Search

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]

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]

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]

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]

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]

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]

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 Illumina iSeq low-capacity sequencing platform was evaluated for use in foodborne disease surveillance and outbreak detection. The platform produced high quality sequence data comparable to that of the Illumina MiSeq and was cost-effective with fast turn-around time in low sample volume environments. • The Illumina iSeq sequencing platform was evaluated against the Illumina MiSeq. • The sequences generated by the iSeq and MiSeq platforms had similar quality metrics. • The iSeq and MiSeq sequences performed equally well in strain characterization. • The iSeq is a cost-efficient platform choice in low sample volume environment. [ABSTRACT FROM AUTHOR]

With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours. This study aimed to determine the ability of the MinION to metagenomically detect and characterise a virus from an infected mosquito. RNA was extracted from an Aedes notoscriptus mosquito infected with Ross River virus (RRV), converted into cDNA and sequenced on the MinION. Bioinformatic analysis of the MinION reads led to detection of full-length RRV, with reads of up to 2.5 kb contributing to the assembly. The cDNA was also sequenced on the MiSeq sequencer, and both platforms recovered the RRV genome with >98% accuracy. This proof of concept study demonstrates the metagenomic detection of an arbovirus, using the MinION, directly from a mosquito with minimal sample purification. [ABSTRACT FROM AUTHOR]

In this study the plankton diversity in 13 environmental samples from Varna Bay (in the western Black Sea) was analyzed using massively parallel sequencing (MPS). This preliminary study was undertaken to assess the potential of this technology for future implementation in monitoring programs in the Black Sea. Amplicon sequences of the 18S rRNA gene (V4-5 regions) were obtained using the Illumina MiSeq 250PE platform. A total of 1137 operational taxonomic units (OTUs) were obtained among which 242 OTUs with >0.990 BLAST top hit similarity (21.3% of all detected OTUs) closely related to sequences belonging to −protists. A large portion (175 OTUs = 72.3%) was identified at the species levels, including species typical for the Bulgarian Black Sea plankton community, as well as many that haven’t been reported earlier in the Bulgarian Black Sea coast (124 OTUs = 51.2%). Dinoflagellates were represented by the highest species number (77 OTUs comprising 31.8% of protist species), with dominant genera Gyrodinium and Heterocapsa . The present survey revealed the presence of 12 species listed as harmful, some of which have been previously overlooked, such as Cochlodinium polykrikoides , Karenia bicuneiformis , and Karlodinium veneficum . Species identification was possible for 10.3–36.0% of the detected OTUs in the six major supergroups. The frequency in Rhizaria was significantly lower than that in other major groups ( p < 0.05–0.01), implying difficulties in the classification from morphology-based observations. The metagenetic data had an insufficient resolution of the 18S rRNA gene for species identification in many genera. These issues may hamper the implementation of MPS-based surveys for plankton monitoring, especially for detecting harmful algal blooms (HAB). The sequencing technology is steadily improving and it is expected that sequence length and quality issues will be resolved in the near future. The ongoing efforts to register taxonomic information and quality controls in the international nucleotide sequence databases (INSDs) will be essential for improving taxonomic identification power. [ABSTRACT FROM AUTHOR]

As DNA databases continue to grow and international cooperation increases, forensic STR loci have expanded to increase the discriminatory power and inter-database compatibility. Current capillary electrophoresis (CE) and/or massively parallel sequencing (MPS)-based commercial STR analysis systems reflect such changing trends of expanding STR loci. Due to the general gains of larger multiplexing and the detection of sequence variation, the application of MPS technology to STR analysis has further improved discrimination and is expected to aid in mixture interpretation by increasing the effective number of alleles. However, high-throughput analysis has rarely been reported for forensic DNA databasing. In this study, we present the sequencing results from 250 Korean samples at 23 commonly used STR loci (D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045) using an in-house assay designed for MPS. All amplicons in the multiplex exhibited a size range of 77 to 217 base pairs, and the barcoded library for the MPS run was easily prepared using a PCR-based library preparation method followed by sequencing on a MiSeq System (Illumina). We compared the STR genotyping results with those obtained using CE and scrutinized the sequence variations in both the targeted STR and flanking regions. MPS results of 23 autosomal STRs were 99.97% concordant with those of CE results. D12S391 and D21S11 exhibited, respectively, the highest number of alleles and genotypes by the MPS analysis. Single nucleotide polymorphisms and insertion and deletions (Indels) were observed in the flanking regions of D1S1656, D2S441, D5S818, D7S820, D13S317, D16S539, D21S11, and Penta D. Consequently, an MPS analysis of an expanded set of STRs, as demonstrated in the population statistics of a Korean population, will be of great practical use in forensic genetics. [ABSTRACT FROM AUTHOR]

Municipal solid waste (MSW) landfills are the most prevalent waste disposal method and constitute one of the largest sources of anthropogenic methane emissions in the world. Microbial activities in disposed waste play a crucial role in greenhouse gas emissions; however, only a few studies have examined metagenomic microbial profiles in landfills. Here, the MiSeq high-throughput sequencing method was applied for the first time to examine microbial diversity of the cover soil and stored waste located at different depths (0–150 cm) in a typical MSW landfill in Yangzhou City, East China. The abundance of microorganisms in the cover soil (0–30 cm) was the lowest among all samples, whereas that in stored waste decreased from the top to the middle layer (30–90 cm) and then increased from the middle to the bottom layer (90–150 cm). In total, 14 phyla and 18 genera were found in the landfill. A microbial diversity analysis showed that Firmicutes , Proteobacteria , and Bacteroidetes were the dominant phyla, whereas Halanaerobium , Methylohalobius , Syntrophomonas , Fastidiosipila , and Spirochaeta were the dominant genera. Methylohalobius (methanotrophs) was more abundant in the cover layers of soil than in stored waste, whereas Syntrophomonas and Fastidiosipila , which affect methane production, were more abundant in the middle to bottom layers (90–150 cm) in stored waste. A canonical correlation analysis showed that microbial diversity in the landfill was most strongly correlated with the conductivity, organic matter, and moisture content of the stored waste. [ABSTRACT FROM AUTHOR]

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements. [ABSTRACT FROM AUTHOR]

Rumen houses a plethora of symbiotic microorganisms empowering the host to hydrolyze plant lignocellulose. In this study, NGS based metagenomic approach coupled with bioinformatic analysis was employed to gain an insight into the deconstruction of lignocellulose by carbohydrate-active enzymes (CAZymes) in Indian crossbred Holstein-Friesian cattle. Cattle rumen metagenomic DNA was sequenced using Illumina-MiSeq and 1.9 gigabases of data generated with an average read length of 871 bp. Analysis of the assembled sequences by Pfam-based Carbohydrate-active enzyme Analysis Toolkit identified 17,164 putative protein-encoding CAZymes belonging to different families of glycoside hydrolases (7574), glycosyltransferases (5185), carbohydrate-binding modules (2418), carbohydrate esterases (1516), auxiliary activities (434) and polysaccharide lyases (37). Phylogenetic analysis of putative CAZymes revealed that a significant proportion of CAZymes were contributed by bacteria belonging to the phylum Bacteroidetes (40%), Firmicutes (30%) and Proteobacteria (10%). The comparative analysis of HF cross rumen metagenome with other herbivore metagenomes indicated that Indian crossbred cattle rumen is endowed with a battery of CAZymes that may play a central role in lignocellulose deconstruction. The extensive catalog of enzymes reported in our study that hydrolyzes plant lignocellulose biomass, can be further explored for the better feed utilization in ruminants and also for different industrial applications. [ABSTRACT FROM AUTHOR]

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements. [ABSTRACT FROM AUTHOR]

Coexistence of heavy metals and organic contaminants in coastal ecosystems may lead to complicated circumstances in ecotoxicological assessment for biological communities due to potential interactions of contaminants. Consequences of metals and polycyclic aromatic hydrocarbons (PAHs) co-contamination on coastal marine microbes at the community level were paid less attention. We chose cadmium (Cd) and phenanthrene (PHE) as representatives of metals and PAHs, respectively, and mimicked contaminations using coastal water microcosms spiked with Cd (1 mg/L), PHE (1 mg/L), and their mixture over two weeks. 16S rRNA gene amplicon sequencing was used to compare individual and cumulative effects of Cd and PHE on temporal succession of bacterioplankton communities. Although we found dramatic impacts of dimethylsulfoxide (DMSO, used as a carrier solvent for PHE) on bacterial α-diversity and composition, the individual and cumulative effects of Cd and PHE on bacterial α-diversity were temporally variable showing an antagonistic pattern at early stage in the presence of DMSO. Temporal succession of bacterial community composition (BCC) was associated with temporal variability of water physicochemical parameters, each of which explained more variation in BCC than two target contaminants did. However, Cd, PHE, and their mixture distinctly altered the successional trajectories of BCC, while only the effect of Cd was retained at the end of experiment, suggesting certain resilience in BCC after the complete dissipation of PHE along the temporal trajectory. Moreover, bacterial assemblages at the genus level associated with the target contaminants were highly time-dependent and more unpredictable in the co-contamination group, in which some genera possessing hydrocarbon-degrading members might contribute to PHE degradation. These results provide preliminary insights into how co-exposure of Cd and PHE phylogenetically alters successional trajectories of bacterioplankton communities in the manipulated coastal water microcosms. [ABSTRACT FROM AUTHOR]

In order to contribute to the description of sourdough LAB composition, MiSeq sequencing and qPCR methods were performed in association with cultural methods. A panel of 16 French organic bakers and farmer-bakers were selected for this work. The lactic acid bacteria (LAB) diversity of their organic sourdoughs was investigated quantitatively and qualitatively combining ( i ) Lactobacillus sanfranciscensis -specific qPCR, ( ii ) global sequencing with MiSeq Illumina technology and ( iii ) molecular isolates identification. In addition, LAB and yeast enumeration, pH, Total Titratable Acidity, organic acids and bread specific volume were analyzed. Microbial and physico-chemical data were statistically treated by Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC). Total yeast counts were 6 log 10 to 7.6 log 10 CFU/g while LAB counts varied from 7.2 log 10 to 9.6 log 10 CFU/g. Values obtained by L. sanfranciscensis -specific qPCR were estimated between 7.2 and 10.3 log 10 CFU/g, except for one sample at 4.4 log 10 CFU/g. HAC and PCA clustered the sixteen sourdoughs into three classes described by their variables but without links to bakers' practices. L. sanfranciscensis was the dominant species in 13 of the 16 sourdoughs analyzed by Next Generation Sequencing (NGS), by the culture dependent method this species was dominant only in only 10 samples. Based on isolates identification, LAB diversity was higher for 7 sourdoughs with the recovery of L. curvatus , L. brevis , L. heilongjiangensis , L. xiangfangensis , L. koreensis , L. pontis , Weissella sp. and Pediococcus pentosaceus , as the most representative species. L. koreensis , L. heilongjiangensis and L. xiangfangensis were identified in traditional Asian food and here for the first time as dominant in organic sourdough. This study highlighted that L. sanfranciscensis was not the major species in 6/16 sourdough samples and that a relatively high LAB diversity can be observed in French organic sourdough. [ABSTRACT FROM AUTHOR]

Illumina library preparation methods for ultra-low input amounts were compared using genomic DNA from two foodborne parasites ( Angiostrongylus cantonensis and Cyclospora cayetanensis ) as examples. The Ovation Ultralow method resulted in libraries with the highest concentration and produced quality sequencing data, even when the input DNA was in the picogram range. [ABSTRACT FROM AUTHOR]

Background Cystic fibrosis (CF) is an autosomal recessive disease characterized by recurrent lung infections. Studies of the lung microbiome have shown an association between decreasing diversity and progressive disease. 454 pyrosequencing has frequently been used to study the lung microbiome in CF, but will no longer be supported. We sought to identify the benefits and drawbacks of using two state-of-the-art next generation sequencing (NGS) platforms, MiSeq and PacBio RSII, to characterize the CF lung microbiome. Each has its advantages and limitations. Methods Twelve samples of extracted bacterial DNA were sequenced on both MiSeq and PacBio NGS platforms. DNA was amplified for the V4 region of the 16S rRNA gene and libraries were sequenced on the MiSeq sequencing platform, while the full 16S rRNA gene was sequenced on the PacBio RSII sequencing platform. Raw FASTQ files generated by the MiSeq and PacBio platforms were processed in mothur v1.35.1. Results There was extreme discordance in alpha-diversity of the CF lung microbiome when using the two platforms. Because of its depth of coverage, sequencing of the 16S rRNA V4 gene region using MiSeq allowed for the observation of many more operational taxonomic units (OTUs) and higher Chao1 and Shannon indices than the PacBio RSII. Interestingly, several patients in our cohort had Escherichia , an unusual pathogen in CF. Also, likely because of its coverage of the complete 16S rRNA gene, only PacBio RSII was able to identify Burkholderia , an important CF pathogen. Conclusion When comparing microbiome diversity in clinical samples from CF patients using 16S sequences, MiSeq and PacBio NGS platforms may generate different results in microbial community composition and structure. It may be necessary to use different platforms when trying to correctly identify dominant pathogens versus measuring alpha-diversity estimates, and it would be important to use the same platform for comparisons to minimize errors in interpretation. [ABSTRACT FROM AUTHOR]

Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6 μM and when yeast densities ranged between 10 6 to 10 7 CFU/mL and bacterial densities were approximately 10 8 CFU/mL. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic pathogen which infects cystic fibrosis and cancer patients with compromised immune systems. LasR is a master regulator which controls the virulence of P. aeruginosa in response to bacterial cell-density and host signals. During infection, lasR is frequently mutated, conferring P. aeruginosa a growth advantage in hosts and enhances resistance to widely used antibiotics. However, the mechanistic basis of lasR mutation is not well understood. We have tested here the hypothesis that transcription strength is a contributory determinant of lasR mutagenesis. P. aeruginosa strains with different lasR transcription strengths were therefore engineered and the lasR mutations were monitored unbiasedly using next-generation sequencing technology. Our results suggest that the strength of transcription could be one of the deterministic factors that drive the mutagenesis of lasR in P. aeruginosa , shedding new insights into bacterial infection and antibiotic resistance. [ABSTRACT FROM AUTHOR]

Background In-vitro gut fermentation systems provide suitable models for studying gut microbiota composition and functionality. However, such methods depend on the availability of donors and the assumption of reproducibility between microbial communities before experimental treatments commence. The aim of this study was to develop a frozen standardised inoculum (FSI) which minimizes inter-individual variation and to determine its stability over time using culture-dependent and culture-independent techniques. Results A method for the preparation difference of a FSI is described which involves pooling the faecal samples, centrifugation and pelleting of the cell biomass and finally homogenising the cell pellets with phosphate buffer and glycerol. Using this approach, no significant difference in total anaerobe cell viability was observed between the fresh standardised inoculum (before freezing) and the 12 days post freezing FSI. Moreover, Quantitative PCR revealed no significant alterations in the estimated bacterial numbers in the FSI preparations for any of the phyla. MiSeq sequencing revealed minute differences in the relative abundance at phylum, family and genus levels between the FSI preparations. Differences in the microbiota denoted as significant were limited between preparations in the majority of cases to changes in percentage relative abundance of ± 0.5%. The independently prepared FSIs revealed a high degree of reproducibility in terms of microbial composition between the three preparations. Conclusions This study provides a method to produce a standardised human faecal inoculum suitable for freezing. Based on culture-dependent and independent analysis, the method ensures a degree of reproducibility between preparations by lessening the effect of inter-individual variation among the donors, thereby making the system more suitable for the accurate interpretation of the effects of experimental treatments. [ABSTRACT FROM AUTHOR]

Current forensic DNA analysis predominantly involves identification of human donors by analysis of short tandem repeats (STRs) using Capillary Electrophoresis (CE). Recent developments in Massively Parallel Sequencing (MPS) technologies offer new possibilities in analysis of STRs since they might overcome some of the limitations of CE analysis. In this study 17 STRs and Amelogenin were sequenced in high coverage using a prototype version of the Promega PowerSeq™ system for 297 population samples from the Netherlands, Nepal, Bhutan and Central African Pygmies. In addition, 45 two-person mixtures with different minor contributions down to 1% were analysed to investigate the performance of this system for mixed samples. Regarding fragment length, complete concordance between the MPS and CE-based data was found, marking the reliability of MPS PowerSeq™ system. As expected, MPS presented a broader allele range and higher power of discrimination and exclusion rate. The high coverage sequencing data were used to determine stutter characteristics for all loci and stutter ratios were compared to CE data. The separation of alleles with the same length but exhibiting different stutter ratios lowers the overall variation in stutter ratio and helps in differentiation of stutters from genuine alleles in mixed samples. All alleles of the minor contributors were detected in the sequence reads even for the 1% contributions, but analysis of mixtures below 5% without prior information of the mixture ratio is complicated by PCR and sequencing artefacts. [ABSTRACT FROM AUTHOR]

Next generation sequencing technologies have vastly changed the approach of sequencing of the 16S rRNA gene for studies in microbial ecology. Three distinct technologies are available for large-scale 16S sequencing. All three are subject to biases introduced by sequencing error rates, amplification primer selection, and read length, which can affect the apparent microbial community. In this study, we compared short read 16S rRNA variable regions, V1-V3, with that of near-full length 16S regions, V1-V8, using highly diverse steer rumen microbial communities, in order to examine the impact of technology selection on phylogenetic profiles. Short paired-end reads from the Illumina MiSeq platform were used to generate V1-V3 sequence, while long "circular consensus" reads from the Pacific Biosciences RSII instrument were used to generate V1-V8 data. The two platforms revealed similar microbial operational taxonomic units (OTUs), as well as similar species richness, Good's coverage, and Shannon diversity metrics. However, the V1-V8 amplified ruminal community resulted in significant increases in several orders of taxa, such as phyla Proteobacteria and Verrucomicrobia ( P < 0.05). Taxonomic classification accuracy was also greater in the near full-length read. UniFrac distance matrices using jackknifed UPGMA clustering also noted differences between the communities. These data support the consensus that longer reads result in a finer phylogenetic resolution that may not be achieved by shorter 16S rRNA gene fragments. Our work on the cattle rumen bacterial community demonstrates that utilizing near full-length 16S reads may be useful in conducting a more thorough study, or for developing a niche-specific database to use in analyzing data from shorter read technologies when budgetary constraints preclude use of near-full length 16S sequencing. [ABSTRACT FROM AUTHOR]

The low number and activity of hydrocarbon-degrading bacteria and the low solubility and availability of hydrocarbons hamper bioremediation of oil-contaminated soils in arid deserts, thus bioremediation treatments that circumvent these limitations are required. We tested the effect of Ethylenediaminetetraacetic acid (EDTA) addition, at different concentrations (i.e. 0.1, 1 and 10 mM), on bacterial respiration and biodegradation of Arabian light oil in bioaugmented (i.e. with the addition of exogenous alkane-degrading consortium) and non-bioaugmented oil-contaminated desert soils. Post-treatment shifts in the soils' bacterial community structure were monitored using MiSeq sequencing. Bacterial respiration, indicated by the amount of evolved CO 2 , was highest at 10 mM EDTA in bioaugmented and non-bioaugmented soils, reaching an amount of 2.2 ± 0.08 and 1.6 ± 0.02 mg-CO 2 g −1 after 14 days of incubation, respectively. GC–MS revealed that 91.5% of the C 14 –C 30 alkanes were degraded after 42 days when 10 mM EDTA and the bacterial consortium were added together. MiSeq sequencing showed that 78–91% of retrieved sequences in the original soil belonged to Deinococci , Alphaproteobacteria , Gammaproteobacteia and Bacilli . The same bacterial classes were detected in the 10 mM EDTA-treated soils, however with slight differences in their relative abundances. In the bioaugmented soils, only Alcanivorax sp. MH3 and Parvibaculum sp. MH21 from the exogenous bacterial consortium could survive until the end of the experiment. We conclude that the addition of EDTA at appropriate concentrations could facilitate biodegradation processes by increasing hydrocarbon availability to microbes. The addition of exogenous oil-degrading bacteria along with EDTA could serve as an ideal solution for the decontamination of oil-contaminated desert soils. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin ( Grampus griseus ) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1–V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii . Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations. [ABSTRACT FROM AUTHOR]

Objectives In recent years, next-generation sequencing (NGS) technologies, which enable high throughput sample processing at relatively lower costs, are adopted in both research and clinical settings. A multiplex PCR-based NGS assay to identify mutations in the ATP7B gene for routine molecular diagnosis of Wilson disease was evaluated in comparison with the gold standard direct Sanger sequencing. Design and methods Five multiplex PCRs to amplify the partial promoter, 5′ untranslated and the entire coding regions of the ATP7B gene were designed. Indexed paired-end libraries were generated from the pooled amplicons using Nextera XT DNA Sample Preparation Kit and subjected to NGS on the MiSeq platform. DNA from the peripheral blood of 12 patients with Wilson disease, 2 B-lymphocyte cell lines and 3 external quality assurance samples were sequenced by the MiSeq and Sanger sequencing. Results Complete coverage was achieved across the targeted bases without any drop-out sequences. The observed read depth in a single run with 20 samples was > 100X. Comparison of the NGS results against Sanger sequencing data on a panel of clinical specimens, cell lines and European Molecular Genetics Quality Networks (EMQN) quality assurance samples showed 100% concordance in identifying pathogenic mutations. Conclusion With the capability of generating relatively higher throughput in a short time period, the NGS assay is a viable alternative to Sanger sequencing for detecting ATP7B mutations causally linked to Wilson disease in the clinical diagnostic laboratory. [ABSTRACT FROM AUTHOR]

Abstract Microbial alpha-diversity, a fundamental ecological concept, is often determined using multiplexed amplicon sequencing approaches. Here we show, using standardized controls and 1879 soil samples, across 21 illumina MiSEQ high throughput sequencing (HTS) runs, that sample alpha-diversity estimates are highly dependent upon the overall diversity of all samples within the sequencing run, a likely result of within sequencing run cross-talk. Cross-talk, therefore, has grave implications for alpha-diversity interpretation in mulitplexed sequencing studies. Highlights • Microbial alpha-diversity, a fundamental ecological concept, is often determined using multiplexed amplicon sequencing approaches. • Alpha-diversity is highly dependent upon the overall diversity of all samples within the sequencing run. [ABSTRACT FROM AUTHOR]

This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms – ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing. [ABSTRACT FROM AUTHOR]

Capillary electrophoresis (CE) and multiplex amplification with fluorescent tagging have been routinely used for STR typing in forensic genetics. However, CE-based methods restrict the number of markers that can be multiplexed simultaneously and cannot detect any intra-repeat variations within STRs. Several studies already have indicated that massively parallel sequencing (MPS) may be another potential technology for STR typing. In this study, the prototype PowerSeq™ Auto System (Promega) containing the 23 STR loci and amelogenin was evaluated using Illumina MiSeq. Results showed that single source complete profiles could be obtained using as little as 62 pg of input DNA. The reproducibility study showed that the profiles generated were consistent among multiple typing experiments for a given individual. The mixture study indicated that partial STR profiles of the minor contributor could be detected up to 19:1 mixture. The mock forensic casework study showed that full or partial profiles could be obtained from different types of single source and mixture samples. These studies indicate that the PowerSeq Auto System and the Illumina MiSeq can generate concordant results with current CE-based methods. In addition, MPS-based systems can facilitate mixture deconvolution with the detection of intra-repeat variations within length-based STR alleles. [ABSTRACT FROM AUTHOR]

Biological technology used to treat flue gas is useful to replace conventional treatment, but there is sulfide inhibition. However, no sulfide toxicity effect was observed in haloalkaliphilic bioreactors. The performance of the ethanol-fed bioreactor was better than that of lactate-, glucose-, and formate-fed bioreactor, respectively. To support this result strongly, Illumina MiSeq paired-end sequencing of 16S rRNA gene was applied to investigate the bacterial communities. A total of 389,971 effective sequences were obtained and all of them were assigned to 10,220 operational taxonomic units (OTUs) at a 97% similarity. Bacterial communities in the glucose-fed bioreactor showed the greatest richness and evenness. The highest relative abundance of sulfate-reducing bacteria (SRB) was found in the ethanol-fed bioreactor, which can explain why the performance of the ethanol-fed bioreactor was the best. Different types of SRB, sulfur-oxidizing bacteria, and sulfur-reducing bacteria were detected, indicating that sulfur may be cycled among these microorganisms. Because high-throughput 16S rRNA gene paired-end sequencing has improved resolution of bacterial community analysis, many rare microorganisms were detected, such as Halanaerobium , Halothiobacillus , Desulfonatronum , Syntrophobacter , and Fusibacter . 16S rRNA gene sequencing of these bacteria would provide more functional and phylogenetic information about the bacterial communities. [ABSTRACT FROM AUTHOR]

Sequence-independent methods for viral discovery have been widely used for whole genome sequencing of viruses. Different protocols for viral enrichment, library preparation and sequencing have increasingly been more available and at lower costs. However, no study to date has focused on optimization of viral sample preparation for commercial deep sequencing. Accordingly, the aim of the present study was to evaluate an In-House enzymatic protocol for double-stranded DNA (dsDNA) synthesis and also compare the use of a commercially available kit protocol (Nextera XT, Illumina Inc, San Diego, CA, USA) and its combination with a library quantitation kit (Kapa, Kapa Biosystems, Wilmington, MA, USA) for deep sequencing (Illumina Miseq). Two RNA viruses (canine distemper virus and dengue virus) and one ssDNA virus (porcine circovirus type 2) were tested with the optimized protocols. The tested method for dsDNA synthesis has shown satisfactory results and may be used in laboratory setting, particularly when enzymes are already available. Library preparation combining commercial kits (Nextera XT and Kapa) has yielded more reads and genome coverage, probably due to a lack of small fragment recovering at the normalization step of Nextera XT. In addition, libraries may be diluted or concentrated to provide increase on genome coverage with Kapa quantitation. [ABSTRACT FROM AUTHOR]

Routine use of massively parallel sequencing (MPS) for forensic genomics is on the horizon. The last few years, several algorithms and workflows have been developed to analyze forensic MPS data. However, none have yet been tailored to the needs of the forensic analyst who does not possess an extensive bioinformatics background. We developed our previously published forensic MPS data analysis framework MyFLq (My-Forensic-Loci-queries) into an open-source, user-friendly, web-based application. It can be installed as a standalone web application, or run directly from the Illumina BaseSpace environment. In the former, laboratories can keep their data on-site, while in the latter, data from forensic samples that are sequenced on an Illumina sequencer can be uploaded to Basespace during acquisition, and can subsequently be analyzed using the published MyFLq BaseSpace application. Additional features were implemented such as an interactive graphical report of the results, an interactive threshold selection bar, and an allele length-based analysis in addition to the sequenced-based analysis. Practical use of the application is demonstrated through the analysis of four 16-plex short tandem repeat (STR) samples, showing the complementarity between the sequence- and length-based analysis of the same MPS data. [ABSTRACT FROM AUTHOR]

The development of molecular tools to detect and report mitochondrial DNA (mtDNA) heteroplasmy will increase the discrimination potential of the testing method when applied to forensic cases. The inherent limitations of the current state-of-the-art, Sanger-based sequencing, including constrictions in speed, throughput, and resolution, have hindered progress in this area. With the advent of next-generation sequencing (NGS) approaches, it is now possible to clearly identify heteroplasmic variants, and at a much lower level than previously possible. However, in order to bring these approaches into forensic laboratories and subsequently as accepted scientific information in a court of law, validated methods will be required to produce and analyze NGS data. We report here on the development of an optimized approach to NGS analysis for the mtDNA genome (mtgenome) using the Illumina MiSeq instrument. This optimized protocol allows for the production of more than 5 gigabases of mtDNA sequence per run, sufficient for detection and reliable reporting of minor heteroplasmic variants down to approximately 0.5–1.0% when multiplexing twelve samples. Depending on sample throughput needs, sequence coverage rates can be set at various levels, but were optimized here for at least 5000 reads. In addition, analysis parameters are provided for a commercially available software package that identify the highest quality sequencing reads and effectively filter out sequencing-based noise. With this method it will be possible to measure the rates of low-level heteroplasmy across the mtgenome, evaluate the transmission of heteroplasmy between the generations of maternal lineages, and assess the drift of variant sequences between different tissue types within an individual. [ABSTRACT FROM AUTHOR]

Abstract: The salmon louse Caligus rogercresseyi is the dominant ectoparasite species affecting the salmon aquaculture industry in the Southern hemisphere, and it is currently the main cause for economic losses in Chilean aquaculture. However, despite the great concern over Caligus infestations, genomic information on this louse is still scarce, even while the need to develop high-resolution molecular markers is growing. This study provides the first deep transcriptome survey to identify thousands of SNP markers from C. rogercresseyi, with a total of 69,466 SNPs identified using the MiSeq platform (Illumina®), 30,605 (52%) of which were found in contigs successfully annotated against known protein databases. Furthermore, in silico gene expression profiles associated with SNP variants were evaluated, and the results evidenced a wide array of genes that were down- and upregulated throughout the developmental stages of C. rogercresseyi. Interestingly, putative KEGG pathways involved in resistance to antiparasitic agents were also identified, where ten pathways were associated with the nervous system and one was related to ABC transporters. Taken together, this information could be highly useful for investigating the molecular underpinnings involved in the susceptibility or resistance of salmon lice to chemical treatments. [Copyright &y& Elsevier]

Abstract: Forensic scientists are currently investigating how to transition from capillary electrophoresis (CE) to massive parallel sequencing (MPS) for analysis of forensic DNA profiles. MPS offers several advantages over CE such as virtually unlimited multiplexy of loci, combining both short tandem repeat (STR) and single nucleotide polymorphism (SNP) loci, small amplicons without constraints of size separation, more discrimination power, deep mixture resolution and sample multiplexing. We present our bioinformatic framework My-Forensic-Loci-queries (MyFLq) for analysis of MPS forensic data. For allele calling, the framework uses a MySQL reference allele database with automatically determined regions of interest (ROIs) by a generic maximal flanking algorithm which makes it possible to use any STR or SNP forensic locus. Python scripts were designed to automatically make allele calls starting from raw MPS data. We also present a method to assess the usefulness and overall performance of a forensic locus with respect to MPS, as well as methods to estimate whether an unknown allele, which sequence is not present in the MySQL database, is in fact a new allele or a sequencing error. The MyFLq framework was applied to an Illumina MiSeq dataset of a forensic Illumina amplicon library, generated from multilocus STR polymerase chain reaction (PCR) on both single contributor samples and multiple person DNA mixtures. Although the multilocus PCR was not yet optimized for MPS in terms of amplicon length or locus selection, the results show excellent results for most loci. The results show a high signal-to-noise ratio, correct allele calls, and a low limit of detection for minor DNA contributors in mixed DNA samples. Technically, forensic MPS affords great promise for routine implementation in forensic genomics. The method is also applicable to adjacent disciplines such as molecular autopsy in legal medicine and in mitochondrial DNA research. [Copyright &y& Elsevier]

Abstract: Identification of a specific genetic cause of early onset dementia (EOD) is important but can be difficult because of pleiotropy, locus heterogeneity and accessibility of gene tests. Here we assess the use of next generation sequencing (NGS) technologies as a quick, accurate and cost effective method to determine genetic diagnosis in EOD. We developed gene panel based technologies to assess 16 genes known to harbour mutations causal of dementia and combined these with PCR based assessments of the C9orf72 hexanucleotide repeat expansion and the octapeptide repeat region of PRNP. In a blinded study of 95 samples we show very high sensitivity and specificity are achievable using either Ion Torrent or MiSeq sequencing platforms. Modifications to the gene panel permit accurate detection of structural variation in APP. In 2/10 samples which had been selected because they possess a variant of uncertain significance the new technology discovered a causal mutation in genes not previously sequenced. A large proportion (23/85) of samples showed genetic variants of uncertain significance in addition to known mutations. The MRC Dementia Gene Panel and similar technologies are likely to be transformational in EOD diagnosis with a significant impact on the proportion of patients in whom a genetic cause is identified. [Copyright &y& Elsevier]

• Improved viral mapping single-stranded DNA viruses by >60-fold for begomoviruses out of plants. • Successful sequencing of viral single-stranded DNA from whiteflies over a wide range of viral loads. • Size selection improved viral read mapping by over 90 %. The ability of begomoviruses to evolve rapidly threatens many crops and underscores the importance of detecting these viruses quickly and to understand their genome diversity. This study presents an improved protocol for the enhanced amplification and enrichment of begomovirus DNA for use in next generation sequencing of the viral genomes. An enhanced rolling circle amplification (RCA) method using EquiPhi29 polymerase was combined with size selection to generate a cost-effective, short-read sequencing method. This improved short-read sequencing produced at least 50 % of the reads mapping to the target viral reference genomes, African cassava mosaic virus and East African cassava mosaic virus. This study provided other insights into common misconceptions about RCA and lessons that could be learned from the sequencing of single-stranded DNA virus genomes. This protocol can be used to examine the viral DNA as it moves from host to vector, thus producing valuable information for viral DNA population studies, and would likely work well with other circular Rep-encoding ssDNA viruses (CRESS) DNA viruses. [ABSTRACT FROM AUTHOR]

• Identification of a novel virus infecting roses, "rosa ilarvirus-1″. • Characterisation of the virus performing high-throughput sequencing (HTS). • Support of the deployment of HTS as a front-line diagnostic tool. • Innovations such as the Flongle are potentially well suited to this application. Roses are one of the most valuable ornamental flowering shrubs grown worldwide. Despite the widespread of rose viruses and their impact on cultivation, they have not been studied in detail in the United Kingdom (UK) since the 1980′s. As part of a survey of rose viruses entering the UK, 35 samples were collected at Heathrow Airport (London, UK) and were tested by RT-qPCR for different common rose viruses. Of the 35 samples tested using RT-qPCR for prunus necrotic ringspot virus (PNRSV; genus Ilarvirus), 10 were positive. Confirmatory testing was performed using RT-PCR with both PNRSV-specific and ilarvirus-generic primers, and diverse results were obtained: One sample was exclusively positive when using the ilarvirus-generic primers, and subsequent sequencing of the RT-PCR product revealed homology to other ilarviruses but not PNRSV. Further work to characterise the virus was performed using high throughput sequencing, both the MinION Flongle and Illumina MiSeq. The sequencing confirmed the presence of a new virus within group 2 of the genus Ilarvirus and we propose the name "rosa ilarvirus-1″ (RIV-1). Here, we describe the identification of a novel virus using the low-cost Flongle flow cell and discuss its potential as a front-line diagnostic tool. [ABSTRACT FROM AUTHOR]

The incessant increase in the demand for food from the world population, which is occurring due to its continuous growth has, as a consequence, led to an increase in the use of fertilisers. Our work aims to analyse the application effect of various fungal strains with urea fertiliser on the structure and functions of bacterial communities, including ammonia-oxidising microorganisms (AOM) in different soil types. By analysing the abundance of ammonia-oxidising archaea (AOA) and bacteria (AOB), we observed that the application of urea fertiliser (UC) changed the soil microbial community, causing a disturbance in soil microorganisms diversity, whereas the addition of fungal strains to urea fertiliser (UA100; UA60) did not have a disordering effect compared to the Control soil (C). This indicates that the addition of fungal strains mitigated the adverse effects of urea on the soil environment. A similar effect was observed for the genes involved in the carbon cycle (bacterial carbon fixation, chitinase, and methanotrophic pathway genes). It was observed that the addition of fungi to the urea fertiliser had a positive effect, as the abundance of functional genes recorded in these treatments was the same as in the Control soil. From the diversity point of view, it is worth emphasizing that the diversity index changed after fungal organisms' addition. We may also observe that the addition of fungal strains to urea protected or even improved the state of the soil macropores which are important for microbial communication in the soil environment. Furthermore, it may be concluded that to avoid a strong negative effect from the application of urea and the mitigation of its influence by fungal strains, we must consider the soil texture, pH, total carbon content, and soil organic matter including humic and fulvic acids. [Display omitted] • Fungal application affected abundance of amoA gene and diversity of soil microbes. • Fungal application mitigated adverse influence of urea on bacterial community function and structure. • Proteobacteria and Actinobacteria predominated in the soil microbiome after urea application. • Fungal strains addition protected or improved the presence of the soil macropores. • High content of cht gene in UA100 treatment was connected with the increase of AOB abundance. [ABSTRACT FROM AUTHOR]

• Four different series of pretreatments were tested on pig fecal samples. • Filtration and enzyme pretreatments do not affect the virome's alpha and beta diversities. • Enzyme pretreatments affect single-stranded RNA virus representation from the fecal virome. The porcine enteric microbiota is currently extensively studied, taking advantage of developments in high-throughput sequencing technologies. However, the viral part of the microbiota, the virome, is being lightly explored, and the impact of the pretreatments used before sequencing the viruses is barely considered. In this study, the impacts of filtration, RNase and DNase treatments on virus reads recovery and diversity after sequencing on a MiSeq platform were assessed on fecal samples individually taken at <3, 5, 12 and 20 weeks from two piglets. None of the four pretreatment series affected the virus read averages or influenced diversity, but the samples with the higher proportion of reads corresponding to an entry in the "nt" database were those receiving the least number of pretreatments. The enzymatic pretreatments affected the detection of the single-stranded RNA viruses of Aichivirus C , porcine astrovirus, Sapovirus and posavirus, which is worrisome, as these viruses can be involved in swine diarrhea. If enzymatic pretreatments are used when sequencing using a high-throughput method, it may impact single-stranded RNA virus recovery, but not the overall virome diversity. Therefore, filtrated samples may be the better option, reducing the amount of bacterial genetic material while preserving the virus reads. [ABSTRACT FROM AUTHOR]

• Equal performance with the MiSeq and the Ion S5. • Reproducible genotypes for low DNA input with the GeneRead assay. • Concordant genotypes with previously tested MPS assays. • Performance for typical forensic case samples was good. A custom GeneRead DNAseq SNP panel with 210 markers was evaluated using the Ion S5 and MiSeq sequencing platforms. Sensitivity, PCR cycle number, and the use of half volume of reagents for target enrichment and library preparation were tested. Furthermore, genotype concordance between results obtained with the different sequencing platforms and with known profiles generated using other sequencing assays was analysed. The GeneRead DNASeq SNP assay gave reproducible results with an input of 200 pg DNA on both platforms. A total of 204 loci were successfully sequenced. Three loci failed completely in the PCR amplification, and three additional loci displayed frequent locus drop-outs due to low read depth or high heterozygote imbalance. Overall, the read depth across the loci was more well-balanced with the MiSeq, while the heterozygote balance was less variable with the Ion S5. Noise levels were low on both platforms (median< 0.2 %). Two simple criteria for genotyping were applied: A minimum threshold of 45 reads and an acceptable heterozygote balance range of 0.3−3.0. Complete concordance between platforms was observed except for three genotypes in one of the poorly performing loci, rs1470637. This locus had relatively low read depths on both platforms, skewed heterozygote balance, and frequent locus drop-outs. There was also full genotype concordance between the results from the GeneRead assay and known profiles generated with the QIAseq and Ion AmpliSeq assays. The few discordant results were either due to locus drop-outs in the poorly performing loci or allele drop-outs in the QIAseq assay. Profiles with a minimum of 179 SNPs were obtained from four challenging case work samples (blood swabs, bone, or blood from a corpse). Overall, the GeneRead DNASeq assay showed considerable potential and could provide a reliable method for SNP genotyping in cases involving identification of individuals, prediction of phenotypic traits, and ancestry inference. [ABSTRACT FROM AUTHOR]