identify metabolites for which evidence indicates relevance to health and disease and to decide on the appropriate course of action to develop a fit-for-purpose reference material.RESULTS:This document represents the consensus opinions of workshop participants and co-authors of this manuscript, and provides additional supporting information. In addition to developing general criteria for metabolite selection and a preliminary list of proposed metabolites, this paper describes some of the strengths and limitations of this initiative given the current state of microbiome research.CONCLUSIONS:Given the rapidly evolving nature of gut microbiome science and the current state of knowledge, an RM (as opposed to a CRM) measured for multiple metabolites is appropriate at this stage. As the science evolves, the RM can evolve to match the needs of the research community. Ultimately, the stool RM may exist in sequential versions. Beneficial to this evolution will be a clear line of communication between NIST and the stakeholder community to ensure alignment with current scientific understanding and community needs.

The pre-Columbian Huecoid and Saladoid cultures were agricultural ethnic groups that supplemented their diets by fishing, hunting and scavenging. Archaeological deposits associated to these cultures contained a variety of faunal osseous remains that hinted at the cultures' diets. The present study identified zoonotic parasites that may have infected these two cultures as a result of their diets. We used metagenomic sequencing and microscopy data from 540-1,400 year old coprolites as well as the zooarchaeological data to recreate the possible interactions between zoonotic parasites and their hosts. Microscopy revealed Diphyllobothrium spp. and Dipylidium caninum eggs along with unidentified cestode and trematode eggs. DNA sequencing together with functional prediction and phylogenetic inference identified reads of Cryptosporidium spp., Giardia intestinalis and Schistosoma spp. The complimentary nature of the molecular, microscopy and zooarchaeology data provided additional insight into the detected zoonotic parasites' potential host range. Network modeling revealed that rodents and canids living in close proximity to these cultures were most likely the main source of these zoonotic parasite infections.

Dysbiosis of the intestinal microbiota has been shown to result in altered immune responses and increased susceptibility to infection; as such, the state of the intestinal microbiome may have profound implications in the perioperative setting. In this first-in-class study, we used 16s ribosomal RNA sequencing and analysis in a mouse model of general anesthesia to investigate the effects of volatile anesthetics on the diversity and composition of the intestinal microbiome. After 4-hour exposure to isoflurane, we observed a decrease in bacterial diversity. Taxonomic alterations included depletion of several commensal bacteria including Clostridiales. These data identify volatile anesthetics as potential contributors to microbial dysbiosis in the postoperative patient.

Malaria is one of the most important human diseases throughout tropical and sub-tropical regions of the world. Global distribution and ample host range have contributed to the genetic diversity of the etiological agent, Plasmodium. Phylogeographical analyses demonstrated that Plasmodium falciparum and Plasmodium vivax follow an Out of Africa (OOA) expansion, having a higher genetic diversity in African populations and a low genetic diversity in South American populations. Modeling the evolutionary rate of conserved genes for both P. falciparum and P. vivax determined the approximate arrival of human malaria in South America. Bayesian computational methods suggest that P. falciparum originated in Africa and arrived in South America through multiple independent introductions by the transatlantic African slave trade; however, in South America, P. vivax could have been introduced through an alternate migratory route. Alignments of P. vivax mitogenomes have revealed low genetic variation between the South American and Southeast Asian populations suggesting introduction through either pre-Columbian human migration or post-colonization events. To confirm the findings of these phylogeographical analyses, molecular methods were used to diagnose malaria infection in archeological remains of pre-Columbian ethnic groups. Immunohistochemistry tests were used and identified P. vivax but not P. falciparum in histologically prepared tissues from pre-Columbian Peruvian mummies, whereas shotgun metagenomics sequencing of DNA isolated from pre-Columbian Caribbean coprolites revealed Plasmodium-homologous reads; current evidence suggests that only P. vivax might have been present in pre-Columbian South America.

espanolEl macizo de Garraf, extensa meseta de roca caliza, esta situado a pocos kilometros al sur de Barcelona y simboliza la cuna y escuela del colectivo espeleologico catalan. Administrativamente es un espacio protegido desde 1986, aunque en su entorno la presion metropolitana ha supuesto serias afecciones al medio natural; la mas importante el emplazamiento del deposito de residuos urbanos de Barcelona (inaugurado en 1974 y donde actualmente se esta explotando el biogas). La fisiografia del endokarst de este macizo, esta caracterizada por modestas simas con un marcado desarrollo vertical, con bocas pequenas y mal ventiladas, donde se generan atmosferas confinadas, muy sensibles a las variaciones de las condiciones ambientales. En este escenario, un grupo de espeleologos e investigadores (GERS Barcelona y UME Gava) respaldados por la Facultad de Ciencias de la Tierra (UB) y por el Consell Catala de l’Esport, desde el ano 2002 y utilizando detectores portatiles de gas, han desarrollado investigaciones dirigidas a la caracterizacion de estas atmosferas, tanto en el espacio como en el tiempo, asi como sus efectos en la fisiologia humana. Los resultados obtenidos en las diferentes campanas emprendidas por este grupo se han concretado en los siguientes estudios: a) Modelo atmosferico en las simas del Garraf, se ha definido un modelo barotermico, donde la presion atmosferica ejerce de motor de la dinamica atmosferica y la temperatura condiciona la homogenizacion o zonacion del aire cavernario. b) Efectos sobre la fisiologia humana, en el macizo del Garraf un numero importante de simas (una treintena de las mas significativas) presentan una atmosfera empobrecida en 02 . Se han constatado las principales sintomatologias, remarcando la evolucion de la saturacion de oxigeno, la capacidad arritmogenica y las alteraciones del sistema nervioso autonomo (simpatico y parasimpatico). c) Mapa atmosferico y calidad atmosferica en los entornos del vertedero, desde el ano 2002 un grupo de espeleologos voluntarios han tomado datos de las simas del Garraf y posteriormente de todo el territorio catalan. Los datos se recogen en un mapa de Google, abierto al publico para su consulta. catalaEl massis del Garraf, extens altipla de roca calcaria, esta situat a pocs kilometres al sud de Barcelona i simbolitzael bressol i escola del col·lectiu espeleologic catala. Administrativament es un espai protegit des del 1986, encaraque el seu entorn la pressio metropolitana ha suposat serioses afeccions al medi natural; la mes importantl’emplacament del diposit de residus urbans de Barcelona (inaugurat el 1974 i on actualment s’esta explotant el biogas). La fisiografia de l’endokarst d’aquest massis, esta caracteritzada per modestos avencs amb un marcat desenvolupament vertical, con boques petites i mal ventilades, on es generen atmosferes confinades, molt sensibles a les variacions de las condicions ambientals. En aquest escenari, un grup d’espeleolegs i investigadors (GERS Barcelona i UME Gava) amb el suport de la Facultat de Ciencies de la Terra (UB) i el Consell Catala de l’Esport, des de l’any 2002 i utilitzant detectors portatils de gas, han desenvolupat investigacions dirigides a la caracteritzacio d’aquestes atmosferes, tant en l’espai, com en el temps, aixi com els seus efectes en la fisiologia humana. Els resultats obtinguts en les diferents campanyes empreses per aquest grup s’han concretat en els seguents estudis: a) Model atmosferic als avencs del Garraf, s’ha definit un model barotermic, on la pressio atmosferica exerceix de motor de la dinamica atmosferica i la temperatura condiciona la homogeneitzacio o zonacio de l’aire cavernari. b) Efectes sobre la fisiologia humana, en el massis del Garraf un numero important d’avencs (una trentena de las mes significatives) presenten una atmosfera empobrida en O2 . S’han constatat les principals simptomatologies, remarcant l’evolucio de la saturacio d’oxigen, la capacitat arritmogenica i les alteracions del sistema nervios autonom (simpatic i parasimpatic). c) Mapa atmosferic i qualitat atmosferica en els entorns de l’abocador, des de l’any 2002 un grup d’espeleolegs voluntaris han pres dades dels avencs del Garraf i posteriorment de tot el territori catala. Les dades es recullen en un mapa de Google, obert al public per a la seva consulta.

The concept of the human oral microbiome was applied to understand health and disease, lifestyles, and dietary habits throughout part of human history. In the present study, we augment the understanding of ancient oral microbiomes by characterizing human dental calculus samples recovered from the ancient Abbey of Badia Pozzeveri (central Italy), with differences in socioeconomic status, time period, burial type, and sex. Samples dating from the Middle Ages (11th century) to the Industrial Revolution era (19th century) were characterized using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. Consistent with previous studies, individuals from Badia Pozzeveri possessed commensal oral bacteria that resembled modern oral microbiomes. These results suggest that members of the oral microbiome are ubiquitous despite differences in geographical regions, time period, sex, and socioeconomic status. The presence of fecal bacteria could be in agreement with poor hygiene practices, consistent with the time period. Respiratory tract, nosocomial, and other rare pathogens detected in the dental calculus samples are intriguing and could suggest subject-specific comorbidities that could be reflected in the oral microbiome.

ABSTRACT Microbiome analysis of environmental samples may represent the next frontier in environmental microbial forensics. Next-generation sequencing technologies significantly increased the available genetic data that could be used as evidentiary material. It is not clear, however, whether the microbiome can scale across institutions using forensic-based evidence due to the data resource requirements and the associated costs of maintaining these databases. A successful microbiome study is impacted by the quality of the information gathered and the steps in sample processing and data analysis. To ascertain the validity of methods and the results obtained, there needs to be a stringent procedure to validate the methods and ensure that the results are comparable and reproducible, not only within the laboratory but also between laboratories conducting similar research. Of primary importance for meaningful microbiome studies is an experimental design that leads to carefully executed, controlled, and reproducible studies. The microbiome literature contains a fair share of anecdotal descriptions of microbial community composition and “diagnostic” relative abundance of the taxa therein. These studies are now being supplemented by experimental designs that feature repeated measurements, error estimates, correlations of microbiota with covariates, and increasingly sophisticated statistical tests that enhance the robustness of data analysis and study conclusions. It is imperative to be careful, especially when carrying out attribution studies, to be fully aware of the possible biases included in a specific sample being analyzed.