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With the emergence of portable DNA sequencers, such as Oxford Nanopore Technology MinION, metagenomic DNA sequencing can be performed in real-time and directly in the field. However, because metagenomic DNA analysis is computationally and memory intensive, and the current methods are designed for batch processing, the current metagenomic tools are not well suited for mobile devices.In this paper, we propose a new memory-efficient method to identify Operational Taxonomic Units (OTUs) in metagenomic DNA streams. Our method is based on finding connected components in overlap graphs constructed over a real-time stream of long DNA reads as produced by MinION platform. We propose an efficient algorithm to maintain connected components when an overlap graph is streamed, and show how redundant information can be removed from the stream by transitive closures. Through experiments on simulated and real-world metagenomic data, we demonstrate that the resulting solution is able to recover OTUs with high precision while remaining suitable for mobile computing devices.

Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? Here, we reveal that metagenomes from no fewer than 20% of the papers found in our literature search, published between 2016 and 2019, were not deposited in a repository or were simply inaccessible. The proportion of inaccessible data within the literature has been increasing year-on-year. Noncompliance with Open Data is best predicted by the scientific discipline of the journal. The number of citations, journal type (e.g., Open Access or subscription journals), and publisher are not good predictors of data accessibility. However, many publications in high–impact factor journals do display a higher likelihood of accessible metagenomic data sets. Twenty-first century science demands compliance with the ethical standard of data sharing of metagenomes and DNA sequence data more broadly. Data accessibility must become one of the routine and mandatory components of manuscript submissions—a requirement that should be applicable across the increasing number of disciplines using metagenomics. Compliance must be ensured and reinforced by funders, publishers, editors, reviewers, and, ultimately, the authors., Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? This Essay reveals that every fifth published metagenome is not available to science, and asks whether data accessibility should be considered a mandatory component of manuscript submission.

The study performed on the stone materials from the Convent of Christ revealed the presence of a complex microbial ecosystem, emphasizing the determinant role of microorganisms on the biodecay of this built cultural heritage. In this case study, the presence of Rubrobacter sp., Arthrobacter sp., Roseomonas sp., and Marinobacter sp. seems to be responsible for colored stains and biofilm formation while Ulocladium sp., Cladosporium sp., and Dirina sp. may be related to structural damages. The implementation of high‐throughput sequencing approaches on the Convent of Christ's biodecay assessment allowed us to explore, compare, and characterize the microbial communities, overcoming the limitations of culture‐dependent techniques, which only identify the cultivable population. The application of these different tools and insights gave us a panoramic view of the microbiota thriving on the Convent of Christ and signalize the main biodeteriogenic agents acting on the biodecay of stone materials. This finding highlighted the importance of performing metagenomic studies due to the improvements and the reduced amount of sample DNA needed, promoting a deeper and more detailed knowledge of the microbiota present on these dynamic repositories that support microbial life. This will further enable us to perform prospective studies in quarry and applied stone context, monitoring biogenic and nonbiogenic agents, and also to define long‐term mitigation strategies to prevent biodegradation/biodeterioration processes., A multianalytical approach based on culture‐dependent methods, high‐throughput sequencing, and scanning electron microscopy was implemented to characterize the microbiota on stone of the Convent of Christ (Tomar, Portugal). The biocolonizer agents detected induce aesthetic damages and structural alterations on these materials, promoting the appearance of stains, biofilms, and detachment of stone fragments. These findings are a starting point for the implementation of a long‐term monitoring plan to prevent the decay of this monument and promote its safeguard.

When compared to other fields of ecological study, cave microbiome is still a growing research area. In recent years, considerable attention has been paid to explore cave microbiomes all over the world to achieve a better understanding of life under unfavorable conditions. With the help of advancing molecular genetic techniques and high-throughput sequencing technologies, this greatly expanded our view of taxonomic distribution from a wide range of cave samples. It is, however, worth noting that different caves have unique physicochemical and biophysical characteristics. These factors have undoubtedly contributed to the variation in taxonomic composition. In addition, with differences in metagenomic DNA extraction protocols, the choice of 16S rRNA variable regions, sequencing technologies, and bioinformatics pipelines, this makes the taxonomic distribution from different caves difficult to compare. Nonetheless, according to the previous studies, it is generally accepted that cave ecosystems usually harbor lower taxonomic diversity than their outside environments. These results strongly suggest that cave environments provide restrictive conditions that filter out nonadaptive taxa. In this review, we present recent progress in taxonomic profiling studies of cave microbiomes based on the integration of metagenomics and high-throughput sequencing. Last but not least, ecological roles of cave-dwelling taxa are also discussed.

Over time, sequencing determination of the unknown isolate has become the routine process to gather information about any piece of the nucleic acid. There have been significant innovations in the sequencing technique, from sequencing one nucleotide at a time to the massively parallel sequencing methods. Over the past three decades, the Sanger sequencing method has been the sole sequencing approach. From the past 15 years, there has been a significant reduction in the DNA sequencing cost which has increased the demand to a revolutionary extent as the newer techniques are providing the inexpensive, robust and precise genomic information. The conventional and next-generation techniques have tremendously progressed and created wealthy opportunities in different research areas, especially in metagenomic approaches, as sequencing play a significant role in its analysis. Therefore, this chapter intends to provide information about past applied and current DNA sequencing approaches and will discuss their limitations and strength. Moreover, the application of these sequencing techniques will also be delineated.

This data article describes the bacterial diversity of the deep sea shark, Centroscyllium fabricii. The data was acquired by metabarcoding using 16S rDNA. Centroscyllium fabricii, a deep sea shark found at depths below 275 m was sampled during Sagar Sampada cruise no 305 in the Indian Ocean and metagenomic DNA was isolated from the gut contents using QIAamp DNA stool minikit. V3 region of 16S rDNA region was amplified and the amplicons were sequenced on Illumina MiSeq system using 151 bp × 2 paired end reads. The data of this metagenome is available in the BioSample Submission Portal as Bio-Project PRJNA431407and Sequence Read Archive (SRA) accession number SRR6507004. Keywords: Centroscyllium fabricii, Gut microbiota, Metagenome, Illumina

Bromeliads tank water or phytotelma is an eutrophic microenvironment where microorganisms have evolved to resist sudden changes in pH and nutritional competition. Metagenomics studies have been poorly studied in bromeliads and environmental DNA (eDNA) characterization for its microenvironment is deficient in Puerto Rico. Therefore, the data represents the microbial communities inhabiting bromeliads phytotelma. eDNA was extracted using Metagenomic DNA Isolation Kit for Water. Next-Generation-Sequencing technology (Illumina MiSeq) was used for sequencing the isolated eDNA. This data provides an insight about diversity and functional depiction of microorganisms inhabiting bromeliads phytotelma. The data of this metagenome is available in the BioSample Submission Portal as Bioproject PRJNA39461 and Sequence Read Archive (SRA) accession number SRP114300. MG-RAST metagenomic analysis server is located under the study ID mgp79812. Keywords: Bromeliads, Phytotelma, DNA, Metagenome, Characterization, Illumina

Growing concerns regarding increasing rates of antibiotic resistance call for global monitoring efforts. Monitoring of environmental media (e.g., wastewater, agricultural waste, food, and water) is of particular interest as these media can serve as sources of potential novel antibiotic resistance genes (ARGs), as hot spots for ARG exchange, and as pathways for the spread of ARGs and human exposure. Next-generation sequence-based monitoring has recently enabled direct access and profiling of the total metagenomic DNA pool, where ARGs are identified or predicted based on the “best hits” of homology searches against existing databases. Unfortunately, this approach tends to produce high rates of false negatives. To address such limitations, we propose here a deep leaning approach, taking into account a dissimilarity matrix created using all known categories of ARGs. Two models, deepARG-SS and deepARG-LS, were constructed for short read sequences and full gene length sequences, respectively. Performance evaluation of the deep learning models over 30 classes of antibiotics demonstrates that the deepARG models can predict ARGs with both high precision (>0.97) and recall (>0.90) for most of the antibiotic resistance categories. The models show advantage over the traditional best hit approach by having consistently much lower false negative rates and thus higher overall recall (>0.9). As more data become available for under-represented antibiotic resistance categories, the deepARG models’ performance can be expected to be further enhanced due to the nature of the underlying neural networks. The deepARG models are available both in command line version and via a Web server athttp://bench.cs.vt.edu/deeparg. Our newly developed ARG database, deepARG-DB, containing predicted ARGs with high confidence and high degree of manual curation, greatly expands the current ARG repository. DeepARG-DB can be downloaded freely to benefit community research and future development of antibiotic resistance-related resources.AbbreviationsARGantibiotic resistance gene

The etiology of sarcoidosis is unknown, but microbial agents are suspected as triggers.We sought to identify bacterial, fungal, or viral lineages in specimens from patients with sarcoidosis enriched relative to control subjects using metagenomic DNA sequencing. Because DNA from environmental contamination contributes disproportionately to samples with low authentic microbial content, we developed improved methods for filtering environmental contamination.We analyzed specimens from subjects with sarcoidosis (n = 93), control subjects without sarcoidosis (n = 72), and various environmental controls (n = 150). Sarcoidosis specimens consisted of two independent sets of formalin-fixed, paraffin-embedded lymph node biopsies, BAL, Kveim reagent, and fresh granulomatous spleen from a patient with sarcoidosis. All specimens were analyzed by bacterial 16S and fungal internal transcribed spacer ribosomal RNA gene sequencing. In addition, BAL was analyzed by shotgun sequencing of fractions enriched for viral particles, and Kveim and spleen were subjected to whole-genome shotgun sequencing.In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, we detected enrichment of several bacterial lineages in sarcoidosis but not Cladosporiaceae. BAL showed limited enrichment of Aspergillus fungi. Several microbial lineages were detected in Kveim and spleen, including Cladosporium. No microbial lineage was enriched in more than one sample type after correction for multiple comparisons.Metagenomic sequencing revealed enrichment of microbes in single types of sarcoidosis samples but limited concordance across sample types. Statistical analysis accounting for environmental contamination was essential to avoiding false positives.

Microbes thrive in diverse habitats. They often form ecological niches with rich species diversity and complex spatial structure. These communities drive biogeochemical cycles in the environment and modulate host health in the human body. Much has been learned about the makeup of human and environmental microbiota via metagenomic DNA sequencing, but information on spatial interactions between microbes and between microbes and their environment remains scarce. Here, we review recent advances in tools to map the biogeography of microbiomes. We discuss methods to spatially map microbial genes, transcripts, and metabolites. We also examine future directions for microbiome mapping technologies that will allow improved understanding of both microbiome structure and function. Finally, we reflect on the impact of these methods in Biomedical Engineering.

Metagenomics is the study of gene pool of an entire community in a particular niche. This provides valuable information about the functionality of host-microbe interaction in a biological ecosystem. Efficient metagenomic DNA extraction is a critical pre-requisite for a successful sequencing run in a metagenomic study. Although isolation of human stool metagenomic DNA is fairly standardized, the same protocol does not work as efficiently in fecal DNA from other organisms. In this study, we report a comparison of manual and commercial DNA extraction methods for diverse samples such as human stool, fish gut and soil. Fishes are known to have variable microbial diversity based on their food habits, so the study included two different varieties of fishes. A modified protocol for effective isolation of metagenomic DNA from human milk samples is also reported, highlighting critical precautions. Recent studies have emphasized the importance of studying functionality of human milk metagenome to understand its influence on infants’ health. While manual method works well with most samples and therefore can be a method of choice for testing new samples, broad-range commercial kit offers advantage of high purity and quality. DNA extraction of different samples would go a long way in unraveling the unexplored association between microbes and host in a biological system.

Recent advances in science have proved that microorganisms exist and thrive in environments that were earlier thought uninhabitable. However, it is well known that only 0.1% of microorganisms are culturable, leaving the remaining 99.9% unexplored. Here metagenomics comes as a boon to science, since it enables the study of all microorganisms, regardless of their ability to be cultured. Analysis of genomic data of various species present in the environment provides a minefield of information regarding the functionality of microbial communities in their natural habitat. It also permits the screening of inaccessible organisms such as those found only under extreme environmental conditions. The metagenomic DNA can be extracted, purified, cloned, and expressed in a heterologous host. Such function-based screenings can be useful for the discovery of novel proteins such as antibiotics. This approach would also help to deal with the problem of drug resistance. Besides, it would also find applications in the field of personalized medicine. Undoubtedly metagenomics holds the key to the discovery of secondary metabolites having the potential to satisfy the urgent demand for novel drugs from natural sources. Hence, we review the recent advances in the field of metagenomics.

A new modified protocol has been developed for extracting pure community inhibitors-free DNA from saline soils, sediments and salts. Amplification of DNA from soil and sediment is often inhibited by copurified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to saline samples. Using a widely used a newly modified direct DNA extraction method proposed in this report, DNA was extracted from samples of Urmia Lake in diverse geological location in Iran and quantity of the DNA were examined. We developed an improved method to extract DNA include the combination of physical, chemical and mechanical lysis methods from saline samples. In the earlier reports, skim milk as an adsorption competitor was added to buffer DNA extract. In current study, we added skim milk to buffer DNA extraction. The results showed that skim milk was useful as an additive for extract DNA from saline samples. This method is applicable to molecular community analysis of saline samples which strongly adsorb DNA. The methods appear to have wide applicability in investigating molecular diversity and exploring functional genes from the total DNA. The extracted DNA was used to successfully amplify 16SrRNA region and functional genes. The amplicons were suitable for further applications such as diversity based analysis by denaturing gradient gel electrophoresis (DGGE) and cloning library.

BackgroundUsing metagenomics to determine animal diet offers a new and promising alternative to current methods. Here we show that rapid and inexpensive diet quantification is possible through metagenomic sequencing with the portable Oxford Nanopore Technologies (ONT) MinION. Using an amplification-free approach, we profiled the stomach contents from wild-caught rats.ResultsWe conservatively identified diet items from over 50 taxonomic orders, ranging across nine phyla that include plants, vertebrates, invertebrates, and fungi. This highlights the wide range of taxa that can be identified using this simple approach. We calibrate the accuracy of this method by comparing the characteristics of reads matching the ground-truth host genome (rat) to those matching diet items, and show that at the family-level, false positive taxon assignments are approximately 97.5% accurate. We also suggest a way to mitigate for database biases in metagenomic approaches. Finally, we implement a constrained ordination analysis and show that we can identify the sampling location of an individual rat within tens of kilometres based on diet content alone.ConclusionsThis work establishes proof-of-principle for long-read metagenomic methods in quantitative diet analysis. We show that diet content can be quantified even with limited expertise, using a simple, amplification free workflow and a relatively inexpensive and accessible next generation sequencing method. Continued increases in the accuracy and throughput of ONT sequencing, along with improved genomic databases, suggests that a metagenomic approach to quantification of animal diets will become an important method in the future.

International audience; AbstractKey messageTwo new efficient, fast and low-cost metagenomic DNA extraction methods were developed for different Persian oak tissues (leaf, stem, root, and rhizospheric) and soil samples.ContextThe new “omics” studies on the genus Quercus are of importance to help finding efficient strategies for overcoming environmental challenges, and to do this, presence of efficient DNA extraction protocols for different Quercus species are very critical.AimsThe objective of the present study was to develop new efficient methods for extraction of metagenomic DNA (mDNA) from of Persian oak (Quercus brantii Lindl.) tissues.MethodsThe efficiency of two newly developed mDNA extraction methods, including indirect SDS-based (ISB or concentrate method) and one spin column-based method (SCB) were compared to that of two classical direct methods, including CTAB-based and SDS-based methods, and two commercial mDNA extraction kits.ResultsThe maximum average yield of mDNA for all samples (leaf, stem, root, bulk, and rhizospheric soils) was obtained by SCB (258 ng/μl) and ISB (189 ng/μl) methods, respectively. Successful PCR amplification for 16SrRNA and ITS sequence was consistently observed for ISB, SCB, and kit-extracted mDNAs, which confirmed the high purity of mDNA extracted by these methods. The new methods showed more than 96% quantitative PCR efficiency, and partial restriction digestion and metagenomic library construction confirmed the high efficiency of the newly developed methods.ConclusionIt could be concluded that two new protocols enhanced efficiency (yield, purity, and cost) of mDNA extraction from different tissues of Persian oak.

Microbial based cleaning products (MBCPs) are a new generation of cleaning products that are gaining greater use in household, institutional, and industrial settings. Little is known about the exact microbial composition of these products because they are not identified in detail on product labels and formulations are often proprietary. To gain a better understanding of their microbial and fungal composition towards risk assessment, the cultivable microorganisms and rDNA was surveyed for microbial content in five different MBCPs manufactured and sold in North America. Individual bacterial and fungal colonies were identified by ribosequencing and fatty acid methyl ester (FAME) gas chromatography. Metagenomic DNA (mDNA) corresponding to each of the products was subjected to amplification and short read sequencing of seven of the variable regions of the bacterial 16S ribosomal DNA. Taken together, the cultivable microorganism and rDNA survey analyses showed that three of the products were simple mixtures of Bacillus species. The two other products featured a mixture of cultivable fungi with Bacilli, and by rDNA survey analysis, they featured greater microbial complexity. This study improves our understanding of the microbial composition of several MBCPs towards a more comprehensive risk assessment.

Despite increasing availability of phylogenomic datasets, strategies to generate genome-scale data from organisms involved in symbiotic relationships remains challenging. Restriction site-associated DNA sequencing (RADseq) can effectively generated reduced representation genomic loci. However, when using metagenomic DNA from inseparable symbiotic organisms, RADseq loci may belong to any number of the organisms involved in these intimate associations. In this study, we explored the potential for a reference-based RADseq approach to generate data for lichen-forming fungi from metagenomic DNA extracted from intact lichens. We simulated RAD data from draft genomes of closely related lichenized fungi to test if RADseq can reconstruct robust evolutionary relationships. Subsequently, we generated empirical RADseq data from metagenomic lichen DNA, with RADseq loci mapped back to a reference genome to exclude loci from other lichen symbionts that are represented in metagenomic libraries. In all cases, phylogenetic reconstructions using RADseq loci recovered diversification histories consistent with a previous study based on more comprehensive genome sampling. Furthermore, RADseq loci were found to resolve relationships among closely related species, which were otherwise indistinguishable using a phylogenetic species recognition criterion. Our studies revealed that a modified, reference-based RADseq approach can successfully be implemented to generate symbiont-specific phylogenomic data from metagenomic reads.

Historically, microbes from the environment have been a reliable source for novel bio-active compounds. Cloning and expression of metagenomic DNA in heterologous strains of bacteria has broadened the range of potential compounds accessible. However, such metagenomic libraries have been under-exploited for applications in mammalian cells because of a lack of integrated methods. We present an innovative platform to systematically mine natural resources for pro-apoptotic compounds that relies on the combination of bacterial delivery and droplet microfluidics. Using the violacein operon from C. violaceum as a model, we demonstrate that E. coli modified to be invasive can serve as an efficient delivery vehicle of natural compounds. This approach permits the seamless screening of metagenomic libraries with mammalian cell assays and alleviates the need for laborious extraction of natural compounds. In addition, we leverage the unique properties of droplet microfluidics to amplify bacterial clones and perform clonal screening at high-throughput in place of one-compound-per-well assays in multi-well format. We also use droplet microfluidics to establish a cell aggregate strategy that overcomes the issue of background apoptosis. Altogether, this work forms the foundation of a versatile platform to efficiently mine the metagenome for compounds with therapeutic potential.

Metagenomic DNA from sediments of selective estuaries of Goa, India was extracted using a simple, fast, efficient and environment friendly method. The recovery of pure metagenomic DNA from our method was significantly high as compared to other well-known methods since the concentration of recovered metagenomic DNA ranged from 1185.1 to 4579.7 µg/g of sediment. The purity of metagenomic DNA was also considerably high as the ratio of absorbance at 260 and 280 nm ranged from 1.88 to 1.94. Therefore, the recovered metagenomic DNA was directly used to perform various molecular biology experiments viz. restriction digestion, PCR amplification, cloning and metagenomic library construction. This clearly proved that our protocol for metagenomic DNA extraction using silica gel efficiently removed the contaminants and prevented shearing of the metagenomic DNA. Thus, this modified method can be used to recover pure metagenomic DNA from various estuarine sediments in a rapid, efficient and eco-friendly manner.

Submitted by YURY TATIANA GRANJA SALCEDO null (yurygranja@hotmail.com) on 2017-03-31T20:41:47Z No. of bitstreams: 1 Tese_Yury_Tatiana_Grnja_Salcedo.pdf: 1406238 bytes, checksum: b2e6edb6a429373b9228ec80d36c204f (MD5) Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-04-06T18:06:01Z (GMT) No. of bitstreams: 1 granjasalcedo_yt_dr_jabo_par.pdf: 829066 bytes, checksum: edc9919d69a5c8f4d32052e4c23ef77c (MD5) Made available in DSpace on 2017-04-06T18:06:01Z (GMT). No. of bitstreams: 1 granjasalcedo_yt_dr_jabo_par.pdf: 829066 bytes, checksum: edc9919d69a5c8f4d32052e4c23ef77c (MD5) Previous issue date: 2017-02-21 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) O objetivo deste estudo foi avaliar o efeito de três métodos e quatro tempos de armazenamento da amostra ruminal, sobre a qualidade e rendimento do DNA metagenômico extraído assim como o efeito sobre a composição da comunidade bacteriana ruminal e avaliar o efeito da glicerina bruta (CG) associada ao óleo de soja (OS) na dieta de Novilhos Nelore sobre o consumo, digestibilidade, fermentação, biohidrogenação (BH), microbiota ruminal e fluxo intestinal de ácidos graxos de cadeia longa. No experimento 1 utilizou-se um novilho Nelore equipado com cânula ruminal de silicone como doador de conteúdo ruminal e compreendeu 11 tratamentos: pellet controle (PC), liofilizado controle (LC), P-20: pellet armazenado a -20 ° C por um período de 3, 6 e 12 meses, P-80: pellet armazenado a -80 ° C durante um período de 3, 6 e 12 meses e L-20: amostra liofilizada armazenada a -20 ° C durante um período de 3, 6 e 12 meses. O método L-20 não conseguiu manter o rendimento de DNA durante o armazenamento. O método P-80 apresentou maior rendimento de DNA após 6 meses de armazenamento. Amostras armazenados como pellets (P-20 e P-80) resultaram em menor riqueza Chao 1, ACE e índice Shannon Wiener quando comparado com PC. Enquanto LC e PC apenas foram diferentes na riqueza ACE. O método de armazenamento e tempo de armazenamento influenciou as proporções de 14 ds 17 filos identificado. No método P-20 a proporção de Cianobactérias, Elusimicrobia, Fibrobacteres, Lentisphaerae, Proteobacteria e Espiroquetas foi menor ao 1%. No método P-80, houve um aumento na proporção do filo Bacteroides (p = 0,010); No entanto, a proporção de Actinobactérias, Chloroflexi, SR1, Synergistetes, TM7, e WPS.2 não mudou quando comparados com PC (p> 0,05). A classe Clostridium foi a mais abundante em todas as amostras armazenadas e aumento especialmente no método L-20. O tempo de armazenamento da amostra rúmen reduz significativamente o rendimento de DNA extraído e pode influenciar a abundância de filos, classes, e as famílias bacterianas estudadas assim como os índices de riqueza e diversidade. No experimento 2: oito Nelore castrados e equipados com cânula ruminal e duodenal foram utilizados em um duplo, quadrado latino 4 × 4 com um arranjo fatorial 2 × 2 (com ou sem SO e com ou sem CG). Animais alimentados com SO sem CG apresentaram menor consumo de MS, CP e FDN e apresentaram a menor digestibilidade de MS e FDN. Foi observado um maior fluxo duodenal de ácidos graxos (FA) monoinsaturados (MUFA), FA poli-insaturados (PUFA) e FA insaturados (UFA) com associação CG e SO na dieta. Houve interação entre CG e SO na taxa de BH ruminal de PUFA, UFA e ácido linolênico, observando-se menor BH com a associação CG e SO, quando comparado com a dieta SO sem CG. Esta interação também foi observada na taxa de BH de MUFA, que foi menor com associação CG e SO. Os novilhos alimentados com a associação de óleo e glicerina na dieta apresentaram maior pH ruminal e menor concentração de N-NH3 ruminal. Dietas com adição de CG reduziram a relação acetato: propionato e aumentaram a proporção de iso-butirato, butirato, iso-valerato e valerato. Novilhos alimentados com dietas contendo SO apresentaram menor excreção de N total e maior retenção de N, expressa em % da ingestão de N. A associação de SO e CG na dieta gerou maior abundância ruminal de Prevotella, Succinivibrio, Ruminococcus, Syntrophococcus e Succiniclasticum. A abundância de Archaea e protozoários ciliados totais foi menor nos animais alimentados com dietas contendo SO. A dieta SO sem CG reduziu a população ruminal de Ruminococcus flavefaciens, R. albus e Fibrobacter succinogenes. CG associada a SO pode ser fonte de energia, útil para a substituição parcial do milho na dieta de bovinos, resultando na redução da excreção total de N e efeitos negativos na abundância de metanogenicos ruminais, sem afetar a ingestão e digestibilidade da dieta, mesmo quando alto teor de lipídios foi adicionado na dieta. Além disso, esta associação limitou a BH de UFA e aumentou o fluxo duodenal destes ácidos sem influenciar as bactérias celulolíticas do rúmen; por tanto, esta associação pode ser uma estratégia nutricional para aumentar a deposição de UFA saudável na carne. The objective of this study was to investigate three methods and four storage times for rumen sampling in terms of quality and yield of extracted metagenomic DNA as well as the composition of the rumen bacterial community, and evaluate the effect of soybean oil (SO) and crude glycerin (CG) association on ruminal fermentation, ruminal biohydrogenation (BH) and ruminal microbial population in Nellore steers. In the experiment 1: One Nellore steer fitted with a ruminal silicone-type cannula was used as a donor of ruminal contents. The experiment comprised 11 experimental groups: pellet control (PC), lyophilized control (LC), P-20: pellet stored frozen at -20 °C for a period of 3, 6, and 12 months, P-80: pellet stored frozen at -80 °C for a period of 3, 6, and 12 months, and L-20: lyophilized sample stored frozen at -20 °C for a period of 3, 6, and 12 months. The L-20 method could not maintain the yield of DNA during storage. In addition, the P-80 group showed a greater yield of metagenomic DNA than the other groups after 6 months of storage. Rumen samples stored as pellets (P-20 and P-80) resulted in lower richness Chao 1, ACE, and Shannon Wiener indices when compared to PC, while LC and PC were only different in richness ACE. The storage method and storage time influenced the proportions of 14 of 17 phyla identified by sequencing. In the P-20 group, the proportion of Cyanobacteria, Elusimicrobia, Fibrobacteres, Lentisphaerae, Proteobacteria, and Spirochaetes phyla identified was lower than 1%. In the P-80 group, there was an increase in the proportion of the Bacteroidetes phylum (p = 0.010); however, the proportion of Actinobacteria, Chloroflexi, SR1, Synergistetes, TM7, and WPS.2 phyla were unchanged compared to the PC group (p > 0.05). The class Clostridium was the most abundant in all stored groups and increased in its proportion, especially in the L-20 group. The rumen sample storage time significantly reduced the yield of metagenomic DNA extracted. Therefore, the storage method can influence the abundance of phyla, classes, and bacterial families studied in rumen samples and affect the richness and diversity index. In the experiment 2: Eight Nellore castrated and fitted with ruminal and duodenal cannulas were used in a double, simultaneous, Latin square design 4 × 4 with a 2 × 2 factorial arrangement of treatments (with or without SO and with or without CG). Steers fed with SO without CG showed lowest intake of DM, CP and NFC and presented lowest DM and NDF digestibility. A higher duodenal flow of monounsaturated fatty acid (MUFA), poly-unsaturated fatty acid (PUFA) and unsaturated fatty acid (UFA) was observed with the CG and SO association diet. There was an interaction between CG and SO on the ruminal BH rate of PUFA, UFA, and linolenic acid, the BH being lower with the CG and SO association diet than with the SO without CG diet. This interaction was also observed to affect the BH rate of MUFA, which was lower with the CG and SO association diet. Steers fed with oil and glycerine association diet had highest rumen pH and lowest ruminal N-NH3 concentration. Diets with CG addition reduced the acetate: propionate ratio and increased the proportion of iso-butirate, butirate, iso-valerate and valerate. Steers fed with both diets contain SO had lower total N excretion and showed higher N retained expressed as % N intake. SO and CG association diet generated higher rumen abundance of Prevotella, Succinivibrio, Ruminococcus, Syntrophococcus and Succiniclasticum. Rumen Archaea abundance and total ciliate protozoa were lower in steers fed with diets contained SO. The SO without CG diet reduced the population of Ruminococcus flavefaciens, R. albus and Fibrobacter succinogenes. CG associated with SO could be used as an energy source, and is a useful partial replacement for corn in cattle diets, resulting in a reduction of the total nitrogen excretion and ruminal methanogen abundance. This is environmentally beneficial, since methane emissions could be reduced without affecting dietary intake and digestibility, even when high lipid content is added to the diet. In addition, this association in the diet limited the BH of UFA, and increased the duodenal flow of these acids without influencing the cellulolytic bacteria of the rumen; therefore, this association may be a nutritional strategy to increase the deposition of healthy UFA in meat. FAPESP: 2013/23851-6

Stenotrophomonas maltophilia is a Gram-negative aerobic bacterium and emerging nosocomial pathogen. Here, we present a draft genome sequence for an S. maltophilia strain assembled from a metagenomic DNA extract isolated from a laboratory stock of the nematode worm Caenorhabditis remanei .

Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing.

Graphical abstract Stages of sample treatment: (a) seeds in fermentation boxes; (b) frozen cupuassu beans; (c) maceration of beans with liquid nitrogen; (d) fat extraction under stirring with petroleum ether; and (e) initial step of DNA extraction process., The pulp surrounding the seeds of some fruits is rich in mucilage, carbohydrates, etc. Some seeds are rich in proteins and polyphenols. Fruit seeds, like cacao (Theobroma cacao) and cupuassu (Theobroma grandiflorum), are subjected to fermentation to develop flavor. During fermentation, ethanol is produced [2–6]. All of these compounds are considered as interfering substances that hinder the DNA extraction [4–8]. Protocols commonly used in the DNA extraction in samples of plant origin were used, but without success. Thus, a protocol for DNA samples under different conditions that can be used for similar samples was developed and applied with success. The protocol initially described for RNA samples by Zeng et al. [9] and with changes proposed by Provost et al. [5] was adapted for extracting DNA samples from those described. However, several modifications have been proposed:•Samples were initially washed with petroleum ether for fat phase removal.•RNAse was added to the extraction buffer, while spermidin was removed.•Additional steps of extraction with 5 M NaCl, saturated NaCl and CTAB (10%) were included and precipitation was carried out with isopropanol, followed by washing with ethanol.

Several reports mention the presence of antibiotic resistance genes in natural and polluted environments, but many studies are based on their detection via polymerase chain reaction (PCR amplification of known genes and not on an activity screening. We constructed a metagenomic fosmid bank from DNA isolated from a polluted river in Brussels, Belgium, the Zenne. A total of 120,000 clones were pooled and plated directly on solid media containing different antibiotics. Several clones were isolated which could grow in the presence of ampicillin. The DNA from several clones was extracted and subjected to restriction analysis and, based on their restriction pattern, two different clones were found. One of the clones was selected for further study as it showed a higher level of resistance to different β-lactams antibiotics (ticarcilline and ceftazidime). To find out which gene is responsible for the resistance, an in vitro transposon mutagenesis was performed and clones having lost the resistance phenotype were analyzed via inverse PCR amplification. Several clones had an insert in a gene encoding a new type of β-lactamase. The amplified fosmid DNA was fully sequenced revealing an insert of 41 kb containing 39 open reading frames (ORFs). Transposon insertions inactivating the resistance to β-lactams were also found in the ORF upstream of the blaA gene, encoding an aminotransferase, suggesting a polar effect on the transcription of the gene downstream. In addition, other genes were found such as histidine biosynthesis genes, which were found to be scattered on the insert, a relA/spoT gene, and genes belonging to type II toxin-antitoxin system. This predicted system was experimentally validated in Escherichia coli using an inducible expression system., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

The vast majority of the Earth's biological diversity is hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is a cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA.

Recent advances in culture-independent studies of microbes had proved to be more reliable and efficient than the conventional ones. The isolation of good quality and quantity of total community DNA are one of the major hurdles in this endeavour. Shearing of DNA during the extraction process and the co-extraction of inhibitory compounds reduce the quality of the isolated nucleic acids making it unsuitable for the construction of large insert metagenomic libraries. In the present study, a multi-level filtration step was brought in which efficiently isolated total bacterial DNA from three different environment samples. The preprocessing method could efficiently improve the 260/230 ratio of the isolated DNA by 2.3–45 % and decreased the protein contamination by 22.5–34.5 % on saltpan and arctic sediment samples, respectively. The more significant part of the experiment was that the DNA obtained was of high quality with minimal shearing making it most suitable for the construction of large insert genomic libraries. PCR amplification of 16S rRNA gene confirmed that the filtration method was effective in the isolation of high-quality DNA.

El presente trabajo tuvo como objetivo la bioprospección de ADN metagenómico derivado de comunidades microbianas asociadas a un agroecosistema de importancia nacional. Este análisis permitió realizar la producción, expresión, purificación y caracterización de una enzima novedosa con actividad esterasa. Esta enzima, denominada LipM, había sido previamente identificada en clones metagenómicos derivados de suelos dedicados al cultivo de papa criolla (Solanum pureja), mediante secuencia de nueva generación y análisis bioinformáticos. La secuencia codificante de la enzima fue clonada en el vector pBADgiii y expresada en E. coli como sistema de expresión, lo que permitió optimizar el proceso de producción recombinante y su posterior purificación.Funcionalmente la enzima presentó una mayor afinidad por sustratos de p-nitrofenil con ácidos grasos de cadena corta (C8). LipM mostró completa funcionalidad a temperaturas entre 30 – 37 ºC y en valores de pH cercanos al fisiológico (entre 7.0 y 8.0). Igualmente, esta enzima exhibió buena estabilidad en presencia de varios iones metálicos, inhibidores y 0.1% (p/v) de SDS. Su alto nivel de estabilidad en presencia de iones metálicos e inhibidores, así como su particular especificidad en cuanto a sustratos, la hacen una enzima óptima para utilización en diferentes aplicaciones biotecnológicas.Palabras clave: metagenómica, enzima esterasa, caracterización, suelos, Lipasa/esterasa The present work had as a main objective to bioprospect metagenomic DNA from microbial communities associated with an agro-ecosystem of national importance. This analysis allowed the production, expression, purification and characterization of a novel enzyme with esterase activity. This enzyme, named here as LipM, was previously identified in metagenomic clones derived from soils dedicated to creole potato (Solanum pureja) crops by means of next-generation sequencing and bioinformatics analyses. The coding sequence of the enzyme was cloned into pBADgiii vector and expressed in E. coli as an expression system, allowing to optimize its recombinant production process and its further purification.The enzyme functionally showed a greater affinity for p-nitrophenyl substrates with short-chain fatty acids (C8). LipM showed full functionality at temperatures between 30 - 37°C and close to physiological pH (between 7.0 and 8.0). This enzyme also exhibited proper stability in the presence of various metal ions, inhibitors and at 0.1% (w/v) of SDS. Its high level of stability in the presence of inhibitors, metal ions and its particular specificity of substrates make it optimal for use in various biotechnology applications.Key words: Metagenomics; esterase enzyme; characterization; soil; lipase/esterase.

The renewal of sequencing technology have promoted the rapid development of metagenomics, which have lead to the sharply increasing of sequenced metagenome DNA sequences number. But most of DNA sequences species are unknown and the current method is to extract nearly ten thousands digital features as the species label, and oversize feature dimension will make large amount of calculation, so how to reduce those large metagenome DNA digital features is a problem that bioinformatics faced. A rough reduction method based on Particle Swarm Optimization (PSO) algorithm is designed which take use of rough theory and combine PSO algorithm. This algorithm have been applied to reduce the metagenome DNA sequences on the level of genera which have approved that it can make effective reduction for metagenome DNA digital features and get more classification accuracy rate than before.

Modern next generation sequencing technologies produce vast amounts of data in the context of large-scale metagenomic studies, in which complex microbial communities can be reconstructed to an unprecedented level of detail. Most prominent examples are human microbiome studies that correlate the bacterial taxonomic profile with specific physiological conditions or diseases.In order to perform these analyses high-throughput computational tools are needed that are able to process these data within a short time while preserving a high level of sensitivity and specificity.Here we present MALT (MEGAN ALignment Tool) a program for the ultrafast alignment and analysis of metagenomic DNA sequencing data. MALT processes hundreds of millions of sequencing reads within only a few hours. In addition to the alignment procedure MALT implements a taxonomic binning algorithm that is able to specifically assign reads to bacterial species. Its tight integration with the interactive metagenomic analysis software MEGAN allows for visualization and further analyses of results.We demonstrate MALT by its application to the metagenomic analysis of two ancient microbiomes from oral cavity and lung samples of the 5,300-year-old Tyrolean Iceman. Despite the strong environmental background, MALT is able to pick up the weak signal of the original microbiomes and identifies multiple species that are typical representatives of the respective host environment.

Archaea constitute one of the three recognized phylogenetic groups of organisms living on the planet, and the latest to be discovered. Most Archaea resist cultivation and are studied using molecular methods. High-throughput amplicon sequencing and metagenomic approaches have been key in uncovering hitherto unknown archaeal diversity, their metabolic potential, and have even provided an insight into genomes of a number of uncultivated members of this group. Here, we summarize protocols describing sampling, molecular, metagenomic, and metatranscriptomic analyses as well as bioinformatics approaches that have proved useful for the study of archaea in natural samples.