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Forensic microbiology is widely applied to identify people at crime scenes and determine the cause and time of deaths by examination of trace evidence. In particular, identification of bacteria from post-mortem specimens is important because the cause of death could be an infection with clinically significant bacteria that results in a fatal outcome, such as sepsis. The MicroSEQ 500 16S rDNA Bacteria Identification system (MicroSeq ID) from Applied Biosystems is a molecular technique for profiling of bacteria isolated by cultures, based on Sanger sequencing of the bacterial 16S rDNA gene. The MiSeq Illumina platform uses next-generation sequencing (NGS) for metagenomic analysis of 16S rDNA and culture-independent identification of bacteria. In this study, we analysed 65 post-mortem specimens by both methods and compared the workflows involved with regard to methodology, time-efficiency, cost-efficiency, and performance to determine the limitations and benefits of these techniques for forensic application. We found that MiSeq is more time-efficient, and exhibits superior cost-efficiency when more than 30 samples are analysed. In addition, MiSeq has a number of other advantages over MicroSeq ID, including a simpler identification of bacteria that are difficult to culture and use of a larger library of 16S rDNA, allowing more precise identification of bacterial species. [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]

Human mastadenoviruses (HAdVs) are DNA viruses that can cause a wide range of clinical diseases, including gastroenteritis, respiratory illnesses, conjunctivitis, and in more severe cases hepatitis, pancreatitis and disseminated diseases. HAdV infections are generally asymptomatic or self-limiting, but can cause adverse outcomes within vulnerable populations. Since most HAdV serotypes replicate within the human gastrointestinal tract, high levels of HAdV DNA are excreted into wastewater systems. In this study, we identified the genetic diversity of HAdV at a population level using wastewater samples collected from Sydney and Melbourne from 2016 to 2017, with the use of next generation sequencing (NGS) technologies. In addition, HAdV DNA levels were quantified using quantitative polymerase chain reaction (qPCR) based methods to better understand the health risks involved if wastewater contamination occurs. An average of 1.8 × 107 genome copies of HAdV DNA was detected in one litre of wastewater collected in Sydney and Melbourne, over the two-year study period. A total of six major groups of HAdV were identified in wastewater samples using MiSeq, which included 19 different serotypes. Of those, the most prevalent was F41 (83.5%), followed by F40 (11.0%) and A31 (3.7%). In contrast, five groups of HAdV were identified in clinical samples with F41 as the most dominant serotype, (52.5% of gastroenteritis cases), followed by C1 and C2 (each responsible for 15.0%), and B3 was the fourth most common serotype (7.5%). This study demonstrated the practicability of using amplicon based NGS to identify HAdV diversity and quantify HAdV genome levels in environmental water samples, as well as broadening our current understanding of circulating HAdV in the Australian population. Unlabelled Image • Viral concentration method developed for virus detection in wastewater, using MS2 bacteriophage as a process control. • Quantification of total human mastadenovirus genome in wastewater • Identification of human mastadenovirus genetic diversity at a population level using wastewater and clinical samples. [ABSTRACT FROM AUTHOR]

Microbial succession during the initial stages of marine biofouling has been rarely studied, especially in the Arabian Gulf. This study was undertaken to follow temporal shifts in biofouling communities in order to identify primary and secondary colonizers. Quantitative analysis revealed a significant increase in total biomass, coverage of macrofoulers, chlorophyll a concentrations, and bacterial counts with time. The relative abundance of the adnate diatoms increased with time, whereas it decreased in the case of the plocon diatoms. Non-metric multidimensional scaling (NMDS) ordination based on MiSeq data placed the bacterial communities in three distinct clusters, depending on the time of sampling. While the relative abundance of Alphaproteobacteria and Flavobacteriia decreased with time, suggesting their role as primary colonizers, the relative abundance of Actinobacteria and Planctomycetia increased with time, suggesting their role as secondary colonizers. Biofouling is a dynamic process that involves temporal quantitative and qualitative shifts in the micro- and macrofouling communities. [ABSTRACT FROM AUTHOR]

Background Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities (“microbiomes”) in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Methods & Results Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus. However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Discussion Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta diversity differed significantly by month, season, water temperature, and salinity. The 2013–2014 dry season (October–April), was warmer and wetter than historical averages. This may have driven significant differences in beta diversity. Increased nitrogen and phosphorous concentrations were observed in these dry season months, possibly creating favorable bacterial growth conditions. Potentially pathogenic genera were present in the PEI. However their relatively low, non-significant abundance levels highlight their relatively low risk for public health concerns. This study represents the first to sample a large port at this sampling scale and sequencing depth. These data can help establish the inlet microbial community baseline and supplement the vital monitoring of local marine and recreational environments, all the more poignant in context of local reef disease outbreaks and worldwide coral reef collapse in wake of a harsh 2014–16 El Niño event. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Hebei University of Science & Technology is the property of Hebei University of Science & Technology, Journal of Hebei University of Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Henan Agricultural Sciences is the property of Editorial Board of Journal of Henan Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Dumping wastes generated from the oil industry to the local waste disposal facilities remains the most common means of oil waste management. In industrial landfills, the most serious ecological problem is the contamination of soil and local groundwater by landfill leachate. Polycyclic aromatic hydrocarbons (PAHs) represent a hazardous group of harmful chemical compounds that threaten human and ecosystem health. This study focuses on a PAH-contaminated soil from a landfill site in New South Wales, Australia. Soil was exposed to two different bioremediation treatments, natural attenuation and biostimulation using pea straw. The bacterial community composition and diversity of the PAH-contaminated soil were also examined using high throughput Illumina MiSeq sequencing. The results revealed that PAHs were degraded naturally by indigenous microorganisms; however, the addition of plant residues led to enhanced degradation (66.6%) at the beginning of the treatment, although in all treatments a degradation plateau occurred between 43 and 102 days of incubation during bioremediation. Quantitative PCR analysis showed an increase in the number of 16S rRNA and ITS gene copies as well as genes associated with Gram-positive PAH-degrading bacteria in the soil amended with pea straw. Next generation sequencing results and diversity indices revealed that a high proportion of the sequences from all soil samples belonged to the Proteobacteria phyla; gammaprotobacteria was the dominant class in all the investigated samples. Proteobacteria , Bacteroidetes , Firmicutes and Cyanobacteria were the most dominant in the natural attenuation and pea straw treated soil bacterial communities. The largest shift in bacterial communities was in the pea straw amended soils with increased abundances by Day 42 of Pseudomonas , Pseudoxanthomonas , Lewinella and Parvibaculum ; these organisms are all known for their abilities to degrade petroleum hydrocarbons. However by Day 102, these organisms were decreased or not detected in both NA and PS treated soil samples at the end of incubation which may explain the degradation plateau. These findings suggest that Gram-negative PAH-degrading bacteria, mainly Pseudomonas sp., may be the key contributor to PAH-degradation in the landfill-polluted soil. Graphical abstract Unlabelled Image Highlights • Biodegradation rate of PAHs in landfill soil was investigated. • Degradation plateau occurred in day 43 and 102. • PS increased the number of 16S rDNA, ITS and Gram-positive PAH-degrading genes. • PS displayed higher diversity of bacterial species. [ABSTRACT FROM AUTHOR]

Whole genome sequencing of bacterial isolates has become a daily task in many laboratories, generating incredible amounts of data. However, data acquisition is not an end in itself; the goal is to acquire high‐quality data useful for understanding genetic relationships. Having a method that could rapidly determine which of the many available run metrics are the most important indicators of overall run quality and having a way to monitor these during a given sequencing run would be extremely helpful to this effect. Therefore, we compared various run metrics across 486 MiSeq runs, from five different machines. By performing a statistical analysis using principal components analysis and a K‐means clustering algorithm of the metrics, we were able to validate metric comparisons among instruments, allowing for the development of a predictive algorithm, which permits one to observe whether a given MiSeq run has performed adequately. This algorithm is available in an Excel spreadsheet: that is, MiSeq Instrument & Run (In‐Run) Forecast. Our tool can help verify that the quantity/quality of the generated sequencing data consistently meets or exceeds recommended manufacturer expectations. Patterns of deviation from those expectations can be used to assess potential run problems and plan preventative maintenance, which can save valuable time and funding resources. [ABSTRACT FROM AUTHOR]

Highlights • Analysis of cytosine-5 methylation in amplicons from bisulfite-treated RNA. • Filtering of bisulfite artifacts in RNA. • Web-based analysis. Abstract RNA cytosine-5 methylation (m5C) has emerged as a key epitranscriptomic mark, which fulfills multiple roles in structural modulation, stress signaling and the regulation of protein translation. Bisulfite sequencing is currently the most accurate and reliable method to detect m5C marks at nucleotide resolution. Targeted bisulfite sequencing allows m5C detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse m5C marks in DNA bisulfite sequencing. However, these methods are not directly applicable to the analysis of RNA m5C marks, because DNA analysis focuses on CpG methylation, and because artifactual unconversion and misamplification in RNA can obscure actual methylation signals. We describe a pipeline designed specifically for RNA cytosine-5 methylation analysis in targeted bisulfite sequencing experiments. The pipeline is directly applicable to Illumina MiSeq (or equivalent) sequencing datasets using a web interface (https://bisamp.dkfz.de), and is defined by optimized mapping parameters and the application of tailored filters for the removal of artifacts. We provide examples for the application of this pipeline in the unambiguous detection of m5C marks in tRNAs from mouse embryonic stem cells and neuron-differentiated stem cells as well as in 28S rRNA from human fibroblasts. Finally, we also discuss the adaptability of BisAMP to the analysis of DNA methylation. Our pipeline provides an accurate, fast and user-friendly framework for the analysis of cytosine-5 methylation in amplicons from bisulfite-treated RNA. [ABSTRACT FROM AUTHOR]

The Gulf of Oman is becoming increasingly polluted with plastics, hence bioplastics have been considered 'a substitute', although their biodegradability in marine environments has not been well investigated. Most research has been performed on cellulose-based bioplastics, whereas starch-based bioplastics have proven to be a suitable, but less researched, alternative. This study is the first of its kind designed to investigate the degradability of two different types of starch-based bioplastic bags, available in the market and labeled as "biodegradable", in the pelagic and benthic zones of one of the warmest marine environment in the world. Fourier-Transform Infrared Spectroscopy (FTIR) showed a clear reduction in the presence of OH, C H, and C O in the bioplastic bags after 5 weeks of immersion. Thermo-Gravimetric Analysis (TGA) indicated degradation of glycerol, starch, and polyethylene. The biofouling bacterial communities on bioplastic surfaces showed distinct grouping based on the immersion zone. Candidaatus saccharibacteria , Verrucomicrobiae , Acidimicrobiia and Planctomycetia sequences were only detectable on bioplastics in the pelagic zone, whereas Actinomyces , Pseudomonas , Sphingobium and Acinetobacter related sequences were only found on bioplastics in the benthic layer. We conclude that starch-based bioplastics are more readily degradable in the Gulf of Oman than conventional plastics, hence could serve as a better environmentally friendly alternative. [Display omitted] • Starch-based bioplastics were readily degradable in the Gulf of Oman. • TGA analysis indicated degradation of glycerol, starch and PE. • Biofouling communities were habitat- but not plastic-specific. • Candidatus saccharibacteria were enriched bioplastics in the planktonic zone. • Starch-based bioplastics could serve as a good alternative to conventional plastics. [ABSTRACT FROM AUTHOR]

Here we compared microbial results for the same Phosphorus (P) biogeochemical cycle genes from a GeoChip microarray and PICRUSt functional predictions from 16S rRNA data for 20 samples in the four spatially separated Gotjawal forests on Jeju Island in South Korea. The high homogeneity of microbial communities detected at each site allows sites to act as environmental replicates for comparing the two different functional analysis methods. We found that while both methods capture the homogeneity of the system, both differed greatly in the total abundance of genes detected, as well as the diversity of taxa detected. Additionally, we introduce a more comprehensive functional assay that again captures the homogeneity of the system but also captures more extensive community gene and taxonomic information and depth. While both methods have their advantages and limitations, PICRUSt appears better suited to asking questions specifically related to microbial community P as we did here. This comparison of methods makes important distinctions between both the results and the capabilities of each method and can help select the best tool for answering different scientific questions.

Background: Over 700 bacterial species reside in human oral cavity, many of which are associated with local or distant site infections. Extensive characterization of the oral microbiome depends on the technologies used to determine the presence and proportions of specific bacterial species in various oral sites. Objective: The objective of this study was to compare the microbial composition of dental plaque at baseline using Human Oral Microbe Identification Microarray (HOMIM) and Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) technologies, which are based on 16S rRNA. Methods: Dental plaque samples were collected from 96 patients at baseline prior to a dental procedure involving manipulation of gingival tissues. The samples were surveyed for 293 and 597 oral bacterial species via HOMIM and HOMINGS, respectively, based on 16S rRNA gene sequences. We determined the concordance between the two technologies for common species. Genus level analysis was performed using HOMINGS-specific genus identification capabilities. Results: HOMINGS detected twice the number of species in the same dental plaque samples compared to HOMIM. For the species detected by both HOMIM and HOMINGS, there was no difference in relative proportions of overall bacterial composition at the species, genus or phylum levels. Additionally, there was no difference in relative proportion for total species per patient between the two technologies. Conclusion: HOMINGS significantly expanded oral bacterial species identification compared to HOMIM. The genus and species probes, combined in HOMINGS, provided a more comprehensive representation of oral bacterial community, critical for future characterization of oral microbes in distant site infections.

The first mitochondrial genome of Ophiocara porocephala was determined by the combination of nextgeneration sequencing (NGS) and Sanger sequencing methods. A complete circular mitogenome of O. porocephala (16,529 bp) consisted of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and two non-coding regions, including a control region (D-loop) and a light strand origin of replication (OL). Two start codons (ATG and GTG) and four stop codons (TAG, TAA, TA–, and T–) were used in all the PCGs. Except for ND6 and eight transfer RNAs (tRNAs), all the other genes were encoded in the heavy strand. Based on phylogenetic analysis, O. porocephala formed a clade with three other species in the subfamily Butinae, while the other 10 made a subfamily Eleotrinae clade. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genomes of four fish species of the commercially important family Latidae were sequenced using the Illumina MiSeq, thereby significantly increasing the mitogenomic resources for the family. Whole mitogenome-based phylogenetic analysis supports the monophyly of the genus Lates and more generally the family Latidae. The mitogenome sequences from this study will be useful for future assessments of the diversity within and between Lates species and studies of phylogenetic relationships within the diverse and taxonomically challenging perciform fishes.

Antimicrobial resistance (AMR) is one of the most prominent public health threats. AMR genes localized on plasmids can be easily transferred between bacterial isolates by horizontal gene transfer, thereby contributing to the spread of AMR. Next-generation sequencing (NGS) technologies are ideal for the detection of AMR genes; however, reliable reconstruction of plasmids is still a challenge due to large repetitive regions. This study proposes a workflow to reconstruct plasmids with NGS data in view of AMR gene localization, i.e., chromosomal or on a plasmid. Whole-genome and plasmid DNA extraction methods were compared, as were assemblies consisting of short reads (Illumina MiSeq), long reads (Oxford Nanopore Technologies) and a combination of both (hybrid). Furthermore, the added value of conjugation of a plasmid to a known host was evaluated. As a case study, an isolate harboring a large, low-copy mcr-1-carrying plasmid (>200 kb) was used. Hybrid assemblies of NGS data obtained from whole-genome DNA extractions of the original isolates resulted in the most complete reconstruction of plasmids. The optimal workflow was successfully applied to multidrug-resistant Salmonella Kentucky isolates, where the transfer of an ESBL-gene-containing fragment from a plasmid to the chromosome was detected. This study highlights a strategy including wet and dry lab parameters that allows accurate plasmid reconstruction, which will contribute to an improved monitoring of circulating plasmids and the assessment of their risk of transfer.

Aside from two samples collected nearly 50 years ago, little is known about the microbial composition of wind tidal flats in the hypersaline Laguna Madre, Texas. These mats account for ~42% of the lagoon’s area. These microbial communities were sampled at four locations that historically had mats in the Laguna Madre, including Laguna Madre Field Station (LMFS), Nighthawk Bay (NH), and two locations in Kenedy Ranch (KRN and KRS). Amplicon sequencing of 16S genes determined the presence of 51 prokaryotic phyla dominated by Bacteroidota, Chloroflexi, Cyanobacteria, Desulfobacteria, Firmicutes, Halobacteria, and Proteobacteria. The microbial community structure of NH and KR is significantly different to LMFS, in which Bacteroidota and Proteobacteria were most abundant. Twenty-three cyanobacterial taxa were identified via genomic analysis, whereas 45 cyanobacterial taxa were identified using morphological analysis, containing large filamentous forms on the surface, and smaller, motile filamentous and coccoid forms in subsurface mat layers. Sample sites were dominated by species in Oscillatoriaceae (i.e., Lyngbya) and Coleofasciculaceae (i.e., Coleofasciculus). Most cyanobacterial sequences (~35%) could not be assigned to any established taxa at the family/genus level, given the limited knowledge of hypersaline cyanobacteria. A total of 73 cyanobacterial bioactive metabolites were identified using ultra performance liquid chromatography-Orbitrap MS analysis from these commu nities. Laguna Madre seems unique compared to other sabkhas in terms of its microbiology.

Although the human gut microbiome is shaped by factors such as diet, environment, and genetic background, most studies investigating the relationship between ethnicity and microbiota have compared groups living in separate geographical locations. To isolate the effects of ethnicity on microbial diversity by minimizing environmental differences, we selected 143 school children from Han, Tibetan, and Hui populations from the same town on the Qinghai−Tibetan Plateau for fecal microbiome 16S rDNA sequencing. We characterized the diversity, identified signature taxa, and performed correlation analysis between diet and community composition. Firmicutes (47.61%) and Bacteroidetes (38.05%) were dominant phyla among the three ethnic groups; seven genera showed significant differences in relative abundance. Tibetan populations had a higher relative abundance of Oscillibacter and Barnesiella, compared with Han and Hui populations. Alpha diversity analyses (observed species, ACE, and Shannon indices) showed that the Tibetan population had the highest diversity compared to the Hui and Han groups, whereas beta diversity analysis revealed no significant differences between groups. The consumption of grains, milk, eggs, and fruits were positively correlated with specific taxa. Under similar environments and diet, ethnic background significantly contributed to differences in alpha diversity but not beta diversity of gut microbiota.

Introduction: Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer.Methods: Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D2 sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer.Results: Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D2 reads.Conclusion: We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses.

Background Similar to natural rivers, manmade inlets connect inland runoff to the ocean. Port Everglades Inlet (PEI) is a busy cargo and cruise ship port in South Florida, which can act as a source of pollution to surrounding beaches and offshore coral reefs. Understanding the composition and fluctuations of bacterioplankton communities (“microbiomes”) in major port inlets is important due to potential impacts on surrounding environments. We hypothesize seasonal microbial fluctuations, which were profiled by high throughput 16S rRNA amplicon sequencing and analysis. Methods & Results Surface water samples were collected every week for one year. A total of four samples per month, two from each sampling location, were used for statistical analysis creating a high sampling frequency and finer sampling scale than previous inlet microbiome studies. We observed significant differences in community alpha diversity between months and seasons. Analysis of composition of microbiomes (ANCOM) tests were run in QIIME 2 at genus level taxonomic classification to determine which genera were differentially abundant between seasons and months. Beta diversity results yielded significant differences in PEI community composition in regard to month, season, water temperature, and salinity. Analysis of potentially pathogenic genera showed presence of Staphylococcus and Streptococcus. However, statistical analysis indicated that these organisms were not present in significantly high abundances throughout the year or between seasons. Discussion Significant differences in alpha diversity were observed when comparing microbial communities with respect to time. This observation stems from the high community evenness and low community richness in August. This indicates that only a few organisms dominated the community during this month. August had lower than average rainfall levels for a wet season, which may have contributed to less runoff, and fewer bacterial groups introduced into the port surface waters. Bacterioplankton beta diversity differed significantly by month, season, water temperature, and salinity. The 2013–2014 dry season (October–April), was warmer and wetter than historical averages. This may have driven significant differences in beta diversity. Increased nitrogen and phosphorous concentrations were observed in these dry season months, possibly creating favorable bacterial growth conditions. Potentially pathogenic genera were present in the PEI. However their relatively low, non-significant abundance levels highlight their relatively low risk for public health concerns. This study represents the first to sample a large port at this sampling scale and sequencing depth. These data can help establish the inlet microbial community baseline and supplement the vital monitoring of local marine and recreational environments, all the more poignant in context of local reef disease outbreaks and worldwide coral reef collapse in wake of a harsh 2014–16 El Niño event.

The intestinal microbiota has been noted to contribute to obesity. Ursolic acid may have potential in the prevention and treatment of obesity, however, little is known about the underlying mechanism. The aim of the present study was to evaluate the effect of the oral intake of ursolic acid on the intestinal microbiota of rats. The results showed that: (i) ursolic acid decreased the microbial diversity in the proximal intestine while having an opposite effect in the distal intestine; (ii) Fat enhanced the effect of ursolic acid on the microbiota in the proximal intestine while attenuating its effect in the distal intestine; (iii) Ursolic acid inhibited colonization by energy harvest-related microbes; and (iv) Ursolic acid may enhance intestinal health by inhibiting colonization by Proteobacteria. Thus, the intestinal microbiota contributes to the function of ursolic acid as a regulator of the nutritional status.

Shiraz disease (SD) is one of the most destructive viral diseases of grapevines in Australia and is known to cause significant economic loss to local growers. Grapevine virus A (GVA) was reported to be the key pathogen associated with this disease. This study aimed to better understand the diversity of GVA variants both within and between individual SD and grapevine leafroll disease (LRD) affected grapevines located at vineyards in South Australia. Amplicon high throughput sequencing (Amplicon-HTS) combined with median-joining networks (MJNs) was used to analyze the variability in specific gene regions of GVA variants. Several GVA II variant groups contain samples from both vineyards studied, suggesting that these GVA II variants were from a common origin. Variant groups analyzed by MJNs using the overall data set denote that there may be a possible relationship between variant groups of GVA and the geographical location of the grapevines.

Pollen is a vector for diversification, fitness-selection, and transmission of plant genetic material. The extent to which the pollen microbiome may contribute to host diversification is largely unknown, because pollen microbiome diversity within a plant species has not been reported, and studies have been limited to conventional short-read 16S rRNA gene sequencing (e.g., V4-MiSeq) which suffers from poor taxonomic resolution. Here we report the pollen microbiomes of 16 primitive and traditional accessions of maize (corn) selected by indigenous peoples across the Americas, along with the modern U.S. inbred B73. The maize pollen microbiome has not previously been reported. The pollen microbiomes were identified using full-length (FL) 16S rRNA gene PacBio SMRT sequencing compared to V4-MiSeq. The Pan-American maize pollen microbiome encompasses 765 taxa spanning 39 genera and 46 species, including known plant growth promoters, insect-obligates, plant pathogens, nitrogen-fixers and biocontrol agents. Eleven genera and 13 species composed the core microbiome. Of 765 taxa, 63% belonged to only four genera: 28% were Pantoea , 15% were Lactococcus , 11% were Pseudomonas, and 10% were Erwinia . Interestingly, of the 215 Pantoea taxa, 180 belonged to a single species, P. ananatis . Surprisingly, the diversity within P. ananatis ranged nearly 10-fold amongst the maize accessions analyzed (those with ≥3 replicates), despite being grown in a common field. The highest diversity within P. ananatis occurred in accessions that originated near the center of diversity of domesticated maize, with reduced diversity associated with the north-south migration of maize. This sub-species diversity was revealed by FL-PacBio but missed by V4-MiSeq. V4-MiSeq also mis-identified some dominant genera captured by FL-PacBio. The study, though limited to a single season and common field, provides initial evidence that pollen microbiomes reflect evolutionary and migratory relationships of their host plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Khalaf, Shrestha, Reid, McFadyen and Raizada.)

Herpotrichia needle browning (HNB) is a disease that affects several species of fir trees in Europe and North America. HNB was first described by Hartig in 1884, who isolated a fungal pathogenic agent identified as responsible for the disease. This fungus was later named Herpotrichia parasitica but is currently named Nematostoma parasiticum . However, the identity of the pathogens causing HNB is regularly questioned and, to date, the true causal agent of this disease has not been definitely established. The present study aimed to identify the fungal populations present in needles of Christmas fir trees ( Abies balsamea ) and to correlate them with needle health status using robust molecular methods. PCR primers specific to N. parasiticum allowed detection of the presence of this fungus in DNA samples from symptomatic needles. Furthermore, high-throughput sequencing (Illumina MiSeq) clearly showed that N. parasiticum was associated with symptomatic needles. However, high-throughput sequencing results revealed that the presence of other species such as Sydowia polyspora and Rhizoctonia sp. may also correlate with the development of HNB. A diagnostic tool, based on quantitative PCR using a probe, was then developed to detect and quantify N. parasiticum in DNA samples. The efficacy of this molecular approach was validated through the detection of the pathogenic agent in symptomatic needle samples as well as in nonsymptomatic needles collected in trees affected by HNB. In contrast, N. parasiticum could not be found in needles from healthy trees. The present study argues for the importance of N. parasiticum in causing HNB symptoms., Competing Interests: The author(s) declare no conflict of interest.

Background: Even though microsatellite loci frequently have been isolated using recently developed next-generation sequencing (NGS) techniques, this task is still difficult because of the subsequent polymorphism screening requires a substantial amount of time. Selecting appropriate polymorphic microsatellites is a critical issue for ecological and evolutionary studies. However, the extent to which assembly strategy, read length, sequencing depth, and library layout produce a measurable effect on microsatellite marker development remains unclear. Here, we use six frog species for genome skimming and two frog species for transcriptome sequencing to develop microsatellite markers, and investigate the effect of different isolation strategies on the yield of microsatellites. Results: The results revealed that the number of isolated microsatellites increases with increased data quantity and read length. Assembly strategy could influence the yield and the polymorphism of microsatellite development. Larger k-mer sizes produced fewer total number of microsatellite loci, but these loci had a longer repeat length, suggesting greater polymorphism. However, the proportion of each type of nucleotide repeats was not affected; dinucleotide repeats were always the dominant type. Finally, the transcriptomic microsatellites displayed lower levels of polymorphisms and were less abundant than genomic microsatellites, but more likely to be functionally linked loci. Conclusions: These observations provide deep insight into the evolution and distribution of microsatellites and how different isolation strategies affect microsatellite development using NGS. [ABSTRACT FROM AUTHOR]

Rising atmospheric CO2 concentration directly stimulates plant productivity and affects nutrient dynamics in the soil. However, the influence of CO2 enrichment on soil bacterial communities remains elusive, likely due to their complex interactions with a wide range of plant and soil properties. Here, we investigated the bacterial community response to a decade long preindustrial‐to‐future CO2 gradient (250–500 ppm) among three contrasting soil types using 16S rRNA gene amplicon sequencing. In addition, we examined the effect of seasonal variation and plant species composition on bacterial communities. We found that Shannon index (H') and Faith's phylogenetic diversity (PD) did not change in response to the CO2 gradient (R2 = 0.01, p > 0.05). CO2 gradient and season also had a negligible effect on overall community structure, although silty clay soil communities were better structured on a CO2 gradient (p < 0.001) among three soils. Similarly, CO2 gradient had no significant effect on the relative abundance of different phyla. However, we observed soil‐specific variation of CO2 effects in a few individual families. For example, the abundance of Pirellulaceae family decreased linearly with CO2 gradient, but only in sandy loam soils. Conversely, the abundance of Micromonosporaceae and Gaillaceae families increased with CO2 gradient in clay soils. Soil water content (SWC) and nutrient properties were the key environmental constraints shaping bacterial community structure, one manifestation of which was a decline in bacterial diversity with increasing SWC. Furthermore, the impact of plant species composition on community structure was secondary to the strong influence of soil properties. Taken together, our findings indicate that bacterial communities may be largely unresponsive to indirect effects of CO2 enrichment through plants. Instead, bacterial communities are strongly regulated by edaphic conditions, presumably because soil differences create distinct environmental niches for bacteria. We investigated the bacterial community response to a decade‐long preindustrial‐to‐future CO2 gradient (250–500 ppm) among three contrasting soil types using 16S rRNA gene amplicon sequencing. We found that bacterial communities may be largely unresponsive to indirect effects of CO2 enrichment through plants. Instead, bacterial communities are strongly regulated by edaphic conditions, presumably because soil differences create distinct environmental niches for bacteria. [ABSTRACT FROM AUTHOR]

Introduction: Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer. Methods: Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D2 sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer. Results: Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D2 reads. Conclusion: We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses. [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]

Graphical abstract Highlights • We provide a proof of concept for identification of helminth communities within museum samples. • A novel metabarcoding approach amplified helminth DNA from various fixed samples. • Multi-locus metabarcoding provides a more holistic view of the shrew gut helminth community. Abstract Natural history collections spanning multiple decades provide fundamental historical baselines to measure and understand changing biodiversity. New technologies such as next generation DNA sequencing have considerably increased the potential of museum specimens to address significant questions regarding the impact of environmental changes on host and parasite/pathogen dynamics. We developed a new technique to identify intestinal helminth parasites and applied it to shrews (Eulipotyphla: Soricidae) because they are ubiquitous, occupy diverse habitats, and host a diverse and abundant parasite fauna. Notably, we included museum specimens preserved in various ways to explore the efficacy of using metabarcoding analyses that may enable identification of helminth symbiont communities from historical archives. We successfully sequenced the parasite communities (using 12S mtDNA, 16S mtDNA, 28S rDNA) of 23 whole gastrointestinal tracts. All gastrointestinal tracts were obtained from the Museum of Southwestern Biology, USA, and from recent field collections, varying both in time since fixation (ranging from 4 months to 16 years) and preservation method (70% or 95% ethanol stored at room temperature, or flash frozen in liquid nitrogen and stored at −80 °C). Our proof of concept demonstrates the feasibility of applying next generation DNA sequencing techniques to authoritatively identify the parasite/pathogen communities within whole gastrointestinal tracts from museum specimens of varying age and fixation, and the value of future preservation of host-associated whole gastrointestinal tracts in public research archives. This powerful approach facilitates future comparative examinations of the distributions and interactions among multiple associated groups of organisms through time and space. [ABSTRACT FROM AUTHOR]

We investigated the impact of a fermented milk product on gut microbiota and their metabolism in 3 different conditions of the colon with a systemic viewpoint. An in vitro semi-continuous anaerobic cultivation was used to assess the colon compartment-specific influence of fermented milk, followed by a multiomics approach combining 16S rDNA amplicon sequencing and nuclear magnetic resonance (NMR) spectroscopy. The microbiome profiling and metabolomic features were significantly different across three colon compartments and after fermented milk treatment. Integrative correlation analysis indicated that the alteration of butyrate-producing microbiota ( Veillonella, Roseburia, Lachnospira, and Coprococcus ) and some primary metabolites (butyrate, ethanol, lactate, and isobutyrate) in the treatment group had a strong association with the fermented milk microorganisms. Our findings suggested that fermented milk treatment significantly affected microbial population in an in vitro cultivation system as well as the colonic metabolome in different ways in each of colon compartment. [ABSTRACT FROM AUTHOR]

This study evaluates the use of non-pretreated thermo-mechanical pulping (TMP) wastewater as a potential substrate for hydrogen production by dark fermentation. Batch incubations were conducted in a temperature gradient incubator at temperatures ranging from 37 to 80 °C, using an inoculum from a thermophilic, xylose-fed, hydrogen-producing fluidised bed reactor. The aim was to assess the short-term response of the microbial communities to the different temperatures with respect to both hydrogen yield and composition of the active microbial community. High throughput sequencing (MiSeq) of the reversely transcribed 16S rRNA showed that Thermoanaerobacterium sp. dominated the active microbial community at 70 °C, resulting in the highest hydrogen yield of 3.6 (±0.1) mmol H 2  g −1 COD tot supplied. Lower hydrogen yields were obtained at the temperature range from 37 to 65 °C, likely due to consumption of the produced hydrogen by homoacetogenesis. No hydrogen production was detected at temperatures above 70 °C. Thermomechanical pulping wastewaters are released at high temperatures (50–80 °C), and thus dark fermentation at 70 °C could be sustained using the heat produced by the pulp and paper plant itself without any requirement for external heating. [ABSTRACT FROM AUTHOR]

Highlights • Concordance assessment of the Precision ID mtDNA whole genome panel on the Ion S5 and the MiSeq FGx. • Estimation of amplicon haplotypes using read-backed phasing. • Detection and mitigation of the effects of nuclear mitochondrial sequences. Abstract Sequencing whole mitochondrial genomes by capillary electrophoresis is a costly and time/labor-intensive endeavor. Many of the previous Sanger sequencing-based approaches generated amplicons that were several kilobases in length; lengths that are likely not amenable for most forensic applications. However, with the advent of massively parallel sequencing (MPS) short-amplicon multiplexes covering the entire mitochondrial genome can be sequenced relatively easily and rapidly. Recently, the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific by Applied Biosystems™) has been introduced. This panel is composed of 162 amplicons (in two multiplexes) that are considerably smaller in length (∼163bp) and thus are more amenable to analyzing challenged samples. This panel was evaluated on both the Ion S5™ System (Thermo Fisher Scientific) and the MiSeq™ FGx Desktop Sequencer (Illumina). A script was developed to extract phased haplotypes associated with these amplicons. Levels of read-depth were compared across sequencing pools and between sequencing technologies and haplotype concordances were assessed. Given modest thresholds on read depth, the haplotypes identified by either technology were consistent. Nuclear mitochondrial sequences (Numts) were also inferred, and the effect of different mapping strategies commonly used to filter out Numts were contrasted. Some Numts are co-amplified with this amplification kit, and while the choice of reference sequence can mitigate some of these effects, some data from the mitochondrial genome were lost in the process in this study. This study demonstrates that the Ion and MiSeq platforms provide consistent haplotype estimation of the whole mitochondrial genome, thus providing further support for the reliability and validity of the Precision ID mtDNA Whole Genome Panel. [ABSTRACT FROM AUTHOR]

The present study compares the performances of an in-house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS-FLX for detecting and quantifying drug-resistant mutations (DRMs) in the human immunodeficiency virus polymerase gene (reverse transcriptase [RT] and protease [PR]). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS-FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson's analysis showed good concordance between the percentage of drug-resistant variants determined by MiSeq and 454 GS-FLX sequencing (p = .77, P < .0001). The MiSeq platform is as accurate as the 454 GS-FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance. [ABSTRACT FROM AUTHOR]

Abstract: Despite the ever increase in rigorous control and monitoring measures to assure safe food along the entire farm‐to‐fork chain, the past decade has also witnessed an increase in microbial food alerts. Hence, research on food safety and quality remain of utmost importance. Complementary, and at least as important, is the necessity to be able to assess the potential microbial risks along the food chain. Risk assessment relies on sound scientific data. Unfortunately, often, quality data are limited if not lacking. High‐throughput tools such as next‐generation sequencing (NGS) could fill this gap. NGS approaches can be used to generate ample qualitative and quantitative data to be used in the risk assessment process. NGS applications are not new in food microbiology with applications ranging from pathogen detection along the food chain, food epidemiology studies, whole genome analysis of food‐associated microorganisms up to describing complete food microbiomes. Yet, its application in the area of microbial risk assessment is still at an early stage and faces important challenges. The possibilities of NGS for risk assessment are ample, but so are the questions on the subject. One of the major strengths of NGS lies in its capacity to generate a lot of data, but to what extend can this wealth be of use in hazard identification, hazard characterisation and exposure assessment to perform a sound risk characterisation, which in turn will make it possible to take substantiated risk management decisions. [ABSTRACT FROM AUTHOR]

Desert wadis are widespread in the Arabian Peninsula and play a vital role in the ecology of the region; nevertheless, these ecosystems are among the least studied. Various types of microbial mats are predominant in wadis, but information on their bacterial diversity and spatial distribution is very scarce. We investigated bacterial diversity, pigments and lipid composition of ten mats located at the down-, mid- and upstream of a desert wadi in Oman. Direct microscopy revealed the existence of different unicellular and filamentous cyanobacteria, with the dominance of the heterocystous genera Calothrix and Scytonema. The majority of MiSeq 16S rRNA sequences (44-76%) were affiliated to Cyanobacteria and Proteobacteria. While Alphaproteobacteria was the most dominant proteobacterial class (10 to 48% of total sequences), Gamma- and Deltaproteobacteria were subdominant. Cluster analysis showed that the mats’ bacterial communities at the different locations along the wadi were different and shared less than 60% of their operational taxonomic units (OTUs). Chlorophyll a and scytonemin were the most predominant pigments in all mats. Different saturated, branched and mono- and poly-unsaturated fatty acids were detected in all mats, with C16 and C18 compounds as most dominant. The detected pigments and fatty acids indicate a major role of cyanobacteria in the wadi mats and the adaptation of microorganisms therein to the harsh wadi environment. Detection of diadinoxanthin and fucoxanthin confirmed the presence of diatoms. We conclude that microbial mats are important elements in wadi ecosystems and exist in a great variety of structure and community composition. [ABSTRACT FROM AUTHOR]

Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus , Micromonas and Ostreococcus , in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus , Ostreococcus and Micromonas were in the ranges of 42–49%, 35–49% and 13–17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas , was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay. [ABSTRACT FROM AUTHOR]

Since two decades, short tandem repeats (STRs) are the preferred markers for human identification, routinely analysed by fragment length analysis. Here we present a novel set of short hypervariable autosomal microhaplotypes (MH) that have four or more SNPs in a span of less than 70 nucleotides (nt). These MHs display a discriminating power approaching that of STRs and provide a powerful alternative for the analysis;1;is of forensic samples that are problematic when the STR fragment size range exceeds the integrity range of severely degraded DNA or when multiple donors contribute to an evidentiary stain and STR stutter artefacts complicate profile interpretation. MH typing was developed using the power of massively parallel sequencing (MPS) enabling new powerful, fast and efficient SNP-based approaches. MH candidates were obtained from queries in data of the 1000 Genomes, and Genome of the Netherlands (GoNL) projects. Wet-lab analysis of 276 globally dispersed samples and 97 samples of nine large CEPH families assisted locus selection and corroboration of informative value. We infer that MHs represent an alternative marker type with good discriminating power per locus (allowing the use of a limited number of loci), small amplicon sizes and absence of stutter artefacts that can be especially helpful when unbalanced mixed samples are submitted for human identification. [ABSTRACT FROM AUTHOR]

Benthic diatoms are ubiquitous in tidal flats and play major roles in maintaining coastal ecosystems. Spatio-temporal variations in diatom diversity have not been well-studied, mainly because of difficulties in morphological identification and the lack of appropriate genetic tools. To overcome these problems, we used the gene encoding the ribulose bisphosphate carboxylase large-subunit (rbcL) as a molecular marker, and sequenced these genes with the aid of the MiSeq platform. In this manner, we explored the genetic diversity of benthic diatoms in tidal flats of Guenso Bay on the west coast of Korea; differences in the spatial distributions of benthic diatoms were evident. The diatom communities were dominated by Nitzschia, Navicula, and Amphora; their relative distributions were affected by the sand proportion, grain size, and air exposure time. Our results suggest that meta-barcoding of the rbcL gene and next-generation sequencing can be used to explore the diversity of benthic diatoms. [ABSTRACT FROM AUTHOR]

The influence of different inoculum pretreatments (pH and temperature shocks) on mesophilic (37 °C) and thermophilic (55 °C) dark fermentative H 2 production from xylose (50 mM) and, for the first time, on the composition of the active microbial community was evaluated. At 37 °C, an acidic shock (pH 3, 24 h) resulted in the highest yield of 0.8 mol H 2 mol −1 xylose. The H 2 and butyrate yield correlated with the relative abundance of Clostridiaceae in the mesophilic active microbial community, whereas Lactobacillaceae were the most abundant non-hydrogenic competitors according to RNA-based analysis. At 55 °C, Clostridium and Thermoanaerobacterium were linked to H 2 production, but only an alkaline shock (pH 10, 24 h) repressed lactate production, resulting in the highest yield of 1.2 mol H 2 mol −1 xylose. This study showed that pretreatments differentially affect the structure and productivity of the active mesophilic and thermophilic microbial community developed from an inoculum. [ABSTRACT FROM AUTHOR]

Background: Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Results: Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. Conclusions: A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results. [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]

Abstract: Microbial ecology has been profoundly advanced by the ability to profile complex microbial communities by sequencing of marker genes amplified from environmental samples. However, inclusion of appropriate controls is vital to revealing the limitations and biases of this technique. “Mock community” samples, in which the composition and relative abundances of community members are known, are particularly valuable for guiding library preparation and data processing decisions. I generated a set of three mock communities using 19 different fungal taxa and demonstrate their utility by contrasting amplicon sequencing data obtained for the same communities under modifications to PCR conditions during library preparation. Increasing the number of PCR cycles elevated rates of chimera formation, and of errors in the final data set. Extension time during PCR had little impact on chimera formation, error rate or observed community structure. Polymerase fidelity impacted error rates significantly. Despite a high error rate, a master mix optimized to minimize amplification bias yielded profiles that were most similar to the true community structure. Bias against particular taxa differed among ITS1 vs. ITS2 loci. Preclustering nearly identical reads substantially reduced error rates, but did not improve similarity to the expected community structure. Inaccuracies in amplicon sequence‐based estimates of fungal community structure were associated with amplification bias and size selection processes, as well as variable culling rates among reads from different taxa. In some cases, the numerically dominant taxon was completely absent from final data sets, highlighting the need for further methodological improvements to avoid biased observations of community profiles. [ABSTRACT FROM AUTHOR]

While fungi are intimately associated with substrates in freshwater systems, the role of fungi in the open water column is less well defined. Using next generation sequencing of 0.2 μm–1 μm filtered water column samples, we detected abundant and diverse fungal sequences across 25 stream and river sites in the Ozark region of Oklahoma and Arkansas. Fungal communities were only weakly related to stream environmental metrics with the exception of total phosphorus (TP). We infer from our results that TP is acting as a proxy for unique catchment effects. We observed patterns of dominant community taxa at higher taxonomic groupings but lower taxonomic groupings were site specific. OTU functional assignment showed the majority of sequences to be related to plant and animal pathogens, and some saprotrophs. The likely allochthonous origin and strong site specificity of these fungi suggest overlooked dispersal via lotic waterways, which may have important biogeographic consequences for fungi. [ABSTRACT FROM AUTHOR]

Dark fermentative biohydrogen production in a thermophilic, xylose-fed (50 mM) fluidised bed reactor (FBR) was evaluated in the temperature range 55–70 °C with 5-degree increments and compared with a mesophilic FBR operated constantly at 37 °C. A significantly higher (p = 0.05) H 2 yield was obtained in the thermophilic FBR, which stabilised at about 1.2 mol H 2 mol −1 xylose (36% of the theoretical maximum) at 55 and 70 °C, and at 0.8 mol H 2 mol −1 xylose at 60 and 65 °C, compared to the mesophilic FBR (0.5 mol H 2 mol −1 xylose). High-throughput sequencing of the reverse-transcribed 16S rRNA, done for the first time on biohydrogen producing reactors, indicated that Thermoanaerobacterium was the prevalent active microorganism in the thermophilic FBR, regardless of the operating temperature. The active microbial community in the mesophilic FBR was mainly composed of Clostridium and Ruminiclostridium at 37 °C. Thermophilic dark fermentation was shown to be suitable for treatment of high temperature, xylose-containing wastewaters, as it resulted in a higher energy output compared to the mesophilic counterpart. [ABSTRACT FROM AUTHOR]

The use of fertilizers to overcome nutrient constraints is standard practice in Paulownia fortunei plantations to increase stem wood production and tree yields. However, little is known about the responses and important role of the soil bacteria involved in the increases of soil fertility and standing volume with different fertilization. The 16S rRNA was used to compare four different fertilizer treatments for soil bacterial communities in three P. fortunei plantations, in Hunan Province, China. The results showed that the soil bacteria richness was significantly higher in plantations fertilized for ten years than for shorter times. The phyla Acidobacteria and Gemmatimonadetes were more prevalent for ten-year old plantations, and Firmicutes significantly increased with organic manure treatment (denoted as OM; p < 0.05). OM significantly increased the ratio of Bacilli -like sequences in Firmicutes to 87%, 97% and 78% in seven-, nine- and ten-year-old plantations, respectively ( p < 0.05), while control and inorganic fertilizer had no effect. Principal coordinate analysis revealed that the soil bacterial communities in OM were different from the control and inorganic fertilizer in the three different plantations. Canonical correspondence analyses showed that available phosphorus and available magnesium were the most strongly related to bacterial community composition. The addition of OM led to soil being more fertile and an increased standing volume compared with the other fertilizer treatments. Bacilli , the most abundant class of Firmicutes may represent the bacterial type that responded most distinctly to organic manure fertilization and could be highly effective bacteria to increase soil fertility and achieve sustainable development of P. fortunei plantations. [ABSTRACT FROM AUTHOR]

The Israeli Mediterranean ecosystem has a distinctive flora with high levels of plant diversity. Previous studies of soil microbial ecology from the region have paid little attention to the possibility of distinctive soil bacterial communities associated with particular habitats. This study looked at bacterial communities present in the bulk soil from the closed canopy of four indigenous plants – Quercus ithaburensis, Calicotome villosa, Sarcopoterium spinosum , and Rhamnus puncata . Soil DNA was amplified for the 16S rRNA gene and sequenced using MiSeq Illumina platform. Results indicated that the bacterial communities differed between open and closed canopy with no significant difference in community structure within closed canopy, with soil moisture and organic matter content significantly influenced the community. Only the relative abundance of Gemmatimonadetes varied significantly with the open canopy having the highest abundance, and the relative abundance of Spartobacteri a and Saprospirae was unusually high compared to other Mediterranean soils. There was no difference in OTU richness between plant species. Additionally, this study revealed that open canopy community between plants was nested within closed canopy bacterial community implying that open canopy community potentially serves as a source for the closed canopy. It is then possible that the rhizospheric influence extends beyond the root-soil interface and to the surrounding bulk soil. Overall, this study indicates that in Israeli Mediterranean ecosystems native plants extend a selection beyond the rhizosphere, and like other studies from Mediterranean ecosystems pH does not seem to play an integral role in structuring bacterial communities. [ABSTRACT FROM AUTHOR]

The article focuses on the evolution in the field of the study of fungi and other microorganisms. Topics discussed include Estonian forest nursery samples for oomycete diversity, a wide range of rRNA gene regions, and taxonomic richness and composition of fungi in samples sequenced using PacBio technology .