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Xichú River is a Mexican river located in an environmental preservation area called Sierra Gorda Biosphere Reserve. Around it, there are tons of abandoned mine residues that represent a serious environmental issue. Sediment samples of Xichú River, visibly contaminated by flows of an acid mine drainage, were collected to study their prokaryotic diversity. The study was based on both cultural and non-cultural approaches. The analysis of total 16S rRNA gene by MiSEQ sequencing allowed to identify 182 Operational Taxonomic Units. The community was dominated by Pseudomonadota, Bacteroidota, “Desulfobacterota” and Acidobacteriota (27, 21, 19 and 16%, respectively). Different culture conditions were used focusing on the isolation of anaerobic bacteria, including sulfate-reducing bacteria (SRB) and arsenate-reducing bacteria (ARB). Finally, 16 strains were isolated. Among them, 12 were phylogenetically identified, with two strains being SRB, belonging to the genus Solidesulfovibrio (“Desulfobacterota”), while ten are ARB belonging to the genera Azospira (Pseudomonadota), Peribacillus (Bacillota), Raineyella and Propionicimonas (Actinomycetota). The isolate representative of Raineyella genus probably corresponds to a new species, which, besides arsenate, also reduces nitrate, nitrite, and fumarate. [ABSTRACT FROM AUTHOR]

Marine sediments are complex ecosystems where structures and functions constantly change due to natural and anthropogenic influences. In this investigation, a comprehensive and comparative analysis of the bacterial communities and their functional potential of the pristine and polluted marine sediments were carried out using MiSeq. The phylum Proteobacteria was dominant in all study sites. Other phyla were Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Chloroflexi, Nitrospirae, Cyanobacteria, Verrucomicrobia, Tenericutes, and Chlorobi. Interestingly, about 50% of genera belong to the unclassified categories. The key genera were identified as Acinetobacter, Bacillus, Pseudomona, Idiomarina, Thalassospira, and Marinobacter, Halomonas, Planctomyces, Psychrobacter, and Vogesella. PICRUSt analysis revealed that major functions are associated with the metabolism category. Additionally, metabolism related to amino acids, carbohydrates, energy generation, xenobiotics degradation, nitrogen, sulfate, and methane were prominent. Similarly, the predicted metabolisms by COG and KEGG were observed in the microbial communities of the marine sediments. To date, a comprehensive description of the microbial life with metabolic potential in these study sites has not been investigated. This study therefore significantly adds to our understanding of the microbiome and its functional attributes of marine sediments. [ABSTRACT FROM AUTHOR]

Fungi belonging to the genus Tuber produce edible ascocarps known as truffles. Tuber magnatum Picco may be the most appreciated truffle species given its peculiar aroma. While its life cycle is not yet fully elucidated, some studies demonstrated an active role of microorganisms. The main goal of this study was to determine how the T. magnatum microbiome varies across space and time. To address this, we characterized microbial communities associated with T. magnatum through high-throughput amplicon sequencing of internal transcribed spacer (ITS) and 16S rDNAs in three productive natural sites in Italy across 2 years. At each site, four truffles were sampled as well as the soil underneath and at 40, 100, and 200 cm from the harvesting points, to assess for microbial variation between substrates, years, and sites. A statistically significant site-related effect on microbial communities was identified, whereas only the prokaryotic community was significantly affected by the distance of soil from the truffle. Significant differences between sampling years were also found, demonstrating a possible relation among rainfall precipitation and Firmicutes and Actinobacteria. Thirty-six bacterial OTUs in truffles and 11 bacterial OTUs in soils beneath truffles were identified as indicator taxa. As shown for other truffle species, the dominance of Bradyrhizobium, Rhizobium, and Ensifer spp. within the truffle fruiting body suggests an evolutionary adaptation of this microorganism to the genus Tuber. The present work offers novel and relevant insights into the microbial ecology of T. magnatum ecosystems and fruiting bodies. The function and role of these bacteria in the truffle microbiome and life cycle are in need of further investigation. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing technology has allowed for the rapid development of microsatellites, neutral polymorphic markers that can be used for the analysis of population structure. Methods and Results: In this study, we performed whole-genome sequencing using the Illumina MiSeq system and de novo assembly to design microsatellite primers for Triops granarius populations in Qatar. The developed microsatellites are suitable for future studies of genetic structuring among geographically isolated freshwater pools. A total of 23 different primer pairs produced typical microsatellite results, with each pair successfully amplified in up to 40 individuals. Only five of the loci produced a significant departure from Hardy-Weinberg equilibrium. Conclusions: Some of the underlying mechanisms regarding the few loci that deviated from HWE may be further investigated to determine the source of deviation. As T. granarius is the most widely distributed species of the family, the development of these molecular markers would be useful for conducting population genetics and biogeographical studies broadly. [ABSTRACT FROM AUTHOR]

This prospective study was aimed to explore the bacterial diversity of marine mucilage developed in the Marmara Sea and the North Aegean Sea by metabarcoding. For this purpose, mucilage samples were collected from five different sampling locations, and the bacterial community structure was analyzed by 16S rRNA gene amplicon sequencing. The results highlighted a diverse bacterial community dominated by Proteobacteria and Bacteroidetes species. A negative and significant correlation between pH level and Campylobacterales, Clostridiales, and Vibronales abundances was detected, while a strong positive correlation was determined between total phosphorus (TP) and Campylobacterales. Results revealed that the bacterial community in the mucilage samples was predominated by particle-attached species preferring high-nutrient concentrations. This is the first study evaluating the bacterial diversity in a mucilage outbreak using a metabarcoding approach. Its results may contribute to this growing area of research and provide a database for further studies. [ABSTRACT FROM AUTHOR]

Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows forensic laboratories to report heteroplasmy on a routine basis. Statistical approaches will be needed to determine the relative frequency of observing an mtDNA haplotype when including the presence of a heteroplasmic site. Here, we examined 1301 control region (CR) sequences, collected from individuals in four major population groups (European, African, Asian, and Latino), and covering 24 geographically distributed haplogroups, to assess the rates of point heteroplasmy (PHP) on an individual and nucleotide position (np) basis. With a minor allele frequency (MAF) threshold of 2%, the data was similar across population groups, with an overall PHP rate of 37.7%, and the majority of heteroplasmic individuals (77.3%) having only one site of heteroplasmy. The majority (75.2%) of identified PHPs had an MAF of 2–10%, and were observed at 12.6% of the nps across the CR. Both the broad and phylogenetic testing suggested that in many cases the low number of observations of heteroplasmy at any one np results in a lack of statistical association. The posterior frequency estimates, which skew conservative to a degree depending on the sample size in a given haplogroup, had a mean of 0.152 (SD 0.134) and ranged from 0.031 to 0.83. As expected, posterior frequency estimates decreased in accordance with 1/n as the sample size (n) increased. This provides a proposed conservative statistical framework for assessing haplotype/heteroplasmy matches when applying an MPS technique in forensic cases and will allow for continual refinement as more data is generated, both within the CR and across the mitochondrial genome. [ABSTRACT FROM AUTHOR]

We tried to estimate individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq. The BAM files produced by MiSeq were viewed using Integrative Genomics Viewer (IGV) to verify mixed bases. By sorting the reads according to base type for each mixed base, partial haplotypes were determined. Then, the BAM files produced by MinKNOW were viewed using IGV. To determine haplotypes with IGV, only mixed bases determined by MiSeq were used as target bases. By sorting the reads according to base type for each target base, each contributor's haplotype was estimated. In mixed samples from two contributors, even a haplotype with a minor contribution of 5% could be distinguished from the haplotype of the major contributor. In mixed samples of three contributors (mixture ratios of 1:1:1 and 4:2:1), each haplotype could also be distinguished. Sequences of C-stretches were determined very inaccurately in the MinION analysis. Although the analysis method was simple, each haplotype was correctly detected in all mixed samples with two or three contributors in various mixture ratios by combining MinION and MiSeq. This should be useful for identifying contributors to mixed samples. [ABSTRACT FROM AUTHOR]

The selection of tree species used for the afforestation of urban forests is very important for maintaining the urban ecosystem, while soil microbe is one of the driving factors of material cycling in the urban forest ecosystem and for health of forests. In this study, the characteristics of surface soil bacterial and fungal community structure in four urban forests (primarily composed of Fraxinus mandshurica (Fm), Quercus mongolica (Qm), Pinus sylvestris var. mongolica (Ps), and Pinus tabulaeformis var. Mukdensis (Pt) as the main dominant tree species, respectively) were investigated by high-throughput sequencing. Our results showed that the alpha diversity of the soil microbial community in the Fm urban forest was the highest, while the lowest was in the Ps urban forest. In the bacterial community, Proteobacteria was the most predominant phylum in soils from Fm, Ps, and Pt urban forests. The most relatively abundant phylum of the Qm urban forest soil was Acidobacteria. The relative abundances of the bacterial communities at the genus level in the soil of four urban forests were significantly different. The soil bacterial communities in Ps and Pt urban forests were more similar, and Qm and Fm were also more similar. In the fungal community, Basidiomycota was the most predominant phylum in soils from Qm, Ps, and Pt urban forests. The phylum with the greatest relative abundance in the Fm urban forest soil was Ascomycota. There were differences in the fungal community between Qm, Fm, Ps, and Pt urban forests. Soil microbial community composition was affected by environmental factors: soil bacterial and fungal community compositions were significantly related to soil electrical conductivity (EC), alkali hydrolysable nitrogen (AHN), total nitrogen (TN), and total phosphorus (TP). In conclusion, the soil microbial community structure was related to both forest's tree species and soil properties. [ABSTRACT FROM AUTHOR]

As bacteria and archaea are key components in the ecosystem, information on their dynamics in soil profiles is important for understanding the biogeochemical cycles in peatlands. However, little is known about the vertical distribution patterns of bacteria and archaea in the Bitahai peatland, or about their relationships with soil chemical properties. Here, bacterial and archaeal abundance, diversity, and composition of the Bitahai peatlands at 0-100 cm soil depths were analyzed by sequencing of 16S rRNA genes (Illumina, MiSeq). Soil pH, total C, N, and P concentrations and stoichiometric ratios were also estimated. The results revealed that total C and total N contents, as well as C:P and N:P ratios, significantly increased with increasing peatland soil depths, while total P decreased. The top three dominant phyla were Proteobacteria (39.64%), Acidobacteria (12.93%), and Chloroflexi (12.81%) in bacterial communities, and were Crenarchaeota (58.67%), Thaumarchaeota (14.34%), and Euryarchaeota (10.82%) in archaeal communities in the Bitahai peatland, respectively. The total relative abundance of methanogenic groups and ammonia-oxidizing microorganisms all significantly decreased with soil depth. Both bacterial and archaeal diversities were significantly affected by the soil depth. Soil C, N, and P concentrations and stoichiometric ratios markedly impacted the community structure and diversity in archaea, but not in bacteria. Therefore, these results highlighted that the microbial community structure and diversity depended on soil depth for the Bitahai peatlands, and the factors affecting bacteria and archaea in the Bitahai peatlands were different. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) is a small but significant part of the human genome, whose applicability potential has gradually increased with the advent of massively parallel sequencing (MPS) technology. Knowledge of the particular workflow, equipment, and reagents used, along with extensive usage of negative controls to monitor all preparation steps constitute the prerequisites for confident reporting of results. In this study, we performed an assessment of Illumina® Human mtDNA Genome assay on MiSeq FGx™ instrument. Through analysis of several types of negative controls, as well as mtDNA positive controls, we established thresholds for data analysis and interpretation, consisting of several components: minimum read depth (220 reads), minimum quality score (41), percentage of minor allele sufficient for analysis (3.0%), percentage of minor allele sufficient for interpretation (6.0%), and percentage of major allele sufficient for homoplasmic variant call (97.0%). Based on these criteria, we defined internal guidelines for analysis and interpretation of mtDNA results obtained by MPS. Our study shows that the whole mtDNA assay on MiSeq FGx™ produces repeatable and reproducible results, independent of the analyst, which are also concordant with Sanger-type sequencing results for mtDNA control region, as well as with MPS results produced by NextSeq®. Overall, established thresholds and interpretation guidelines were successfully applied for the sequencing of complete mitochondrial genomes from high-quality samples. The underlying principles and proposed methodology on the definition of internal laboratory guidelines for analysis and interpretation of MPS results may be applicable to similar MPS workflows, e.g. targeting good-quality samples in forensic genetics and molecular diagnostics. [ABSTRACT FROM AUTHOR]

The effects of platinum (Pt) and gold (Au) and on the soil bacterial community was evaluated in four different Australian soil types (acidic Burn Grounds (BGR), organic matter–rich Fox Lane, high silt/metal Pinpinio (PPN), and alkali Minnipa (MNP) spiked with either Pt or Au at 1, 25, and 100 mg kg−1 using a next-generation sequencing approach (amplicon-based, MiSeq). Soil type and metal concentrations were observed to be key drivers of Pt and Au effects on soil microbial community structure. Different trends were therefore observed in the response of the bacterial community to Pt and Au amendments; however in each soil type, Pt and Au amendment caused a detectable shift in community structure that in most samples was positively correlated with increasing metal concentrations. New dominant groups were only observed in BGR and PPN soils at 100 mg kg−1 (Kazan-3B-28 and Verrucomicrobia groups (BGR, Pt) and Firmicutes and Caldithrix groups (PPN, Pt) and WS2 (BGR, Au). The effects of Pt on soil microbial diversity were largely adverse at 100 mg kg−1 and were pronounced in acidic, basic, and metal/silt-rich soils. However, this effect was concentration-related; Au appeared to be more toxic to soil bacterial communities than Pt at 25 mg kg−1 but Pt was more toxic at 100 mg kg−1. More bacterial groups such as those belonging to Burkholderiales/Burkholderiaceae, Alicyclobacillaceae, Rubrobacteraceae, Cytophagaceae, Oxalobacteraceae were selectively enriched by Pt compared to Au (Sphingomonadaceae and Rhodospirillaceae) amendments irrespective of soil type. The research outcomes have important implications in the management (remediation) of Pt- and Au-contaminated environments. [ABSTRACT FROM AUTHOR]

Although many primers have been proposed for arbuscular mycorrhizal fungi (AMF)–specific sequencing, only a few studies have evaluated suitable primer pairs for MiSeq analysis. Here, we analyzed AMF community compositions using several primer pairs and evaluated which pair can detect a more diverse AMF community. Three AMF-specific primer pairs targeting the 18S and 28S rRNA genes and a fungal universal primer pair targeting the ITS region were examined using soil DNA as the template. The effect of nested PCR was also evaluated. Only a few AMF sequences were obtained from a fungal universal primer targeting the ITS region. On the other hand, use of AMF-specific primers improved AMF detection rates (35–98%). Particularly, almost all of the sequences obtained with the nested PCR were AMF-related sequences. However, the AMF community compositions were differentiated among the primer pairs. In addition, the number of AMF taxa and diversity decreased with the nested PCR, suggesting that it affected AMF community analysis. [ABSTRACT FROM AUTHOR]

Illegal logging and trade of timber are major worldwide concerns, resulting in biodiversity and economic losses. Tropical tree species in the genus Cedrela, which have historically been heavily exploited, are still often illegally traded and there is an urgent need to develop tools to verify the origin of Cedrela products. A set of 351 SNP loci for Cedrela species from Bolivia, Brazil, Costa Rica, Cuba, Ecuador, French Guiana, Mexico, and Peru was developed using restriction associated DNA sequencing (RADSeq) and low coverage MiSeq genome sequencing, and adapted for MassARRAY genotyping. After screening of 94 individuals covering most of the distribution of Cedrela, a final set of 136 SNP loci which included 92 nuclear SNPs, 22 chloroplast markers (20 SNPs and 2 INDELs), and 22 mitochondrial markers (19 SNPs and 3 INDELs) was selected and tested for potential to verify Cedrela timber origin. [ABSTRACT FROM AUTHOR]

Although marine biofouling has been widely studied on different substrates, information on biofouling on plastics in the Arabian Gulf is limited. Substrate- and location-specific effects were investigated by comparing the microbial communities developed on polyethylene terephthalate (PET) and polyethylene (PE) with those on steel and wood, at two locations in the Sea of Oman. Total biomass was lower on PET and PE than on steel and wood. PET had the highest bacterial abundance at both locations, whereas chlorophyll a concentrations did not vary between substrates. MiSeq 16S ribosomal RNA sequencing revealed comparable operational taxonomic unit (OTU) richness on all substrates at one location but lower numbers on PET and PE at the other location. Non-metric multidimensional scaling (NMDS) showed distinct clusters of the bacterial communities based on substrate (analysis of similarity (ANOSIM), R = 0.45–0.97, p < 0.03) and location (ANOSIM, R = 0.56, p < 0.0001). The bacterial genera Microcystis and Hydrogenophaga and the diatoms Licmophora and Mastogloia were specifically detected on plastics. Desulfovibrio and Pseudomonas spp. exhibited their highest abundance on steel and Corynebacterium spp. on wood. Scanning electron microscopy (SEM) revealed fissure formation on PET and PE, indicating physical degradation. The presence of free radicals on PET and carbonyl bonds (C=O) on PE, as revealed by Fourier transform infrared (FTIR) spectroscopy, indicated abiotic degradation while hydroxyl groups and spectral peaks for proteins and polysaccharides on PE indicated biotic degradation. We conclude that fouling microbial communities are not only substrate-specific but also location-specific and microbes developing on plastics could potentially contribute to their degradation in the marine environment. [ABSTRACT FROM AUTHOR]

Virulent Newcastle disease viruses (NDV) have been present in Mexico since 1946, and recently, multiple outbreaks have been reported in the country. Here, we characterized eleven NDV isolated from apparently healthy wild birds and backyard chickens in three different locations of Jalisco, Mexico in 2017. Total RNA from NDV was reverse-transcribed, and 1285 nucleotides, which includes 3/4 of the fusion gene, was amplified and sequenced using a long-read MinION sequencing method. The sequences were 99.99–100% identical to the corresponding region obtained using the Illumina MiSeq. Phylogenetic analysis using MinION sequences demonstrated that nine virulent NDV from wild birds belonged to sub-genotypes Vc and VIn, and two backyard chicken isolates were of sub-genotype Vc. The sub-genotype Vc viruses had nucleotide sequence identity that ranged from 97.7 to 98% to a virus of the same sub-genotype isolated from a chicken in Mexico in 2010. Three viruses from pigeons had 96.3–98.7% nucleotide identity to sub-genotype VIn pigeon viruses, commonly referred to as pigeon paramyxovirus, isolated in the USA during 2000–2016. This study demonstrates that viruses of sub-genotype Vc are still present in Mexico, and the detection of this sub-genotype in both chickens and wild birds suggests that transmission among these species may represent a biosecurity risk. This is the first detection and complete genome sequencing of genotype VI NDV from Mexico. In addition, the utilization of an optimized long-read sequencing method for rapid virulence and genotype identification using the Oxford nanopore MinION system is demonstrated. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is a nutrient of primary importance in all living systems, and it is especially important in streams and rivers which are sensitive to anthropogenic P inputs and eutrophication. Microbes are accepted as the primary mineralizers and solubilizers of P improving bioavailability for organisms at all trophic levels. Here, we use a genomics approach with metagenome sequencing of 24 temperate streams and rivers representing a total P (TP) gradient to identify relationships between functional genes, functional gene groupings, P, and organisms within the P biogeochemical cycle. Combining information from network analyses, functional groupings, and system P levels, we have constructed a System Relational Overview of Gene Groupings (SROGG) which is a cohesive system level representation of P cycle gene and nutrient relationships. Using SROGG analysis in concert with other statistical approaches, we found that the compositional makeup of P cycle genes is strongly correlated to environmental P whereas absolute abundance of P genes shows no significant correlation to environmental P. We also found orthophosphate (PO43−) to be the dominant factor correlating with system P cycle gene composition with little evidence of a strong organic phosphorous correlation present even in more oligotrophic streams. [ABSTRACT FROM AUTHOR]

In an effort to determine genus- or species-level taxonomic profiles and diversity of bacterial consortia in the Tama River around urban Tokyo, next-generation sequencing technology targeting a 16S ribosomal RNA (rRNA) gene amplicon was employed. Metagenomic analysis performed by an Ion Personal Genome Machine after sequentially filtering samples through 5-, 0.8- and 0.2-μm filters yielded 1.48 Gb of 16S sequences (average 2.38 M reads/sample). The results indicated that half of the bacterial sequences belonged to Proteobacteria, followed by Bacteroidetes, Actinobacteria and Cyanobacteria. Flavobacterium (Bacteroidetes), possibly including a potential fish pathogen, was the most numerous genera in the Tama River metagenome, and accounted for ~ 16% of assigned 16S reads, followed by Mycobacterium. Other dominant bacterial genera including Zoogloea, Sediminibacterium, Hyphomicrobium, Sphingopyxis, Thiothrix and Lysobacter, were thought to be associated with waste water and sludge. MiSeq metagenomic analysis revealed that environmental factors, particularly water temperature, influenced the bacterial composition throughout the year, with a strong negative correlation observed for Proteobacteria and a positive correlation for Bacteroidetes. In terms of bacterial genera, Flavobacterium was positively correlated with temperature, while Polaromonas, Pseudomonas and Bradyrhizobium were negatively correlated with this, suggesting dynamic change in the free-living bacterial population throughout the year and versatile adaptation strategies in relation to environmental factors. [ABSTRACT FROM AUTHOR]

Phosphorus (P) is a key biological element with important and unique biogeochemical cycling in natural ecosystems. Anthropogenic phosphorus inputs have been shown to greatly affect natural ecosystems, and this has been shown to be especially true of freshwater systems. While the importance of microbial communities in the P cycle is widely accepted, the role, composition, and relationship to P of these communities in freshwater systems still hold many secrets. Here, we investigated combined bacterial and archaeal communities utilizing 16S ribosomal RNA (rRNA) gene sequencing and computationally predicted functional metagenomes (PFMs) in 25 streams representing a strong P gradient. We discovered that 16S rRNA community structure and PFMs demonstrate a degree of decoupling between structure and function in the system. While we found that total phosphorus (TP) was correlated to the structure and functional capability of bacterial and archaeal communities in the system, turbidity had a stronger, but largely independent, correlation. At TP levels of approximately 55 μg/L, we see sharp differences in the abundance of numerous ecologically important taxa related to vegetation, agriculture, sediment, and other ecosystem inhabitants. [ABSTRACT FROM AUTHOR]

Low coverage MiSeq genome sequencing and restriction associated DNA sequencing (RADseq) were used to identify nuclear and plastid SNP and INDEL genetic markers in Carapa guianensis. 261 genetic markers including 237 nuclear SNPs, 22 plastid SNPs, and 2 plastid INDELs are described based on 96 genotyped individuals from French Guiana, Brazil, Peru, and Bolivia. The best 117 SNPs for identifying population structure and performing individual assignment are assembled into four multiplexes for MassARRAY genotyping. [ABSTRACT FROM AUTHOR]

Nuclear and plastidial single nucleotide polymorphism (SNP) and INDEL markers were developed using restriction associated DNA sequencing (RADSeq) and low coverage MiSeq genome sequencing for population genetics and timber tracking purposes in the Neotropical timber species Jacaranda copaia. We used 407 nuclear SNPs, 29 chloroplast, and 31 mitochondrial loci to genotype 92 individuals from Brazil, Bolivia, French Guiana, and Peru. Based on high amplification rates and genetic differentiation among populations, 113 nuclear SNPs, 11 chloroplast, and 4 mitochondrial loci were selected, and their use validated for genetic tracking of timber origin. [ABSTRACT FROM AUTHOR]

We developed nuclear and chloroplastic single nucleotide polymorphism (SNP) and INDEL (insertion/deletion) markers using restriction associated DNA sequencing (RADSeq) and low coverage MiSeq genome sequencing to set up a genetic tracking method of the geographical origin of Hymenaea sp. From two initial sets of 358 and 32 loci used to genotype at least 94 individuals, a final set of 75 nSNPs, 50 cpSNPs and 6 INDELs identifying significant population structure was developed. [ABSTRACT FROM AUTHOR]

Amplicon-based next-generation sequencing (NGS) of the 16S ribosomal RNA (16S) regions is a culture-free method used to identify and analyze Procaryota occurring within a given sample. The prokaryotic 16S rRNA gene contains conserved regions and nine variable regions (V1-V9) frequently used for phylogenetic classification of genus or species in diverse microbial populations. This work compares the accuracy and efficacy of two platforms, iSeq and MiSeq from Illumina, used in sequencing 16S rRNA. The most important similarities and differences of 16S microbiome sequencing in 20 fecal rat samples were described. Genetic libraries were prepared according to 16S Metagenomic Sequencing Library Preparation (Illumina) for the V3 and V4 regions of the 16S. The species richness obtained using iSeq technology was lower compared to MiSeq. At the second taxonomy level (L2), the abundance of taxa was comparable for both platforms. At the L7, the taxa abundance was significantly different, and the number of taxa was higher for the MiSeq. The alpha diversity was lower for iSeq than for MiSeq, starting from the order to the species level. The beta diversity estimation revealed statistically significant differences in microbiota diversity starting from the class level to the species level in samples sequenced on two investigated platforms. This work disclosed that the iSeq platform could be used to evaluate the bacterial profile of the samples to characterize the overall profile. The MiSeq System seems to be better for a detailed analysis of the differences in the microbiota composition.• iSeq platform allows to shorten the sequencing time three times compared to the MiSeq.• iSeq can only be used for an initial and quick microbiome assessment.• MiSeq is better for a detailed analysis of the differences in the microbiota composition.Key points: Amplicon-based next-generation sequencing (NGS) of the 16S ribosomal RNA (16S) regions is a culture-free method used to identify and analyze Procaryota occurring within a given sample. The prokaryotic 16S rRNA gene contains conserved regions and nine variable regions (V1-V9) frequently used for phylogenetic classification of genus or species in diverse microbial populations. This work compares the accuracy and efficacy of two platforms, iSeq and MiSeq from Illumina, used in sequencing 16S rRNA. The most important similarities and differences of 16S microbiome sequencing in 20 fecal rat samples were described. Genetic libraries were prepared according to 16S Metagenomic Sequencing Library Preparation (Illumina) for the V3 and V4 regions of the 16S. The species richness obtained using iSeq technology was lower compared to MiSeq. At the second taxonomy level (L2), the abundance of taxa was comparable for both platforms. At the L7, the taxa abundance was significantly different, and the number of taxa was higher for the MiSeq. The alpha diversity was lower for iSeq than for MiSeq, starting from the order to the species level. The beta diversity estimation revealed statistically significant differences in microbiota diversity starting from the class level to the species level in samples sequenced on two investigated platforms. This work disclosed that the iSeq platform could be used to evaluate the bacterial profile of the samples to characterize the overall profile. The MiSeq System seems to be better for a detailed analysis of the differences in the microbiota composition.• iSeq platform allows to shorten the sequencing time three times compared to the MiSeq.• iSeq can only be used for an initial and quick microbiome assessment.• MiSeq is better for a detailed analysis of the differences in the microbiota composition. [ABSTRACT FROM AUTHOR]

Background and aims To assess the impacts of soil microbes and plant genotype on the composition of maize associated bacterial communities. Methods Two genotypes of Brazilian maize were planted indoors on sterile sand, a deep underground subsoil, and a nutrient-rich topsoil from the Amazon jungle (terra preta). DNA was extracted from rhizospheres, phyllospheres, and surface sterilized roots for 16S rDNA fingerprinting and next generation sequencing. Results Neither plant genotype nor soil type appeared to influence bacterial diversity in phyllospheres or endospheres. Rhizospheres showed strikingly similar 16S rDNA ordination of both fingerprinting and sequencing data, with soil type driving grouping patterns and genotype having a significant impact only on sterile sand. Rhizospheres grown in non-sterile soils contained greater bacterial diversity than sterile-sand grown ones, however the dominant OTUs (species of Proteobacteria and Bacteroidetes) were found in all rhizospheres suggesting seeds as a common source of inoculum. Rhizospheres of the commercial hybrid appeared to contain less bacterial diversity than the landrace. Conclusions Maize rhizospheres receive diverse bacteria from soil, are influenced by the genotype or treatment of the seed, and are dominated by species of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. As many dominant 16S rDNA sequences were observed in rhizospheres grown in both sterile and non-sterile substrate, we conclude that the most common bacterial cells in juvenile maize rhizospheres are seed transmitted. [ABSTRACT FROM AUTHOR]

The recent advances in the next-generation sequencing (NGS) technology have enabled fast, accurate, and cost-effective genetic testing. Here, we evaluated the performance of a targeted NGS panel for BRCA1/2 sequencing and confirmed its applicability in routine clinical diagnostics. We tested samples from 88 patients using the TruSeq custom panel (Illumina Inc, USA) and a MiSeq sequencer (Illumina) and compared the results to the outcomes of conventional Sanger sequencing. All 1015 sequence variations identified by Sanger sequencing were detected by NGS, except for one missense variant that might have been missed due to a rare mutation on a primer-binding site. One deletion variation, c.1909 + 12delT of BRCA2, was falsely called in all samples due to a homopolymer error. In addition, seven different single-nucleotide substitutions with low variant frequencies (range: 16.2-33.3 %) were falsely called by NGS. In a separate batch, 10 different false-positive variations were found in five samples. The overall sensitivity and positive predictive value of NGS were estimated to be 99.9 and 87.5 %, respectively. The false-positive results could be excluded by setting quality and alternative allele ratio filters and/or by visual inspection using the IGV software. Targeted NGS panel for BRCA1 and BRCA2 showed an excellent agreement with Sanger sequencing results. We therefore conclude that this NGS panel can be used for routine diagnostic method in a clinical genetic laboratory. [ABSTRACT FROM AUTHOR]

The use of organic wastes in bioremediation of oil-contaminated desert soils has received little attention, although their use is cost-effective. We evaluated the use of spent mushroom compost (SMC), poultry manure (PM), and urea in the stimulation of respiration activities and oil degradation in a polluted desert soil. Moreover, we followed post treatment shifts in bacterial community structure using MiSeq sequencing. The addition of SMC and PM resulted in a significant increase in the evolved CO from 8.7 ± 1.9 to 25.7 ± 1.6 and to 23.4 ± 1.2 mg CO g soil after 96 days of incubation, respectively. In contrast, changes in respiration activities after the addition of urea were insignificant. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that most of the alkanes (C-C) were degraded in all biostimulated soils at a rate of 0.12-0.19 mg g soil day, which was significantly higher than in the untreated soil ( P < 0.05). Bacterial community analysis showed that 87-94 % of total sequences in the original soil belonged to Firmicutes, Actinobacteria, and Proteobacteria. While the relative abundance of Firmicutes remained unchanged after the addition of PM (37-48 % of total sequences), it increased in the urea treatment (44-87 %) and dramatically decreased in the SMC treatment (0.5-4.5 %). The remaining bacterial groups were still detectable after the treatments, although no clear treatment-related shifts could be observed, due to the large difference in the relative abundance of the same bacterial groups among the same replicates. We conclude that the use of organic wastes could be one of the ways of combating petroleum pollution in desert soils. [ABSTRACT FROM AUTHOR]

Recent obesity studies in humans and rodents have suggested that host weight gain is significantly associated with energy harvesting efficiency which is regulated by gut microbiota. Antibiotic growth promoters have been banned as feed additives in many countries. In this study, we aimed to provide knowledge of swine fecal microbiota by analyzing bacterial 16S rRNA gene sequences. Our results showed that swine fecal bacterial composition varied at each growth stage. Bacteroidetes decreased as the swine gained weight and unclassified genera significantly increased at later growth stages. Operational taxonomic unit (OTU) distribution analysis showed that the bacterial community difference was most significant between growers and finishers, while analysis of shared OTUs indicated a greater proportion of common species between growers and finishers. The differential abundance test between growers and finishers detected that nearly half of the species were shared OTUs, suggesting that differential abundance of each bacterial species predominantly controls bacterial community differences. Although functions of these bacteria are yet to be identified, understanding differences in fecal microbiota between each growth stage will provide additional insights for further studies related to swine gut microbiota. [ABSTRACT FROM AUTHOR]

Background: The cystic fibrosis transmembrane conductance regulator ( CFTR) gene, responsible for the development of cystic fibrosis, is known as a pancreatitis susceptibility gene. Direct DNA sequencing of PCR-amplified CFTR gene segments is a first-line method to detect unknown mutations, but it is a tedious and labor-intensive endeavor given the large size of the gene (27 exons, 1,480 amino acids). Next-generation sequencing (NGS) is becoming standardized, reducing the cost of DNA sequencing, and enabling the generation of millions of reads per run. We here report a comprehensive analysis of CFTR variants in Japanese patients with chronic pancreatitis using NGS coupling with target capture. Methods: Exon sequences of the CFTR gene from 193 patients with chronic pancreatitis (121 idiopathic, 46 alcoholic, 17 hereditary, and nine familial) were captured by HaloPlex target enrichment technology, followed by NGS. Results: The sequencing data covered 91.6 % of the coding regions of the CFTR gene by ≥20 reads with a mean read depth of 449. We could identify 12 non-synonymous variants including three novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and seven synonymous variants including three novel ones in the exonic regions. The frequencies of the c.4056G>C (p.Q1352H) and the c.3468G>T (p.L1156F) variants were higher in patients with chronic pancreatitis than those in controls. Conclusions: Target sequence capture combined with NGS is an effective method for the analysis of pancreatitis susceptibility genes. [ABSTRACT FROM AUTHOR]

The cost of DNA sequencing has decreased due to advancements in Next Generation Sequencing. The number of sequences obtained from the Illumina platform is large, use of this platform can reduce costs more than the 454 pyrosequencer. However, the Illumina platform has other challenges, including bioinformatics analysis of large numbers of sequences and the need to reduce erroneous nucleotides generated at the 3′-ends of the sequences. These erroneous sequences can lead to errors in analysis of microbial communities. Therefore, correction of these erroneous sequences is necessary for accurate taxonomic identification. Several studies that have used the Illumina platform to perform metagenomic analyses proposed curating pipelines to increase accuracy. In this study, we evaluated the likelihood of obtaining an erroneous microbial composition using the MiSeq 250 bp paired sequence platform and improved the pipeline to reduce erroneous identifications. We compared different sequencing conditions by varying the percentage of control phiX added, the concentration of the sequencing library, and the 16S rRNA gene target region using a mock community sample composed of known sequences. Our recommended method corrected erroneous nucleotides and improved identification accuracy. Overall, 99.5% of the total reads shared 95% similarity with the corresponding template sequences and 93.6% of the total reads shared over 97% similarity. This indicated that the MiSeq platform can be used to analyze microbial communities at the genus level with high accuracy. The improved analysis method recommended in this study can be applied to amplicon studies in various environments using high-throughput reads generated on the MiSeq platform. [ABSTRACT FROM AUTHOR]

A greenhouse experiment was conducted to investigate the effects of zucchini ( Cucurbita pepo L.), inoculated with the arbuscular mycorrhizal (AM) species Acaulospora laevis, Glomus caledonium, and Glomus mosseae, on the soil bacterial community responsible for Aroclor 1242 dissipation. The dissipation rates of Aroclor 1242 and soil bacteria abundance were much higher with the A. laevis and G. mosseae treatments compared to the non-mycorrhizal control. The biphenyl dioxygenase ( bphA) and Rhodococcus-like 2,3-dihydroxybiphenyl dioxygenase ( bphC) genes were more abundant in AM inoculated soils, suggesting that the bphA and Rhodococcus-like bphC pathways play an important role in Aroclor 1242 dissipation in the mycorrhizosphere. The soil bacterial communities were dominated by classes Betaproteobacteria and Actinobacteria, while the relative proportion of Actinobacteria was significantly ( F = 2.288, P < 0.05) correlated with the PCB congener profile in bulk soil. Our results showed that AM fungi could enhance PCB dissipation by stimulating bph gene abundance and the growth of specific bacterial groups. [ABSTRACT FROM AUTHOR]

Kudzu ( Pueraria montana var. lobata) is a perennial vine native to eastern Asia and an invasive in the United States, representing a significant threat to biodiversity. We characterized 15 polymorphic microsatellite loci in four populations from both the native and invasive ranges of kudzu. The number of alleles per locus ranged from 2 to 8 and observed heterozygosity ranged from 0.0 to 0.9. Consistent with vegetative reproduction, loci were initially not in Hardy-Weinberg equilibrium (HWE) and in linkage disequilibrium but when clones were removed, markers were in HWE and in linkage equilibrium. These loci provide valuable tools to study the diversity and invasion history of an important invasive species. [ABSTRACT FROM AUTHOR]

A new strain of Ageratum latent virus (AGLV) was found in symptomless leaves of Veronica bollonsii, V. breviracemosa and V. dieffenbachii - the native shrubs in New Zealand. The partial genome sequence of the AGLV from V. bollonsii has 85.9–89.1% amino acid similarity to the Australian isolate AGLV-1998. This is the first record of the AGLV in Veronica species and in New Zealand. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GCs) are an important anti-inflammatory drug, used widely, regardless of its side effects. GCs can affect intestinal flora directly or indirectly, though few studies have focused on the changes of gut microbiota composition. In this study, ICR mice were randomly divided into three groups, gavage administration with saline, and different doses of dexamethasone (DEX): 0.1 mg/kg and 1 mg/kg. Five days later, the microbial diversity of the colon contents was analyzed. A significant loss in weight was observed in the DEX1.0 group as compared with the control group (P = 0.011). The gut microbiota richness (ACE, P = 0.01; Chao, P = 0.013) and diversity (Shannon, P = 0.035; Simpson, P = 0.032) were decreased in DEX group. The proportions of genus Butyricicoccus, Oscillibacter, Anaerotruncus, Ruminiclostridium, Ruminococcaceae, and Lachnospiraceae were the most abundant and predominant followed by Lactobacillus, Pseudomonas, and Enterorhabdus. Dex administration led to changes in the liver/body ratio and spleen/body ratio. The results obtained from our study indicate that DEX can decrease the level of WBC and change the structure of the gut microbiota composition; moreover, the results of this study provide new insight into alleviating the clinical side effects of GC therapy.

The microbiology of many cassava products and the wastes generated during the processing have been reported; however, majority of these reports used culture-dependent methods. This has resulted in a dearth of information on the bacterial diversity of cassava peels and peel heaps. Large amounts of cassava peels generated during the processing of cassava root are usually discharged on land or water as wastes and are allowed to rot in the open, especially in some developing countries. Culture-independent methods such as PCR-based amplification and sequencing of 16S rRNA genes, among others have been used in recent times to study the diversity of microbes in different environmental samples. In this study, bacterial isolates were screened for cellulase and xylanase enzyme activities on minimal agar and genomic DNA was isolated from cassava peel samples; metagenomics was carried out using MiSeq 2 × 300 with primers specific for V3–V4 bacterial region. Samples collected from Nigeria (AAG) had more species compared with samples from India (JHA) with 793 and 525 observed OTUs (operational taxonomic units), respectively. Five bacterial isolates from cassava peel-heap samples obtained from Ogbomoso, Nigeria showed no ability to produce cellulase enzyme, seven isolates from the Nigeria samples and three from Jorhat samples were positive for xylanase production; the highest amylase activity was shown by isolate AG18 (10,055 U/mL), while the lowest was recorded for isolate JA2 (2333 U/mL) with a significant difference observed in the amylase activities of isolates (p ≤ 0.05). Comparing the most abundant taxonomy for each of the samples at different taxonomic levels, the most abundant for sample AAG were phylum Firmicutes (42.11%), class Bacilli (41.27%), order Lactobacillales (33.11%), family Acetobacteraceae (31.30%), genus Acetobacter (30.02%) and unclassified species of Acetobacter (29.88%), while sample JHA had Actinobacteria (47.47%) as the highest phylum and class, order Actinomycetales (47.47%), family Brevibacteriaceae (46.97%), genus Brevibacterium (46.97%) and unclassified species of Brevibacterium (46.89%). This study provides an insight into the vast diversity of the bacteria associated with cassava peel heaps and the ability of some of the bacteria to produce selected extracellular enzymes. [ABSTRACT FROM AUTHOR]

There are relatively few molecular resources for amphibians in the tropics where widespread habitat loss, disease, and contamination threaten the world's most diverse assemblages. We used Illumina sequencing to develop microsatellite primers for a dominant amphibian species in Costa Rica that has recently experienced population declines. We characterized 12 polymorphic loci and have provided sequences for over 200 additional primers. These novel molecular resources will be useful for future studies of population genetic structure and may help explain recent declines in one of many species that are decreasing in Central America. [ABSTRACT FROM AUTHOR]