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

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

The genomic surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) is based on sequencing of the ORF5 gene of the virus, which covers only 4% of the entire viral genome. It is expected that PRRSV whole-genome sequencing (WGS) will improve PRRSV genomic data and allow better understanding of clinical discrepancies observed in the field when using ORF5 sequencing. Our main objective was to implement an efficient method for WGS of PRRSV from clinical samples. The viral genome was purified using a poly(A)-tail viral genome purification method and sequenced using Illumina technology. We tested 149 PRRSV-positive samples: 80 sera, 33 lungs, 33 pools of tissues, 2 oral fluids, and 1 processing fluid (i.e., castration liquid). Overall, WGS of 67.1% of PRRSV-positive cases was successful. The viral load, in particular for tissues, had a major impact on the PRRSV WGS success rate. Serum was the most efficient type of sample to conduct PRRSV WGS poly(A)-tail assays, with a success rate of 76.3%, and this result can be explained by improved sequencing reads dispersion matching throughout the entire viral genome. WGS was unsuccessful for all pools of tissue and lung samples with Cq values > 26.5, whereas it could still be successful with sera at Cq ≤ 34.1. Evaluation of results of highly qualified personnel confirmed that laboratory skills could affect PRRSV WGS efficiency. Oral fluid samples seem very promising and merit further investigation because, with only 2 samples of low viral load (Cq = 28.8, 32.8), PRRSV WGS was successful.

Temperate bat species use extended torpor to conserve energy when ambient temperatures are low and food resources are scarce. Previous research suggests that migratory bat species and species known to roost in thermally unstable locations, such as those that roost in trees, are more likely to remain active during winter. However, hibernating colonies of cave roosting bats in the southeastern United States may also be active and emerge from caves throughout the hibernation period. We report what bats are eating during these bouts of winter activity. We captured 2,044 bats of 10 species that emerged from six hibernacula over the course of 5 winters (October–April 2012/2013, 2013/2014, 2015/2016, 2016/2017, and 2017/2018). Using Next Generation sequencing of DNA from 284 fecal samples, we determined bats consumed at least 14 Orders of insect prey while active. Dietary composition did not vary among bat species; however, we did record variation in the dominant prey items represented in species’ diets. We recorded Lepidoptera in the diet of 72.2% of individual Corynorhinus rafinesquii and 67.4% of individual Lasiurus borealis. Diptera were recorded in 32.4% of Myotis leibii, 37.4% of M. lucifugus, 35.5% of M. sodalis and 68.8% of Perimyotis subflavus. Our study is the first to use molecular genetic techniques to identify the winter diet of North American hibernating bats. The information from this study is integral to managing the landscape around bat hibernacula for insect prey, particularly in areas where hibernating bat populations are threatened by white-nose syndrome.

Fast and effective methods are needed for sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome to track genetic mutations and to identify new and emerging variants during the ongoing pandemic. The objectives were to assess the performance of the SARS-CoV-2 AmpliSeq research panel and S5 plug-in analysis tools for whole-genome sequencing analysis of SARS-CoV-2 and to compare the results with those obtained with the MiSeq-based ARTIC analysis pipeline, using metrics such as depth, coverage, and concordance of single-nucleotide variant (SNV) calls. A total of 191 clinical specimens and a single cultured isolate were extracted and sequenced with AmpliSeq technology and analysis tools. Of the 191 clinical specimens, 83 (with threshold cycle [CT] values of 15.58 to 32.54) were also sequenced using an Illumina MiSeq-based method with the ARTIC analysis pipeline, for direct comparison. A total of 176 of the 191 clinical specimens sequenced on the S5XL system and prepared using the SARS-CoV-2 research panel had nearly complete coverage (>98%) of the viral genome, with an average depth of 5,031×. Similar coverage levels (>98%) were observed for 81/83 primary specimens that were sequenced with both methods tested. The sample with the lowest viral load (CT value of 32.54) achieved 89% coverage using the MiSeq method and failed to sequence with the AmpliSeq method. Consensus sequences produced by each method were identical for 81/82 samples in areas of equal coverage, with a single difference present in one sample. The AmpliSeq approach is as effective as the Illumina-based method using ARTIC v3 amplification for sequencing SARS-CoV-2 directly from patient specimens across a range of viral loads (CT values of 15.56 to 32.54 [median, 22.18]). The AmpliSeq workflow is very easily automated with the Ion Chef and S5 instruments and requires less training and experience with next-generation sequencing sample preparation than the Illumina workflow.

Phytate represents an organic pool of phosphorus in soil that requires hydrolysis by phytase enzymes produced by microorganisms prior to its bioavailability by plants. We tested the ability of a microbial suspension made from an old growth maple forest’s undisturbed soil to mineralize phytate in a greenhouse trial on soybean plants inoculated or non-inoculated with the suspension. MiSeq Amplicon sequencing targeting bacterial 16S rRNA gene and fungal ITS was performed to assess microbial community changes following treatments. Our results showed that soybean nodulation and shoot dry weight biomass increased when phytate was applied to the nutrient-poor substrate mixture. Bacterial and fungal diversities of the root and rhizosphere biotopes were relatively resilient following inoculation by microbial suspension, however, bacterial community structure was significantly influenced. Interestingly, four arbuscular mycorrhizal fungi (AMF) were identified as indicator species, including Glomus sp., Claroideoglomus etunicatum, Funneliformis mosseae and an unidentified AMF taxon. We also observed that an ericoid mycorrhizal taxon Sebacina sp. and three Trichoderma spp. were among indicator species. Non-pathogenic Planctobacteria members highly dominated the bacterial community as core and hub taxa for over 80% of all bacterial datasets in root and rhizosphere biotopes. Overall, our study documented that inoculation with a microbial suspension and phytate amendment improved soybean plant growth.

Made available in DSpace on 2019-10-06T15:52:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Illegal logging and trade of timber are major worldwide concerns, resulting in biodiversity and economic losses. Tropical tree species in the genus Cedrela, which have historically been heavily exploited, are still often illegally traded and there is an urgent need to develop tools to verify the origin of Cedrela products. A set of 351 SNP loci for Cedrela species from Bolivia, Brazil, Costa Rica, Cuba, Ecuador, French Guiana, Mexico, and Peru was developed using restriction associated DNA sequencing (RADSeq) and low coverage MiSeq genome sequencing, and adapted for MassARRAY genotyping. After screening of 94 individuals covering most of the distribution of Cedrela, a final set of 136 SNP loci which included 92 nuclear SNPs, 22 chloroplast markers (20 SNPs and 2 INDELs), and 22 mitochondrial markers (19 SNPs and 3 INDELs) was selected and tested for potential to verify Cedrela timber origin. Carrera de Ingeniería Forestal Laboratorio de Dendrocronología Facultad de Ciencias Agrícolas Universidad Autónoma Gabriel René Moreno, Km 9 carretera al Norte, El Vallecito Thünen Institute of Forest Genetics, Sieker Landstrasse 2 Instituto de Investigaciones de la Amazonía Peruana (IIAP), Av. José A. Quiñones km 2.5 Instituto Florestal de São Paulo, CP 1322 Faculdade de Engenharia Universidade Estadual Paulista (Unesp) INRA UMR0745 EcoFoG AgroParisTech Cirad CNRS Université des Antilles Université de Guyane, Campus Agronomique, Avenue de France Centre for Ecology & Hydrology Edinburgh, Bush Estate Faculdade de Engenharia Universidade Estadual Paulista (Unesp)

The pretreatment of lignocellulosic substrates with cattle rumen fluid was successfully developed to increase methane production. In the present study, a 16S rRNA gene-targeted amplicon sequencing approach using the MiSeq platform was applied to elucidate the effects of the rumen fluid treatment on the microbial community structure in laboratory-scale batch methane fermenters. Methane production in fermenters fed rumen fluid-treated rapeseed (2,077.3 mL CH4 reactor−1 for a 6-h treatment) was markedly higher than that in fermenters fed untreated rapeseed (1,325.8 mL CH4 reactor−1). Microbial community profiling showed that the relative abundance of known lignocellulose-degrading bacteria corresponded to lignocellulose-degrading enzymatic activities. Some dominant indigenous cellulolytic and hemicellulolytic bacteria in seed sludge (e.g., Cellulosilyticum lentocellum and Ruminococcus flavefaciens) and rumen fluid (e.g., Butyrivibrio fibrisolvens and Prevotella ruminicola) became undetectable or markedly decreased in abundance in the fermenters fed rumen fluid-treated rapeseed, whereas some bacteria derived from seed sludge (e.g., Ruminofilibacter xylanolyticum) and rumen fluid (e.g., R. albus) remained detectable until the completion of methane production. Thus, several lignocellulose-degrading bacteria associated with rumen fluid proliferated in the fermenters, and may play an important role in the degradation of lignocellulosic compounds in the fermenter.

Although co-culture of paddy fields with aquatic animals is lucrative, the ecological impacts of high-protein content entering the agricultural soil via animal pellet feed and feces have not been well studied. Moreover, the effects of dietary protein on soils and soil microbes remain unclear. To elucidate this, we examined soil bacterial and fungal community composition and temporal changes in paddy fields subjected to different protein-content diets via 16S/18S rRNA gene amplicon sequencing analysis with a high-throughput next-generation sequencer. MiSeq sequencing revealed that protein content significantly impacted fungal community structure. High-protein diets reduced bacterial community diversity and relative abundance in both July and October. The phylum-level bacterial taxonomic composition was not affected by diet treatment, while in fungi, a major phylum-level shift was evident. Hierarchically clustered analysis showed that high-protein diets significantly reduced the relative abundance of Brevundimonas in both July and October. Saprotrophic macrofungal diversity was negatively related to dietary protein content. Considering microbial community structure and environmental factors, ca. 15% protein content is appropriate for the rice-crab co-culture system that we studied.

The first mitochondrial genome of Ophiocara porocephala was determined by the combination of next-generation 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.

The structure and diversity of human gut microbiota are directly related to diet, though less is known about the influences of ethnicity and diet-related behaviors, such as fasting (intermittent caloric restriction). In this study, we investigated whether fasting for Ramadan altered the microbiota in Chinese and Pakistani individuals. Using high-throughput 16S rRNA gene sequencing and self-reported dietary intake surveys, we determined that both the microbiota and dietary composition were significantly different with little overlap between ethnic groups. Principal Coordinate Analyses (PCoA) comparison of samples collected from both groups before and after fasting showed partial separation of microbiota related to fasting in the Pakistani group, but not in the Chinese group. Measurement of alpha diversity showed that Ramadan fasting significantly altered the coverage and ACE indices among Chinese subjects, but otherwise incurred no changes among either group. Specifically, Prevotella and Faecalibacterium drove predominance of Bacteroidetes and Firmicutes in the Pakistani group, while Bacteroides (phylum Bacteroidetes) were the most prevalent among Chinese participants both before and after fasting. We observed significant enrichment of some specific taxa and depletion of others in individuals of both populations, suggesting that fasting could affect beta diversity. Notably, Dorea, Klebsiella, and Faecalibacterium were more abundant in the Chinese group after fasting, while Sutterella, Parabacteroides, and Alistipes were significantly enriched after fasting in the Pakistani group. Evaluation of the combined groups showed that genera Coprococcus, Clostridium_XlV, and Lachnospiracea were all significantly decreased after fasting. Analysis of food intake and macronutrient energy sources showed that fat-derived energy was positively associated with Oscillibacter and Prevotella, but negatively associated with Bacteroides. In addition, the consumption of sweets was significantly positively correlated with the prevalence of Akkermansia. Our study indicated that diet was the most significant influence on microbiota, and correlated with ethnic groups, while fasting led to enrichment of specific bacterial taxa in some individuals. Given the dearth of understanding about the impacts of fasting on microbiota, our results provide valuable inroads for future study aimed at novel, personalized, behavior-based treatments targeting specific gut microbes for prevention or treatment of digestive disorders.

We determined the complete mitochondrial genome sequence of nurse shark Ginglymostoma cirratum. The circular DNA of 16692 bp comprises 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, a non-coding control region and a non-coding origin of light strand replication with typical gene order of vertebrates. The nurse shark formed a well-supported clade that included whale shark Rhincodon typus and zebra shark Stegostoma fasciatum within the Orectolobiformes in a phylogenetic tree constructed with other published mitochondrial genomes of sharks.

The entire mitochondrial genome sequence of Sphyraena pinguis collected from Korean water was determined by the Next Generation Sequencing (NGS) technology. Its total length was 16,620 bps in length, which possessed the canonical 37 genes in the eukaryotes. Unusual start codon was exclusively found in COX1(GTG), while incomplete stop codons (TA–/T—) were identified in ATP6, COX2, ND3, ND4, and Cyt b. A phylogenetic analysis with currently identified full mitogenomes in Perciformes, S. pinguis was most closely related to S. barracuda (76.87%) and S. jello (76.84%). This mitogenome sequence would explain the evolution of genus Sphyraena.

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.

Individual age estimation can be applied to criminal, legal, and anthropological investigations. DNA methylation has been established as the biomarker of choice for age prediction, since it was observed that specific CpG positions in the genome show systematic changes during an individual’s lifetime, with progressive increases or decreases in methylation levels. Subsequently, several forensic age prediction models have been reported, providing average age prediction error ranges of ±3–4 years, using a broad spectrum of technologies and underlying statistical analyses. DNA methylation assessment is not categorical but quantitative. Therefore, the detection platform used plays a pivotal role, since quantitative and semi-quantitative technologies could potentially result in differences in detected DNA methylation levels. In the present study, we analyzed as a shared sample pool, 84 blood-based DNA controls ranging from 18 to 99 years old using four different technologies: EpiTYPER®, pyrosequencing, MiSeq, and SNaPshotTM. The DNA methylation levels detected for CpG sites from ELOVL2, FHL2, and MIR29B2 with each system were compared. A restricted three CpG-site age prediction model was rebuilt for each system, as well as for a combination of technologies, based on previous training datasets, and age predictions were calculated accordingly for all the samples detected with the previous technologies. While the DNA methylation patterns and subsequent age predictions from EpiTYPER®, pyrosequencing, and MiSeq systems are largely comparable for the CpG sites studied, SNaPshotTM gives bigger differences reflected in higher predictive errors. However, these differences can be reduced by applying a z-score data transformation.

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii , occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii , and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean = 3.2) and 0.13 to 0.82 (mean = 0.45), respectively. All loci were in Hardy–Weinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.

During the 2014–15 influenza season, 13/168 respiratory samples from students with influenza-like illness (ILI) at a college in New York, USA, were positive for human adenovirus (HAdV); 4/13 samples were positive for HAdV-B14p1. During influenza season, HAdV should be included in the differential diagnostic panel used to determine the etiology of ILI.

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 (TM) (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. (C) 2016 The Author(s). Published by Elsevier B.V.

A complete mitochondrial genome sequence was determined for a member of the Caridina indistincta species complex known as C. indistincta ‘sp. A’. The 15,461 bp sequence (GenBank: MH189850) was obtained via genome skimming, and contains 13 protein coding genes, 22 tRNA genes, 2 rRNA genes, and a 646 bp control region arranged in the pancrustacean ground pattern. Caridina indistincta sp. A is a freshwater macroinvertebrate important for ecosystem health monitoring in Australia and this reference will be a useful resource for metabarcoding and eDNA studies.

The fisheries resources of the Yangtze Estuary and its adjacent waters have undergone dramatic declines as a consequence of environmental changes and human activities, with traditional ecological investigations demonstrating progressive decreases in species diversity and annual numbers in the fisheries resource. Environmental DNA (eDNA) technology has been demonstrated as an effective tool by many studies for detecting fish species, monitoring fish biodiversity, and indicating the abundance of fish. In the present study, we chose the Yangtze Estuary and its adjacent waters as a primary research area to investigate fish assemblage structure using eDNA technology. A total of 50 eDNA samples were collected in the estuary in 2018. The results showed that 41 operational taxonomic units were identified from three seasons, with 18, 12, and 33 fish species associated specifically with spring, summer and autumn, respectively. The fish assemblage differed significantly among seasons. Canonical correspondence analysis showed that water temperature, salinity, and dissolved oxygen were the main environmental factors affecting structure of the seasonal assemblages. Results of the present study indicate that eDNA technology can be an effective tool not only for fisheries monitoring, but might also importantly assist marine resources conservation, sustainable exploitation of fisheries, the aquatic products processing industry, eco-friendly development, and socioeconomic stability.

Microbial mats are rarely reported for chromium-polluted ecosystems, hence information on their bacterial diversity and role in chromium removal are very scarce. We investigated the role of nine microbial mats, collected from three quarry sumps of chromium mining sites, in the removal of hexavalent chromium (Cr(VI)). Bacterial diversity in these mats and community shifts after incubation with Cr(VI) have been investigated using MiSeq sequencing. In nature, a chromium content of 1,911±100 mg kg-1 was measured in the microbial mats, constituting the third highest source of environmentally available chromium. The mats were able to remove 1 mg l-1 of Cr(VI) in 7 days under aerobic and anaerobic conditions. MiSeq sequencing of the original mats yielded 46-99% of the sequences affiliated to Proteobacteria, Firmicutes and Actinobacteria. When the mats were incubated with Cr(VI), the bacterial community shifted in the favor of Alphaproteobacteria and Verrucomicrobiae. We conclude that microbial mats in the quarry sumps harbor diverse microorganisms with the ability to remove toxic Cr(VI), hence these mats can be potentially used to remove chromium from polluted waters.

Phlebopusportentosus (Berk. and Broome) Boedijin is a popular edible mushroom found in China and Thailand. To date, P. portentosus is the only species in the order Boletales that can be successfully cultivated worldwide. The use of a casing layer or casing soil overlaying the substrate is a crucial step in the production of this mushroom. In this study, bacterial profiling and dynamic succession analyses of casing soil during the cultivation of P. portentosus were performed. One hundred and fifty samples were collected, and MiSeq sequencing of the V3-V4 region of the 16S rRNA gene was conducted. After performing a decontamination procedure, only 38 samples were retained, including 6 casing soil-originated samples (OS), 6 casing soil samples (FHCS) and 5 upper substrate samples (FHCU) from the period of complete colonization by mycelia; 6 casing soil samples (PCS) and 5 upper substrate samples (PCU) from the primordium period; and 6 casing soil samples (FCS) and 4 upper substrate samples (FCU) from fruit body period. The results revealed that bacterial diversity increased sharply from the hyphal to the primordium stage and then decreased during harvesting. The non-metric multidimensional scaling (NMDS) ordination and analysis of similarities (ANOSIM) analysis suggested that the community composition during different stages was significantly different in casing soil. The most abundant phyla in all of the samples were Proteobacteria, Chloroflexi, Acidobacteria, Actinobacteria, Saccharibacteria, and Bacteroidetes. Burkholderia was the most abundant genus in all the samples except the OS samples. The relative abundance of Burkholderia in the FHCS samples (55.79%) decreased to 35.14% in the PCS samples and then increased to 45.60% in the FCS samples. The abundances of Acidobacterium, Rhizobium, Acidisphaera, Bradyrhizobium, and Bacillus increased from the FHCS to PCS samples. The linear discriminant analysis (LDA) effect size (LEfSe) suggested that Acidobacterium and Acidisphaera are micromarkers for PCS, whereas Bradyrhizobium, Roseiarcus, and Pseudolabrys were associated with fruit body stages. The network analyses resulted in 23 edges, including 4 negative and 19 positive edges. Extensive mutualistic interactions may occur among casing soil bacteria. Furthermore, these bacteria play important roles in mycelial elongation, primordium formations, and the production of increased yields.

Soil bacterial community compositions and temporal changes in organic paddy fields were elucidated using a 16S rRNA gene amplicon sequencing analysis with a high-throughput next generation sequencer. At transplanting, bacterial community compositions in organic and conventional paddy fields were mostly similar despite differences in field management. The bacterial community composition in organic fields differed from that under conventional management during the rice growth period, possibly as a result of the decomposition process of organic fertilizers. However, differences in the frequency of tillage and photosynthetic bacterial inoculations in the organic plots had less of an impact on bacterial communities.

Tricholoma matsutake is an ectomycorrhizal fungus that has obligate symbiotic relationships with Pinus densiflora. Its fruiting body has a distinctive flavor and is traded at a high price. Thus, it has been a significant source of income for rural communities in Korea. We hypothesized that biotic factors considerably influence the formation of the T. matsutake mushroom, and the soils producing T. matsutake share similar microbial characteristics. Therefore, the present study aimed to detect the specific fungal and bacterial groups in T. matsutake production soils (shiro+) and nonproduction soils (shiro−) of the Bonghwa and Yanyang regions via next-generation sequencing. In a total of 15 phyla, 36 classes, 234 genera of bacteria, six phyla, 29 classes, and 164 genera of fungi were detected from four samples at both sites. The species diversity of shiro+ soils was lower than the shiro− samples in both the fungal and bacterial groups. In addition, we did not find high similarities in the microbial communities between the shiro+ soils of the two regions. However, in the resulting differences between the fungal communities categorized by their trophic assembly, we found a distinguishable compositional pattern in the fungal communities from the shiro+ soils and the shiro− soils of the two sites. Thus, the similarity among the microbial communities in the forest soils may be due to the fact that the microbial communities in the T. matsutake dominant soils are closely associated with biotic factors and abiotic factors such as soil properties.

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.

Metagenome studies have provided us with insights into the complex interactions of microorganisms with their environments and hosts. Few studies have focused on microalgae-associated metagenomes, and no study has addressed aquatic microalgae and their bacterial communities in open pond raceways (OPRs). This study explored the possibility of using microalgal biomasses from OPRs for biodiesel and biofertilizer production. The fatty acid profiles of the biomasses and the physical and chemical properties of derived fuels were evaluated. In addition, the phenotype-based environmental adaptation ability of soybean plants was assessed. The growth rate, biomass, and lipid productivity of microalgae were also examined during mass cultivation from April to November 2017. Metagenomics analysis using MiSeq identified ∼127 eukaryotic phylotypes following mass cultivation with (OPR 1) or without (OPR 3) a semitransparent film. Of these, ∼80 phylotypes were found in both OPRs, while 23 and 24 phylotypes were identified in OPRs 1 and 3, respectively. The phylotypes belonged to various genera, such as Desmodesmus, Pseudopediastrum, Tetradesmus, and Chlorella, of which, the dominant microalgal species was Desmodesmus sp. On average, OPRs 1 and 3 produced ∼8.6 and 9.9 g m−2 d−1 (0.307 and 0.309 DW L−1) of total biomass, respectively, of which 14.0 and 13.3 wt% respectively, was lipid content. Fatty acid profiling revealed that total saturated fatty acids (mainly C16:0) of biodiesel obtained from the microalgal biomasses in OPRs 1 and 3 were 34.93% and 32.85%, respectively; total monounsaturated fatty acids (C16:1 and C18:1) were 32.40% and 31.64%, respectively; and polyunsaturated fatty acids (including C18:3) were 32.68% and 35.50%, respectively. Fuel properties determined by empirical equations were within the limits of biodiesel standards ASTM D6751 and EN 14214. Culture solutions with or without microalgal biomasses enhanced the environmental adaptation ability of soybean plants, increasing their seed production. Therefore, microalgal biomass produced through mass cultivation is excellent feedstock for producing high-quality biodiesel and biofertilizer.

A large number of new microhaplotype loci were identified in the human genome by applying a directed search with selection criteria emphasizing short haplotype length (

The complete mitochondrial genome of Pogonophryne albipinna was determined by the MiSeq platform, which was the first report in the family Artedidraconidae. The circular form of its mitochondrial genome was 17,086 bp, which contained the canonical eukaryotic 37 genes. The gene orders of P. albipinna was identical to the other icefish species, in which there was additional non-coding region and translocation of ND6 gene. Except for ATP6 gene and COI (GTG), 11 genes begin with the typical start codon, while incomplete stop codons (T– –) were identified in COII, ND4, and CytB. Phylogenetic tree with the currently known mitogenomes in suborder Notothenioidei showed that P. albipinna was located distinctly from those in Bathydraconidae and Nototheniidae forming a unique cluster as Artedidraconidae. The first complete mitochondrial genome of P. albipinna would be the fundamental data to understand the evolutional relationship of icefish species in the Antarctic Oceans.

Microbial populations associated with microbial enhanced oil recovery (MEOR) and their abundance in the Xinjiang Luliang water-flooding petroleum reservoir were investigated using 16S rRNA, nitrate reductases, dissimilatory sulfate reductase, and methyl coenzyme-M reductase-encoded genes to provide ecological information for the potential application of MEOR. 16S rRNA gene miseq sequencing revealed that this reservoir harbored large amounts of taxa, including 155 bacterial and 7 archeal genera. Among them, Arcobacter, Halomonas, Marinobacterium, Marinobacter, Sphingomonas, Rhodococcus, Pseudomonas, Dietzia, Ochrobactrum, Hyphomonas, Acinetobacter, and Shewanella were dominant, and have the potential to grow using hydrocarbons as carbon sources. Metabolic gene clone libraries indicated that the nitrate-reducing bacteria (NRB) mainly belonged to Pseudomonas, Azospirillum, Bradyrhizobium, Thauera, Magnetospirillum, Sinorhizobium, Azoarcus, and Rhodobacter; the sulfate-reducing bacteria (SRB) were Desulfarculus, Desulfomonile, Desulfosarcina, Desulfotignum, Desulfacinum, Desulfatibacillum, Desulfatibacillum, Desulfomicrobium, and Desulfovibrio; while the methanogens were archaea and belonged to Methanomethylovorans, Methanosaeta, Methanococcus, Methanolobus, and Methanobacterium. Real-time quantitative PCR analysis indicated that the number of bacterial 16S rRNA reached 106 copies/mL, while the metabolic genes of NRB, SRB, and methanogens reached 104 copies/mL. These results show that the Luliang reservoir has abundant microbial populations associated with oil recovery, suggesting that the reservoir has potential for MEOR.

Background Knowledge on the capacity of Australian ticks to carry Borrelia species is currently limited or missing. To evaluate the potential of ticks to carry bacterial pathogens and their DNA, it is imperative to have a robust workflow that maximises recovery of bacterial DNA within ticks in order to enable accurate identification. By exploiting the bilateral anatomical symmetry of ticks, we were able to directly compare two DNA extraction methods for 16S rRNA gene diversity profiling and pathogen detection. We aimed to assess which combination of DNA extraction and 16S rRNA hypervariable region enables identification of the greatest bacterial diversity, whilst minimising bias, and providing the greatest capacity for the identification of Borrelia spp. Results We collected Australian endemic ticks (Bothriocroton undatum), isolated DNA from equal tick halves using two commercial DNA extraction methods and sequenced samples using V1-V3 and V3-V4 16S rRNA gene diversity profiling assays. Two distinct Borrelia spp. operational taxonomic units (OTUs) were detected using the V1-V3 16S rRNA hypervariable region and matching Borrelia spp. sequences were obtained using a conventional nested-PCR. The tick 16S rRNA gene diversity profile was dominated by Rickettsia spp. (98–99%), while the remaining OTUs belonged to Proteobacteria (51–81%), Actinobacteria (6–30%) and Firmicutes (2–7%). Multiple comparisons tests demonstrated biases in each of the DNA extraction kits towards different bacterial taxa. Conclusions Two distinct Borrelia species belonging to the reptile-associated Borrelia group were identified. Our results show that the method of DNA extraction can promote bias in the final microbiota identified. We determined an optimal DNA extraction method and 16S rRNA gene diversity profile assay that maximises detection of Borrelia species. Electronic supplementary material The online version of this article (doi: 10.1186/s13071-017-2579-5) contains supplementary material, which is available to authorized users.

Livestock waste composts with minimum inorganic fertilizer as a soil amendment in low-input intensive farming are a feasible agricultural practice to improve soil fertility and productivity and to mitigate soil degradation. The key benefits of the practice rely on the activities of soil microorganisms. However, the role of different livestock composts [composted cattle manure (CCM) vs. composted swine manure (CSM)] on soil microbes, their activities and the overall impact on soil fertility and productivity in a flooded paddy remains elusive. This study compares the effectiveness of CCM and CSM amendment on bacterial communities, activities, nutrient availability, and crop yield in a flooded rice cropping system. We used deep 16S amplicon sequencing and soil enzyme activities to decipher bacterial communities and activities, respectively. Both CCM and CSM amendment significantly increased soil pH, nutrient availability (C, N, and P), microbial biomass, soil enzyme activities indicative for C and N cycles, aboveground plant biomass and grain yield. And the increase in above-mentioned parameters was more prominent in the CCM treatment compared to the CSM treatment. The CCM amendment increased species richness and stimulated copiotrophic microbial groups (Alphaproteobacteria, Betaproteobacteria, and Firmicutes) which are often involved in degradation of complex organic compounds. Moreover, some dominant species (e.g., Azospirillum zeae, Azospirillum halopraeferens, Azospirillum rugosum, Clostridium alkalicellulosi, Clostridium caenicola, Clostridium termitidis, Clostridium cellulolyticum, Magnetospirillum magnetotacticum, Pleomorphomonas oryzae, Variovorax boronicumulans, Pseudomonas xanthomarina, Pseudomonas stutzeri, and Bacillus niacini) which have key roles in plant growth promotion and/or lignocellulose degradation were enhanced under CCM treatment compared to CSM treatment. Multivariate analysis revealed that soil pH and available carbon (C) and nitrogen (N) were the major, while total organic carbon (TOC), total nitrogen (TN), and available phosphorus (P) were the minor drivers of variation in bacterial communities. Overall, our observations suggest that CCM amendment is better than CSM amendment to improve soil fertility and crop yield in a submerged rice cropping system.

Primula reinii (Primulaceae), a perennial herb belonging to the Primula section Reinii, occurs on wet, shaded rocky cliffs in the mountains of Japan. This threatened species comprises four varieties; these plants are very localized and rare in the wild. In this study, 43 microsatellite markers were developed using MiSeq sequencing to facilitate conservation genetics of these critically endangered primroses. We developed novel microsatellite markers for three varieties of P. reinii, and tested its polymorphism and genetic diversity using natural populations. These novel markers displayed relatively high polymorphism; the number of alleles and expected heterozygosities ranged from 2 to 6 (mean = 3.2) and 0.13 to 0.82 (mean = 0.45), respectively. All loci were in Hardy–Weinberg equilibrium. These microsatellite markers will be powerful tools to assess P. reinii genetic diversity and develop effective conservation and management strategies.

White clover is a short-lived perennial whose persistence is greatly affected by abiotic stresses, particularly drought. The aim of this work was to characterize its molecular response to water deficit and recovery following re-hydration to identify targets for the breeding of tolerant varieties. We created a white clover reference transcriptome of 16,193 contigs by deep sequencing (mean base coverage 387x) four Suppression Subtractive Hybridization (SSH) libraries (a forward and a reverse library for each treatment) constructed from young leaf tissue of white clover at the onset of the response to drought and recovery. Reads from individual libraries were then mapped to the reference transcriptome and processed comparing expression level data. The pipeline generated four robust sets of transcripts induced and repressed in the leaves of plants subjected to water deficit stress (6,937 and 3,142, respectively) and following re-hydration (6,695 and 4,897, respectively). Semi-quantitative polymerase chain reaction was used to verify the expression pattern of 16 genes. The differentially expressed transcripts were functionally annotated and mapped to biological processes and pathways. In agreement with similar studies in other crops, the majority of transcripts up-regulated in response to drought belonged to metabolic processes, such as amino acid, carbohydrate, and lipid metabolism, while transcripts involved in photosynthesis, such as components of the photosystem and the biosynthesis of photosynthetic pigments, were up-regulated during recovery. The data also highlighted the role of raffinose family oligosaccharides (RFOs) and the possible delayed response of the flavonoid pathways in the initial response of white clover to water withdrawal. The work presented in this paper is to our knowledge the first large scale molecular analysis of the white clover response to drought stress and re-hydration. The data generated provide a valuable genomic resource for marker discovery and ultimately for the improvement of white clover.

Background: Male breast cancer (MBC) is still poorly understood with a large proportion arising in families with a history of breast cancer. Genomic studies have focused on germline determinants of MBC risk, with minimal knowledge of somatic changes in these cancers. Methods: Using a TruSeq amplicon cancer panel, this study evaluated 48 familial MBCs (3 BRCA1 germline mutant, 17 BRCA2 germline mutant and 28 BRCAX) for hotspot somatic mutations and copy number changes in 48 common cancer genes. Results: Twelve missense mutations included nine PIK3CA mutations (seven in BRCAX patients), two TP53 mutations (both in BRCA2 patients) and one PTEN mutation. Common gains were seen in GNAS (34.1%) and losses were seen in GNAQ (36.4%), ABL1 (47.7%) and ATM (34.1%). Gains of HRAS (37.5% vs 3%, P=0.006), STK11 (25.0% vs 0%, P=0.01) and SMARCB1 (18.8% vs 0%, P=0.04) and the loss of RB1 (43.8% vs 13%, P=0.03) were specific to BRCA2 tumours. Conclusions: This study is the first to perform high-throughput somatic sequencing on familial MBCs. Overall, PIK3CA mutations are most commonly seen, with fewer TP53 and PTEN mutations, similar to the profile seen in luminal A female breast cancers. Differences in mutation profiles and patterns of gene gains/losses are seen between BRCA2 (associated with TP53/PTEN mutations, loss of RB1 and gain of HRAS, STK11 and SMARCB1) and BRCAX (associated with PIK3CA mutations) tumours, suggesting that BRCA2 and BRCAX MBCs may be distinct and arise from different tumour pathways. This has implications on potential therapies, depending on the BRCA status of MBC patients.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. Most NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5’ end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (eight forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.

We present high-throughput amplicon sequence (HTS) datasets of the microbial metacommunity DNA of the gut tissue and the gut digesta of naturally occurring (n = 3) and laboratory aquaculture (n = 2) green sea urchins, Lytechinus variegatus. The HTS datasets were generated on an Illumina MiSeq by targeting the amplicons of the V4 region of the 16S rRNA gene. After the raw sequences were quality checked and filtered, 88% of the sequence reads were subjected to bioinformatics analyses to generate operation taxonomic units (OTUs), which were then verified for saturation by using rarefaction analysis at a 3% sequence variation. Further, the OTUs were randomly subsampled to the minimum sequence count values. Then, the FASTA-formatted representative sequences of the microbiota were assigned taxonomic identities through multiple databases using the SILVA ACT: Alignment, Classification and Tree Service (www.arb-silva.de/aligner). The HTS datasets of this metagenome can be accessed from the BioSample Submission Portal (https://www.ncbi.nlm.nih.gov/bioproject/) under the BioProject IDs PRJNA291441 and PRJNA326427. Keywords: Echinoderm, Arcobacter, PhyloToAST, QIIME, MiSeq

This Data in Brief article is a supporting information for the research article entitled “Protistan community composition in anoxic sediments from three salinity-disparate Japanese lakes” by Kataoka and Kondo (2019) [1]. Summary of 18S rRNA gene sequences originated from anoxic sediment of three lakes in two seasons using high throughput sequencing techniques (MiSeq, Illumina) was shown in this data article. Supergroup-level taxonomy was compared between the SILVA search for SILVA database and BLASTn search for the PR2 database. Alpha diversity was calculated in each sample, and beta-diversity was calculated among the six amplicon libraries. Partial sequence length between the primer set of 574*f and 1132R Hugerth et al., 2015 was compared between the forward read and the combined read. Keywords: Protists, 18S rRNA gene, High throughput sequencing (HTS), MiSeq, V4–V5 hypervariable region

Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed.

Octocorals are sources of novel but understudied microbial diversity. Conversely, scleractinian or reef-building coral microbiomes have been heavily examined in light of the threats of climate change. Muricea californica and Muricea fruticosa are two co-occurring species of gorgonian octocoral abundantly found in the kelp forests of southern California, and thus provide an excellent basis to determine if octocoral microbiomes are host specific. Using Illumina MiSeq amplicon sequencing and replicate samples, we evaluated the microbiomes collected from multiple colonies of both species of Muricea to measure both inter- and intra-colony microbiome variabilities. In addition, microbiomes from overlying sea water and nearby zoanthids (another benthic invertebrate) were also included in the analysis to evaluate whether bacterial taxa specifically associate with octocorals. This is also the first report of microbiomes from these species of Muricea. We show that microbiomes isolated from each sample type are distinct, and specifically, that octocoral species type had the greatest effect on predicting the composition of the Muricea microbiome. Bacterial taxa contributing to compositional differences include distinct strains of Mycoplasma associated with either M. californica or M. fruticosa, an abundance of Spirochaetes observed on M. californica, and a greater diversity of γ-Proteobacteria associated with M. fruticosa. Many of the bacterial taxa contributing to these differences are known for their presence in photosymbiont-containing invertebrate microbiomes.

Objective The aim of this study was to examine shifts in the composition of the bacterial population in the intestinal tracts (ITs) of weaning piglets by antibiotic treatment using high-throughput sequencing. Methods Sixty 28-d-old weaning piglets were randomly divided into two treatment groups. The Control group was treated with a basal diet without antibiotics. The Antibiotic group’s basal diet contained colistin sulfate at a concentration of 20 g per ton and bacitracin zinc at a concentration of 40 g per ton. All of the pigs were fed for 28 days. Then, three pigs were killed, and the luminal contents of the jejunum, ileum, cecum, and colon were collected for DNA extraction and high-throughput sequencing. Results The results showed that the average daily weight gain of the antibiotic group was significantly greater (p0.05), than the control group. A total of 812,607 valid reads were generated. Thirty-eight operational taxonomic units (OTUs) that were found in all of the samples were defined as core OTUs. Twenty-one phyla were identified, and approximately 90% of the classifiable sequences belonged to the phylum Firmicutes. Forty-two classes were identified. Of the 232 genera identified, nine genera were identified as the core gut microbiome because they existed in all of the tracts. The proportion of the nine core bacteria varied at the different tract sites. A heat map was used to understand how the numbers of the abundant genera shifted between the two treatment groups. Conclusion At different tract sites the relative abundance of gut microbiota was different. Antibiotics could cause shifts in the microorganism composition and affect the composition of gut microbiota in the different tracts of weaning piglets.

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.

peer-reviewed Background Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. Results The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. Conclusions Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required. This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Numbers SFI/12/RC/2273 and 11/PI/1137 and by FP7 funded CFMATTERS (Cystic Fibrosis Microbiome-determined Antibiotic Therapy Trial in Exacerbations: Results Stratified, Grant Agreement no. 603038).

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 (HOMI NGS ) 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 HOMI NGS , respectively, based on 16S rRNA gene sequences. We determined the concordance between the two technologies for common species. Genus level analysis was performed using HOMI NGS -specific genus identification capabilities. Results : HOMI NGS detected twice the number of species in the same dental plaque samples compared to HOMIM. For the species detected by both HOMIM and HOMI NGS , 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 : HOMI NGS significantly expanded oral bacterial species identification compared to HOMIM. The genus and species probes, combined in HOMI NGS , provided a more comprehensive representation of oral bacterial community, critical for future characterization of oral microbes in distant site infections. Keywords: HOMI NGS; HOMIM; MiSeq; ProbeSeq; dental plaque; oral microbiome (Published: 8 April 2016) Citation: Journal of Oral Microbiology 2016, 8: 30379 - http://dx.doi.org/10.3402/jom.v8.30379

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.

Complete mitochondrial genome sequences were determined for two lineages (“CEQ” and “SEQ”) of the Australian smelt, Retropinna semoni. Both mitogenomes contain the typical vertebrate arrangement of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and control region. A conventional start codon for ND2 was not present in either lineage; instead CTG (Leucine) was present at this position. These sequences will be a useful resource for evolutionary studies of a significant species complex in the Australian freshwater fish fauna.

Rapid determination of full genomes of influenza A viruses from clinical samples is vital to outbreak preparedness. We benchmarked Ion Torrent PGM and Illumina MiSeq to the standard Sanger sequencing on two real clinical samples and demonstrated their performance for full-genome determination and polymorphism identification.

Background The growing interest in the role of epigenetic modifications in human health and disease has led to the development of next-generation sequencing methods for whole genome analysis of DNA methylation patterns. However, many projects require targeted methylation analysis of specific genes or genomic regions. We have developed an approach, termed BiSulfite Amplicon Sequencing (BSAS), for hypothesis driven and focused absolute DNA methylation analysis. This approach is applicable both to targeted DNA methylation studies as well as to confirmation of genome-wide studies. Results BSAS uses PCR enrichment of targeted regions from bisulfite-converted DNA and transposome-mediated library construction for rapid generation of sequencing libraries from low (1 ng) sample input. Libraries are sequenced using the Illumina MiSeq benchtop sequencer. Generating high levels of sequencing depth (>1,000 ×) provides for quantitatively precise and accurate assessment of DNA methylation levels with base specificity. Dual indexing of sequencing libraries allows for simultaneous analysis of up to 96 samples. We demonstrate the superior quantitative accuracy of this approach as compared to existing Sanger sequencing methods. Conclusions BSAS can be applied to any genomic region from any DNA source, including tissue and cell culture. Thus, BSAS provides a new validation approach for rapid and highly quantitative absolute CpG methylation analysis of any targeted genomic regions in a high throughput manner.