Holed up, but thriving: Impact of multitrophic cryoconite communities on glacier elemental cycles.

Cryoconite holes (water and sediment-filled depressions), found on glacier surfaces worldwide, serve as reservoirs of microbes, carbon, trace elements, and nutrients, transferring these components downstream via glacier hydrological networks. Through targeted amplicon sequencing of carbon and nitrogen cycling genes, coupled with functional inference-based methods, we explore the functional diversity of these mini-ecosystems within Antarctica and the Himalayas. These regions showcase distinct environmental gradients and experience varying rates of environmental change influenced by global climatic shifts. Analysis revealed a diverse array of photosynthetic microorganisms, including Stramenopiles, Cyanobacteria, Rhizobiales, Burkholderiales, and photosynthetic purple sulfur Proteobacteria. Functional inference highlighted the high potential for carbohydrate, amino acid, and lipid metabolism in the Himalayan region, where organic carbon concentrations surpassed those in Antarctica by up to 2 orders of magnitude. Nitrogen cycling processes, including fixation, nitrification, and denitrification, are evident, with Antarctic cryoconite exhibiting a pronounced capacity for nitrogen fixation, potentially compensating for the limited nitrate concentrations in this region. Processes associated with the respiration of elemental sulfur and inorganic sulfur compounds such as sulfate, sulfite, thiosulfate, and sulfide suggest the presence of a complete sulfur cycle. The Himalayan region exhibits a higher potential for sulfur cycling, likely due to the abundant sulfate ions and sulfur-bearing minerals in this region. The capability for complete iron cycling through iron oxidation and reduction reactions was also predicted. Methanogenic archaea that produce methane during organic matter decomposition and methanotrophic bacteria that utilize methane as carbon and energy sources co-exist in the cryoconite, suggesting that these niches support the complete cycling of methane. Additionally, the presence of various microfauna suggests the existence of a complex food web. Collectively, these results indicate that cryoconite holes are self-sustaining ecosystems that drive elemental cycles on glaciers and potentially control carbon, nitrogen, sulfur, and iron exports downstream.

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata.

BACKGROUND: Biofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach. RESULTS: Functional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements. CONCLUSIONS: The study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

Correlation of severity & clinical outcomes of COVID-19 with virus variants: A prospective, multicentre hospital network study.

BACKGROUND OBJECTIVES: The clinical course of COVID-19 and its prognosis are influenced by both viral and host factors. The objectives of this study were to develop a nationwide platform to investigate the molecular epidemiology of SARS-CoV-2 (Severe acute respiratory syndrome Corona virus 2) and correlate the severity and clinical outcomes of COVID-19 with virus variants. METHODS: A nationwide, longitudinal, prospective cohort study was conducted from September 2021 to December 2022 at 14 hospitals across the country that were linked to a viral sequencing laboratory under the Indian SARS-CoV-2 Genomics Consortium. All participants (18 yr and above) who attended the hospital with a suspicion of SARS-CoV-2 infection and tested positive by the reverse transcription-PCR method were included. The participant population consisted of both hospitalized as well as outpatients. Their clinical course and outcomes were studied prospectively. Nasopharyngeal samples collected were subjected to whole genome sequencing to detect SARS-CoV-2 variants. RESULTS: Of the 4972 participants enrolled, 3397 provided samples for viral sequencing and 2723 samples were successfully sequenced. From this, the evolution of virus variants of concern including Omicron subvariants which emerged over time was observed and the same reported here. The mean age of the study participants was 41 yr and overall 49.3 per cent were female. The common symptoms were fever and cough and 32.5 per cent had comorbidities. Infection with the Delta variant evidently increased the risk of severe COVID-19 (adjusted odds ratio: 2.53, 95% confidence interval: 1.52, 4.2), while Omicron was milder independent of vaccination status. The independent risk factors for mortality were age >65 yr, presence of comorbidities and no vaccination. INTERPRETATION CONCLUSIONS: The authors believe that this is a first-of-its-kind study in the country that provides real-time data of virus evolution from a pan-India network of hospitals closely linked to the genome sequencing laboratories. The severity of COVID-19 could be correlated with virus variants with Omicron being the milder variant.

Sieving out non-celiac gluten sensitivity amongst patients with irritable bowel syndrome.

OBJECTIVES: It is challenging to make diagnosis of non-celiac gluten sensitivity/non-celiac wheat sensitivity (NCGS/NCWS) in clinical practice, since there is no biomarker and diagnosis is based on response to gluten-free-diet (GFD). We used anti-gliadin antibody (AGA) for screening patients with IBS for gluten-sensitivity. METHODS: 492 Adult-patients with IBS underwent screening for celiac disease and gluten-sensitivity using IgA anti-tissue transglutaminase antibody and IgA-AGA and IgG-AGA, respectively. Patients with positive AGA (IgA and/or IgG) were invited to follow GFD, those willing were put on GFD for 6-weeks. Responsive patients were given gluten re-challenge. Diagnosis of NCGS was confirmed if they had recurrence of symptoms. RESULTS: Of 492 patients with IBS, AGA was positive in 61(12.4 %), hence suspected to have gluten-sensitivity. Of 31 who agreed to participate and followed GFD for 6-weeks, 17 (54.8 %) had complete (>30 % improvement) and 10(32.2 %) had partial (>20 % improvement) response. All 17 complete-responders were given gluten re-challenge for 6-weeks, symptoms recurred in all and hence were confirmed to have NCGS/NCWS. Significant decrease in AGA levels occurred almost in all GFD-responders. CONCLUSIONS: 12.4 % IBS patients have biological evidence of gluten/wheat-sensitivity. Almost 87 % patients with IBS having AGA responded to GFD. The value of AGA may further be explored as a biomarker for screening for the presence of NCGS, before recommending this test for the clinical practice.

Recent advances in production and applications of ectoine, a compatible solute of industrial relevance.

Extremophilic bacteria growing in saline ecosystems are potential producers of biotechnologically important products including compatible solutes. Ectoine/hydroxyectoine are two such solutes that protect cells and associated macromolecules from osmotic, heat, cold and UV stress without interfering with cellular functions. Since ectoine is a high value product, overviewing strategies for improving yields become relevant. Screening of natural isolates, use of inexpensive substrates and response surface methodology approaches have been used to improve bioprocess parameters. In addition, genome mining exercises can aid in identifying hitherto unreported microorganisms with a potential to produce ectoine that can be exploited in the future. Application wise, ectoine has various biotechnological (protein protectant, membrane modulator, DNA protectant, cryoprotective agent, wastewater treatment) and biomedical (dermatoprotectant and in overcoming respiratory and hypersensitivity diseases) uses. The review summarizes current updates on the potential of microorganisms in the production of this industrially relevant metabolite and its varied applications.

Bacterial Communities and Diversity of Western Ghats Soil: A Study of a Biodiversity Hotspot.

The Western Ghats is one of India's mega-diversity hotspots and an ecologically and geologically important area for the diversity of endemic plants and animals. The present study provides insights into the aerobic bacterial diversity and composition of the soils of North Western Ghats located in Maharashtra state (NWGM), India. The samples for the culture-dependent study were collected from 6 different locations namely Malshej Ghat, Bhimashankar, Lonavala, Mulshi, Tail-Baila, and Mahabaleshwar. A total of 173 isolates were obtained from the different samples, which belonged to Proteobacteria (43%), Firmicutes (36%), and Actinobacteria (19%). Sequences of 15 strains shared ≤ 98.7% similarity (a species cut-off) which represent potential novel species. Metagenomic analysis revealed the presence of Actinobacteria and Proteobacteria as the most dominant phyla at both MB and MG. However, both sites showed variation in the composition of rare phyla and other dominant phyla. This difference in bacterial community composition could be due to differences in altitude or other physicochemical properties. The functional prediction from the amplicon sequencing showed the abundance of carbohydrate, protein, and lipid metabolism which was corroborated by screening the isolated bacterial strains for the same. The present study has a unique take on microbial diversity and defines the importance of community assembly processes such as drift, dispersal, and selection. Such processes are relatively important in controlling community diversity, distribution, as well as succession. This study has shown that the microbial community of NWGM is a rich source of polysaccharide degrading bacteria having biotechnological potential.

Prokaryotic communities adapted to microhabitats on the Indian lotus (Nelumbo nucifera) growing in the high-altitude urban Dal Lake.

Indian lotus (Nelumbo nucifera) is one of the dominant aquatic plants cultivated in Dal Lake, situated at 1586 m above mean sea level (MSL) in the northeast of Srinagar, Kashmir. Despite their economic and ecological role, the microbial communities associated with the lotus plant are still unexplored. In this study, we investigated the prokaryotic communities on surfaces of different lotus microhabitats (roots, rhizome, leaves, flowers, and fruits), lake water, and sediments using 16S rRNA gene amplicon sequencing. Overall, prokaryotic diversity decreased significantly on the surface of lotus microhabitats in comparison to the lake water and sediments. Among the microhabitats of lotus, roots and leaves harbored more diverse communities in comparison to rhizomes, fruits, and flowers. A total of 98 genera were shared by lotus and the Dal Lake sediments and water. However, significant differences were found in their relative abundance; for example, Pseudomonas was the most dominant genus on the majority of lotus microhabitats. On the other hand, Flavobacterium was highly abundant in the lake water, while a higher abundance of Acinetobacter was recorded in sediments. Additionally, we also noted the presence of potential human pathogenic genera including Escherichia-Shigella, Enterobacter, Pantoea, Raoultella, Serratia, and Sphingomonas on the lotus microhabitats. Predicted functions of prokaryotic communities revealed a higher abundance of genes associated with nutrient uptake in the microhabitats of the lotus. This study offered first-hand information on the prokaryotic communities harbored by lotus plants and water and sediments of the Dal Lake and demonstrated the adaptation of diverse communities to microhabitats of lotus.

Indian sewage microbiome has unique community characteristics and potential for population-level disease predictions.

Sewage wastewater pollutes water and poses a public health issue but it could also prove useful in certain research domains. Sewage is a complex niche relevant for research concerning 'one-health', human health, pollution and antibiotic resistance. Indian gut microbiome is also understudied due to sampling constraints and sewage could be used to explore it. Ostensibly, Indian sewage needs to be studied and here, we performed a cross-sectional pan-India sewage sampling to generate the first comprehensive Indian sewage microbiome. Indian sewage showed predominance of Burkholderiaceae, Rhodocyclaceae, Veillonellaceae, Prevotellaceae, etc. and has high representation of gut microbes. The identified gut microbes have overrepresentation of Veillonellaceae, Rikenellaceae, Streptococcaceae, and Bacillaceae. Imputed metagenomics of sewage microbiome indicated dominance of transport, motility, peptidases, amino acid metabolism, and antibiotic resistance genes. Microbiome-disease associations drawn using simple decision tree and random forest analysis identified specific microbes as potential predictors of diabetes and obesity in a city. Altogether, we generated the first Indian sewage microbiome and our non-invasive, high-throughput workflow could be emulated for future research, wastewater-based epidemiology and designing policies concerning public health.

Diversity of resistant determinants, virulence factors, and mobile genetic elements in Acinetobacter baumannii from India: A comprehensive in silico genome analysis.

Introduction: The frequency of infections associated with multidrug resistant A. baumannii has risen substantially in India. The use of next-generation sequencing (NGS) techniques combined with comparative genomics has great potential for tracking, monitoring, and ultimately controlling the spread of this troublesome pathogen. Here, we investigated the whole genome sequences of 47 A. baumannii from India. Methods: In brief, A. baumannii genomes were analyzed for the presence of antibiotic resistance genes (ARGs), virulence factors genes (VFGs), and mobile genetic elements (MGEs) using various in silico tools. The AbaR-type resistance islands (AbaRIs) were detected by examining the genetic environment of the chromosomal comM gene. Multilocus sequence types were determined using the Pasteur scheme. The eBURST and whole genome SNPs-based phylogenetic analysis were performed to analyze genetic diversity between A. baumannii genomes. Results and discussion: A larger number of A. baumannii isolates belonging to the ST2 genotype was observed. The SNPs-based phylogenetic analysis showed a diversity between compared genomes. The predicted resistome showed the presence of intrinsic and acquired ARGs. The presence of plasmids, insertion sequences, and resistance islands carrying putative ARGs conferring resistance to antibiotics, quaternary ammonium compounds, and heavy metals was predicted in 43 (91%) genomes. The presence of putative VFGs related to adherence, biofilm formation and iron uptake was observed in the study. Overall, the comprehensive genome analysis in this study provides an essential insight into the resistome, virulome and mobilome of A. baumannii isolates from India.

Whole-Genome Sequencing of the Tropical Marine Bacterium Nocardiopsis dassonvillei NCIM 5124, Containing the Ectoine Biosynthesis Gene Cluster ectABC.

The genome sequence (7,057,619 bp; GC content, 72.07%) of a tropical marine isolate, Nocardiopsis dassonvillei NCIM 5124, containing the biomedically and biotechnologically important gene cluster ectABC is reported here.

Dataset of ileum bacterial diversity in mice after heart failure due to pressure overload.

We recently reported the correlation of gut bacterial diversity with heart failure using a mouse model of heart failure due to pressure overload induced by transverse aortic constriction (TAC). We found that gut the bacterial diversity is significantly altered and is directly correlated to the severity of heart failure (Heart Failure Severity Closely Correlates with Intestinal Dysbiosis and Subsequent Metabolomic Alterations (Spehlmann, 2022). In addition, stool samples that were collected for the gut microbial diversity analysis, we dissected ileum from the mice after 42 days of TAC. The total DNA was extracted to identify the bacterial diversity resided in ileum using 16S rRNA gene amplicon shotgun sequencing and downstream bioinformatics analysis to determine if it is correlated to the heart failure.

False-positive detection of Group B Streptococcus (GBS) in chromogenic media (Strep B Carrot Broth) due to presence of Enterococcus faecalis in High Vaginal swabs.

Introduction. Vaginal colonization of Group B Streptococcus (GBS) is associated with preterm births and neonatal sepsis. Thus routine screening of GBS in prenatal care is recommended.Hypothesis. Chromogenic media (carrot broth) aids in specific and rapid detection of GBS.Aim. To investigate the efficiency of Strep B Carrot Broth for detection of GBS in high vaginal swabs from pregnant women.Methods. In this study 201 vaginal swab samples were collected from pregnant women. Swabs were inoculated in chromogenic media (Strep B Carrot Broth). The positive and negative cultures were inoculated on blood agar and crome agar plates. The colonies were subjected to 16S rRNA sequencing and gene-specific PCR for confirmation. The Christie Atkins Munch Peterson (CAMP) and bile esculin agar (BEA) tests were used for biochemical confirmation. PCR was performed on genomic DNA isolated from uncultured vaginal swabs.Results. It was found that 20/201 (9.9 %) vaginal swab samples were positive in the Strep B Carrot Broth and 17/20 (85 %) and 19/20 (95 %) of these samples yielded colonies on blood agar and crome agar, respectively. Of the 181 carrot broth-negative samples, 1 (0.5 %) and 38 (20.9 %) yielded colonies on blood agar and crome agar plates, respectively. However, 16 s rRNA sequencing revealed that none of the 20 carrot broth-positive cultures were GBS and had sequence similarities to Enterococcus faecalis. This was also confirmed by using gene-specific PCR and BEA positivity. Furthermore, E. faecalis was detected by PCR in DNA isolated from 57 uncultured vaginal swabs samples, GBS could only be detected by PCR in four samples.Conclusion. Carrot broth-based culture can lead to false-positive detection due to the presence of E. faecalis. Thus GBS-positive results in carrot broth must be confirmed by the other molecular and biochemical tests before making a final diagnosis.

Characterization of Bordetella pertussis Strains Isolated from India.

Despite high level vaccination and the availability of two different types of vaccines, whole cell (wP) and acellular vaccines (aP), the resurgence of pertussis has been reported in many countries. Antigenic variation within circulating and vaccine strains is the most documented reason reported for the resurgence of pertussis. Research on genetic divergence among circulating and vaccine strains has largely been reported in countries using aP vaccines. There are inadequate data available for antigenic variation in B. pertussis from wP-using countries. India has used wP for more than 40 years in their primary immunization program. The present study reports five clinical isolates of B. pertussis from samples of pediatric patients with pertussis symptoms observed in India. Genotypic and phenotypic characterization of clinical isolates were performed by serotyping, genotyping, whole genome analyses and comparative genomics. All clinical isolates showed serotype 1, 2 and 3 based on the presence of fimbriae 2 and 3. Genotyping showed genetic similarities in allele types for five aP genes within vaccine strains and clinical isolates reported from India. The presence of the ptxP3 genotype was observed in two out of five clinical isolates. Whole-genome sequencing was performed for clinical isolates using the hybrid strategy of combining Illumina (short reads) and oxford nanopore (long reads) sequencing strategies. Clinical isolates (n = 5) and vaccine strains (n = 7) genomes of B. pertussis from India were compared with 744 B. pertussis closed genomes available in the public databases. The phylogenomic comparison of B. pertussis genomes reported from India will be advantageous in better understanding pertussis resurgence reported globally with respect to pathogen adaptation.

Paenibacillus oleatilyticus sp. nov., isolated from soil.

A gram-stain-negative, endo-spore forming, facultatively anaerobic, motile, rod-shaped bacterial strain SM69T, isolated from soil samples of Rohtak, Haryana, India was characterized using polyphasic approach. White colonies were 2-3 mm, in diameter and growth occurred between 20 and 55 °C, pH 6.0-10.0 with 0-2.0% (w/v) NaCl. Based on 16S rRNA gene sequence similarity the strain is placed in the genus Paenibacillus as it is closely related to 'Paenibacillus tyrfis MSt1T' (99.7%) and P. elgii SD17T (99.6%). The cell wall peptidoglycan contained meso-diaminopimelic acid. The dominant fatty acids included anteiso-C15: 0 (50%), C16: 0 (12%) and anteiso-C17: 0 (10%). Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The size of the draft genome was 7,848,017 bp, with 53.1% G+C content. dDDH (51.6%) and ANI (93.5%) of strain SM69T with its close relatives indicates that it represents a novel species, for which the name Paenibacillus oleatilyticus sp. nov. (Type strain SM69T = MCC 3064T = JCM 33981T = KACC 21649T) is proposed.

A Microcosm Model for the Study of Microbial Community Shift and Carbon Emission from Landfills.

The landfill is an inexpensive way of municipal solid waste (MSW) management and contributes extensively to the total carbon budget and global climate change. Three landfills from two geographically distinct metro- cities of India were taken as model systems to create microcosms and study their physiochemistry, microbiology, and carbon emission. The microcosm experiments revealed that facultative anaerobic bacterial community showing the dominance in the beginning but with the progression of anoxia and anaerobic conditions, methanogenesis prevailed, resulting in a clear shift towards the abundance of methanogens especially the members of Methanosarcina, Methanocorpusculum, and Methanoculleus (70-90% of the total microbial population). Geochemical data showed a wide range of heterogeneity in landfills' composition located even in the same city. In past, greenhouse gas emission from landfills is mainly estimated using different models which lack accuracy. As limited information is available as of now, this study can elicit researcher interest for in-depth characterization of microbial diversity and carbon emission from landfills. The microcosm model presented in the current study is a robust and straightforward method of accurate estimation of amounts of different types of gases release from landfill. It can also be extrapolate for estimation of different gases release from actual landfill sites by setting the on-site experiments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00995-7.

Heart Failure Severity Closely Correlates with Intestinal Dysbiosis and Subsequent Metabolomic Alterations.

Growing evidence suggests an altered gut microbiome in patients with heart failure (HF). However, the exact interrelationship between microbiota, HF, and its consequences on the metabolome are still unknown. We thus aimed here to decipher the association between the severity and progression of HF and the gut microbiome composition and circulating metabolites. Using a mouse model of transverse aortic constriction (TAC), gut bacterial diversity was found to be significantly lower in mice as early as day 7 post-TAC compared to Sham controls (p = 0.03), with a gradual progressive decrease in alpha-diversity on days 7, 14, and 42 (p = 0.014, p = 0.0016, p = 0.0021) compared to day 0, which coincided with compensated hypertrophy, maladaptive hypertrophy, and overtly failing hearts, respectively. Strikingly, segregated analysis based on the severity of the cardiac dysfunction (EF < 40% vs. EF 40−55%) manifested marked differences in the abundance and the grouping of several taxa. Multivariate analysis of plasma metabolites and bacterial diversity produced a strong correlation of metabolic alterations, such as reduced short-chain fatty acids and an increase in primary bile acids, with a differential abundance of distinct bacteria in HF. In conclusion, we showed that HF begets HF, likely via a vicious cycle of an altered microbiome and metabolic products.

Corrigendum: Diversity of resistant determinants, virulence factors, and mobile genetic elements in Acinetobacter baumannii from India: A comprehensive in silico genome analysis.

[This corrects the article DOI: 10.3389/fcimb.2022.997897.].

Rectal administration of buttermilk processed with medicinal plants alters gut microbiome in obese individuals.

OBJECTIVE: To evaluate the effect of rectal administration of buttermilk processed with medicinal plants on gut microbial composition and thereby on weight in obese individuals. METHODS: With ethics committee approval, 16 obese individuals in the age group 20-50 years (BMI ≥30 kg/m2) were recruited who received a course of 15-enemas over 15-days. Of these, 1st, 8th and 15th enemas were of sesame-oil administered after food, while other enemas were of buttermilk processed with medicinal plants administered before food. Outcome variables viz. anthropometry, body composition, blood glucose, insulin and lipid profile were evaluated on day 0, 16 and 45. Also, microbial composition of buttermilk preparation and faecal samples of patients collected on day 0, 16 and 45 were studied with the help of 16S rRNA gene sequencing. RESULTS: The circumferential measures and skinfold-thickness showed a decrease on day 16, which remained lower as compared to baseline till day 45. A gradual decrease in blood-glucose was seen, which was statistically significant on day 45, while insulin levels increased on day 16 and fell to baseline on day 45. There was an overall increase in bacterial diversity on day 16 that settled back to its original composition by day 45. CONCLUSION: Our findings suggest that buttermilk administration per rectum is effective for a specific period and may have to be repeated for sustained benefits. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40200-021-00879-z.

MicFunPred: A conserved approach to predict functional profiles from 16S rRNA gene sequence data.

The 16S rRNA gene amplicon sequencing is a popular technique that provides accurate characterization of microbial taxonomic abundances but does not provide any functional information. Several tools are available to predict functional profiles based on 16S rRNA gene sequence data that use different genome databases and approaches. As variable regions of partially-sequenced 16S rRNA gene cannot resolve taxonomy accurately beyond the genus level, these tools may give inflated results. Here, we developed 'MicFunPred', which uses a novel approach to derive imputed metagenomes based on a set of core genes only, thereby minimizing false-positive predictions. On simulated datasets, MicFunPred showed the lowest False Positive Rate (FPR) with mean Spearman's correlation of 0.89 (SD = 0.03), while on seven real datasets the mean correlation was 0.75 (SD = 0.08). MicFunPred was found to be faster with low computational requirements and performed better or comparable when compared with other tools.

Benchmarking of 16S rRNA gene databases using known strain sequences.

16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at various taxonomic levels using various methods and databases.