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ImportanceAmplicon sequencing of targeted genes is the predominant approach to estimate the membership and structure of microbial communities. However, accurate reconstruction of community composition is difficult due to sequencing errors, and other methodological biases and effective approaches to overcome these challenges are essential. Using a mock community of 33 phylogenetically diverse strains, this study evaluated the effect of GC content on sequencing results and tested different approaches to improve overall sequencing accuracy while characterizing the pros and cons of popular amplicon sequence data processing approaches. The sequencing results from this study can serve as a benchmarking data set for future algorithmic improvements. Furthermore, the new insights on sequencing error, chimera formation, and GC bias from this study will help enhance the quality of amplicon sequencing studies and support the development of new data analysis approaches.

ABSTRACTObjective Next generation sequencing is commonly used to characterize the microbiome structure. MiSeq is most commonly used to analyze the microbiome due to its relatively long read length. Illumina also introduced the 250 × 2 chip for NovaSeq. The purpose of this study was to compare the performance of MiSeq and NovaSeq in the context of oral microbiome study.Methods Total read count, read quality score, relative bacterial abundance, community diversity, and correlation between two platforms were analyzed. Phylogenetic trees were analyzed for Streptococcus and periodontopathogens.Results NovaSeq produced significantly more read counts and assigned more operational taxonomic units (OTUs) compared to MiSeq. Community diversity was similar between MiSeq and NovaSeq. NovaSeq were able to detect more unique OTUs compared to MiSeq. When phylogenetic trees were constructed for Streptococcus and periodontopathogens, both platforms detected OTUs for most of the clades.Conclusion Taken together, while both MiSeq and NovaSeq platforms effectively characterize the oral microbiome, NovaSeq outperformed MiSeq in terms of read counts and detection of unique OTUs, highlighting its potential as a valuable tool for large scale oral microbiome studies.

Pulp-paper mills (PPMs) are known for consistently generating a wide variety of pollutants, that are often unidentified and highly resistant to environmental degradation. The current study aims to investigate the changes in the indigenous bacterial communities profile grown in the sediment co-contaminated with organic and inorganic pollutants discharged from the PPMs. The two sediment samples, designated PPS-1 and PPS-2, were collected from two different sites. Physico-chemical characterization of PPS-1 and PPS-2 revealed the presence of heavy metals (mg kg-1) like Cu (0.009-0.01), Ni (0.005-0.002), Mn (0.078-0.056), Cr (0.015-0.009), Pb (0.008-0.006), Zn (0.225-0.086), Fe (2.124-0.764), Al (3.477-22.277), and Ti (99.792-45.012) along with high content of chlorophenol, and lignin. The comparative analysis of organic pollutants in sediment samples using gas chromatography-mass spectrometry (GC-MS) revealed the presence of major highly refractory compounds, such as stigmasterol, β-sitosterol, hexadecanoic acid, octadecanoic acid; 2,4-di-tert-butylphenol; heptacosane; dimethyl phthalate; hexachlorobenzene; 1-decanol, 2-hexyl; furane 2,5-dimethyl, etc in sediment samples which are reported as a potential toxic compounds. Simultaneously, high-throughput sequencing targeting the V3-V4 hypervariable region of the 16S rRNA genes, resulted in the identification of 1,249 and 1,345 operational taxonomic units (OTUs) derived from a total of 115,665 and 119,386 sequences read, in PPS-1 and PPS-2, respectively. Analysis of rarefaction curves indicated a diversity in OTU abundance between PPS-1 (1,249 OTUs) and PPS-2 (1,345 OTUs). Furthermore, taxonomic assignment of metagenomics sequence data showed that Proteobacteria (55.40%; 56.30%), Bacteoidetes (11.30%; 12.20%), and Planctomycetes (5.40%; 4.70%) were the most abundant phyla; Alphproteobacteria (20.50%; 23.50%), Betaproteobacteria (16.00%; 12.30%), and Gammaproteobacteria were the most recorded classes in PPS-1 and PPS-2, respectively. At the genus level, Thiobacillus (7.60%; 4.50%) was the most abundant genera grown in sediment samples. The results indicate significant differences in both the diversity and relative abundance of taxa in the bacterial communities associated with PPS-2 when compared to PPS-1. This study unveils key insights into contaminant characteristics and shifts in bacterial communities within contaminated environments. It highlights the potential for developing efficient bioremediation techniques to restore ecological balance in pulp-paper mill waste-polluted areas, stressing the importance of identifying a significant percentage of unclassified genera and species to explore novel genes. [ABSTRACT FROM AUTHOR]

Zooplankton and ichthyoplankton community assessments depend on species diagnostics, yet morphological identifications are time-consuming, require taxonomic expertise, and are hampered by a lack of diagnostic characters, particularly for larval stages. Metabarcoding can identify multiple species in communities from short DNA sequences in comparison to reference databases. To evaluate species resolution across phylogenetic groups and food webs of zooplankton and ichthyoplankton, we compare five metabarcode mitochondrial (mt)DNA markers from gene regions of (a) cytochrome c oxidase subunit I, (b) cytochrome b, (c) 16S ribosomal RNA, and (d) 12S ribosomal RNA for DNA extracted from net tows in the Northeastern Pacific Ocean's Salish Sea across seven sites and two seasons. Species resolved by metabarcoding are compared to invertebrate morphological identifications and biomass estimates. Results indicate that species resolution for different zooplankton and ichthyoplankton taxa can markedly vary among gene regions and markers in comparison to morphological identifications. Thus, researchers seeking "universal" metabarcoding should take caution that several markers and gene regions likely will be needed; all will miss some taxa and yield incomplete overlap. Species resolution requires careful attention to taxon marker selection and coverage in reference sequence repositories. In summary, combined multi-marker metabarcoding and morphological approaches improve broadscale zooplankton diagnostics. [ABSTRACT FROM AUTHOR]

Genotypic testing is often recommended to improve the management of patients infected with human immunodeficiency virus (HIV). To help combat this major pandemic, next-generation sequencing (NGS) techniques are widely used to analyse resistance to antiretroviral drugs. In this study, we used a Vela Sentosa kit (Vela Diagnostics, Kendall, Singapore), which is usually used for the Ion Torrent personal genome machine (PGM) platform, to sequence HIV using the Illumina Miseq platform. After RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR), minor modifications were applied to the Vela Sentosa kit to adapt it to the Illumina Miseq platform. Analysis of the results showed the same mutations present in the samples using both sequencing platforms. The total number of reads varied from 185,069 to 752,343 and from 642,162 to 2,074,028 in the Ion Torrent PGM platform and the Illumina Miseq platform, respectively. The average depth was 21,955 and 46,856 for Ion Torrent PGM and Illumina Miseq platforms, respectively. The cost of sequencing a run of eight samples was quite similar between the two platforms (about USD 1790 for Illumina Miseq and about USD 1833 for Ion Torrent PGM platform). We have shown for the first time that it is possible to adapt and use the Vela Sentosa kit for the Illumina Miseq platform to obtain high-quality results with a similar cost. [ABSTRACT FROM AUTHOR]

Lichens are not only fungal–algal symbiotic associations but also matrices for association with bacteria, and the bacterial diversity linked to lichens has been receiving more attention in studies. This study compares the diversity and possible metabolism of lichen-associated bacteria from saxicolous foliose and fruticose taxa Alectoria, Canoparmelia, Crocodia, Menegazzia, Usnea, and Xanthoparmelia from the Venezuelan Guiana Shield and the South African Highveld Plateau. We used DNA extractions from the lichen thalli to amplify the eukaryotic 18S rRNA gene (rDNA) and the V3–V4 region of the bacterial 16S rDNA, of which amplicons were then Sanger- and MiSeq-sequenced, respectively. The V3–V4 sequences of the associated bacteria were grouped into operational taxonomic units (OTUs) ascribed to twelve bacterial phyla previously found in the rock tripe Umbilicaria lichens. The bacterial OTUs emphasized the uniqueness of each region, while, at the species and higher ranks, the regional microbiomes were shown to be somewhat similar. Nevertheless, regional biomarker OTUs were screened to predict relevant metabolic pathways, which implicated different regional metabolic features. [ABSTRACT FROM AUTHOR]

Zooplankton and ichthyoplankton community assessments depend on species diagnostics, yet morphological identifications are time-consuming, require taxonomic expertise, and are hampered by a lack of diagnostic characters, particularly for larval stages. Metabarcoding can identify multiple species in communities from short DNA sequences in comparison to reference databases. To evaluate species resolution across phylogenetic groups and food webs of zooplankton and ichthyoplankton, we compare five metabarcode mitochondrial (mt)DNA markers from gene regions of (a) cytochrome c oxidase subunit I, (b) cytochrome b, (c) 16S ribosomal RNA, and (d) 12S ribosomal RNA for DNA extracted from net tows in the Northeastern Pacific Ocean’s Salish Sea across seven sites and two seasons. Species resolved by metabarcoding are compared to invertebrate morphological identifications and biomass estimates. Results indicate that species resolution for different zooplankton and ichthyoplankton taxa can markedly vary among gene regions and markers in comparison to morphological identifications. Thus, researchers seeking “universal” metabarcoding should take caution that several markers and gene regions likely will be needed; all will miss some taxa and yield incomplete overlap. Species resolution requires careful attention to taxon marker selection and coverage in reference sequence repositories. In summary, combined multi-marker metabarcoding and morphological approaches improve broadscale zooplankton diagnostics.

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

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

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION's versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic. [ABSTRACT FROM AUTHOR]

Pulp-paper mills (PPMs) are known for consistently generating a wide variety of pollutants, that are often unidentified and highly resistant to environmental degradation. The current study aims to investigate the changes in the indigenous bacterial communities profile grown in the sediment co-contaminated with organic and inorganic pollutants discharged from the PPMs. The two sediment samples, designated PPS-1 and PPS-2, were collected from two different sites. Physico-chemical characterization of PPS-1 and PPS-2 revealed the presence of heavy metals (mg kg−1) like Cu (0.009–0.01), Ni (0.005–0.002), Mn (0.078–0.056), Cr (0.015–0.009), Pb (0.008–0.006), Zn (0.225–0.086), Fe (2.124–0.764), Al (3.477–22.277), and Ti (99.792–45.012) along with high content of chlorophenol, and lignin. The comparative analysis of organic pollutants in sediment samples using gas chromatography–mass spectrometry (GC–MS) revealed the presence of major highly refractory compounds, such as stigmasterol, β-sitosterol, hexadecanoic acid, octadecanoic acid; 2,4-di-tert-butylphenol; heptacosane; dimethyl phthalate; hexachlorobenzene; 1-decanol,2-hexyl; furane 2,5-dimethyl, etc in sediment samples which are reported as a potential toxic compounds. Simultaneously, high-throughput sequencing targeting the V3–V4 hypervariable region of the 16S rRNA genes, resulted in the identification of 1,249 and 1,345 operational taxonomic units (OTUs) derived from a total of 115,665 and 119,386 sequences read, in PPS-1 and PPS-2, respectively. Analysis of rarefaction curves indicated a diversity in OTU abundance between PPS-1 (1,249 OTUs) and PPS-2 (1,345 OTUs). Furthermore, taxonomic assignment of metagenomics sequence data showed that Proteobacteria (55.40%; 56.30%), Bacteoidetes (11.30%; 12.20%), and Planctomycetes (5.40%; 4.70%) were the most abundant phyla; Alphproteobacteria (20.50%; 23.50%), Betaproteobacteria (16.00%; 12.30%), and Gammaproteobacteria were the most recorded classes in PPS-1 and PPS-2, respectively. At the genus level, Thiobacillus (7.60%; 4.50%) was the most abundant genera grown in sediment samples. The results indicate significant differences in both the diversity and relative abundance of taxa in the bacterial communities associated with PPS-2 when compared to PPS-1. This study unveils key insights into contaminant characteristics and shifts in bacterial communities within contaminated environments. It highlights the potential for developing efficient bioremediation techniques to restore ecological balance in pulp-paper mill waste-polluted areas, stressing the importance of identifying a significant percentage of unclassified genera and species to explore novel genes.

Indian lilac or neem (Azadirachta indica) is found in tropical and subtropical regions of the Indian subcontinent. Each part of the tree is a source of various phytochemicals. Neem gum is an exudate from mature parts of the plant stem. Biochemically, it has an acidic pH range (5–6) and is composed of monosaccharides, saponins, phenols, and tannins. This study aimed to elucidate the diversity of neem gum-associated microflora through high throughput metagenomics approach using 16S rRNA variable region sequencing. The bacterial community of neem gum was dominated by Firmicutes (~82%), Proteobacteria (~18%), and Actinobacteria (~0.02%). Among the genera, Lactococcus was found to be the most dominant bacterium. The predominance of Lactococcus in neem gum is probably due to its acidic nature, which provides a suitable microenvironment for its proliferation. In addition, Lactococcus and beneficial microorganisms such as Pseudomonas, Burkholderia, Pantoea, Klebsiella, and Methylobacterium were also present in the gum. This study highlights the fact that neem gum can be exploited as a unique source of microorganisms for biotechnological and agricultural applications.

ABSTRACT Targeted amplicon sequencing is widely used in microbial ecology studies. However, sequencing artifacts and amplification biases are of great concern. To identify sources of these artifacts, a systematic analysis was performed using mock communities comprised of 16S rRNA genes from 33 bacterial strains. Our results indicated that while sequencing errors were generally isolated to low-abundance operational taxonomic units, chimeric sequences were a major source of artifacts. Singleton and doubleton sequences were primarily chimeras. Formation of chimeric sequences was significantly correlated with the GC content of the targeted sequences. Low-GC-content mock community members exhibited lower rates of chimeric sequence formation. GC content also had a large impact on sequence recovery. The quantitative capacity was notably limited, with substantial recovery variations and weak correlation between anticipated and observed strain abundances. The mock community strains with higher GC content had higher recovery rates than strains with lower GC content. Amplification bias was also observed due to the differences in primer affinity. A two-step PCR strategy reduced the number of chimeric sequences by half. In addition, comparative analyses based on the mock communities showed that several widely used sequence processing pipelines/methods, including DADA2, Deblur, UCLUST, UNOISE, and UPARSE, had different advantages and disadvantages in artifact removal and rare species detection. These results are important for improving sequencing quality and reliability and developing new algorithms to process targeted amplicon sequences.IMPORTANCEAmplicon sequencing of targeted genes is the predominant approach to estimate the membership and structure of microbial communities. However, accurate reconstruction of community composition is difficult due to sequencing errors, and other methodological biases and effective approaches to overcome these challenges are essential. Using a mock community of 33 phylogenetically diverse strains, this study evaluated the effect of GC content on sequencing results and tested different approaches to improve overall sequencing accuracy while characterizing the pros and cons of popular amplicon sequence data processing approaches. The sequencing results from this study can serve as a benchmarking data set for future algorithmic improvements. Furthermore, the new insights on sequencing error, chimera formation, and GC bias from this study will help enhance the quality of amplicon sequencing studies and support the development of new data analysis approaches.

Cyanobacteria are favored by climate change and global warming; however, to date, most research and monitoring programs have focused on planktic cyanobacteria. Benthic cyanobacteria blooms also increase and pose a risk to animal and human health; however, there is limited knowledge of their occurrence, distribution and the toxins involved, especially in relation to their planktic conspecifics. Therefore, we analyzed the benthic and planktic life forms of cyanobacterial communities in 34 lakes in Germany, including a monitoring of cyanotoxins. Community analyses were based on microscopic examination and Illumina sequencing of the 16S rRNA gene. The analyses of cyanotoxins were carried out using LC-MS/MS and ELISA. Observed benthic mats containing cyanobacteria consisted mainly of Nostocales and Oscillatoriales, being present in 35% of the lakes. Anatoxin was the most abundant cyanotoxin in the benthic samples, reaching maximum concentrations of 45,000 µg/L, whereas microcystin was the predominate cyanotoxin in the open-water samples, reaching concentrations of up to 18,000 µg/L. Based on the results, specific lakes at risk of toxic cyanobacteria could be identified. Our findings suggest that monitoring of benthic cyanobacteria and their toxins should receive greater attention, ideally complementing existing open-water sampling programs with little additional effort. [ABSTRACT FROM AUTHOR]

Abstract Background Microbial communities in recirculating aquaculture systems (RAS) play a role in system success, nutrient cycling, and water quality. Considering the increasing socio-economic role of fish farming, e.g., regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective, especially regarding the growth, development, and welfare of the farmed animal. However, the current data on the composition of microbial communities within RAS is patchy, which is partly attributable to diverging method choices that render comparative analyses challenging. Therefore, there is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems. Results We compared sequencing approach performances (3 types of 16S short amplicon sequencing, PacBio long-read amplicon sequencing, and amplification-free shotgun metagenomics) in the characterization of microbial communities in two commercial RAS fish farms. Results showed that 16S primer choice and amplicon length affect some values (e.g., diversity measures, number of assigned taxa or distinguishing ASVs) but have no impact on spatio-temporal patterns between sample types, farms and time points. This implies that 16S rRNA approaches are adequate for community studies. The long-read amplicons underperformed regarding the quantitative resolution of spatio-temporal patterns but were suited to identify functional services, e.g., nitrification cycling and the detection of pathogens. Finally, shotgun metagenomics extended the picture to fungi, viruses, and bacteriophages, opening avenues for exploring inter-domain interactions. All sequencing datasets agreed on major prokaryotic players, such as Actinobacteriota, Bacteroidota, Nitrospirota, and Proteobacteria. Conclusion The different sequencing approaches yielded overlapping and highly complementary results, with each contributing unique data not obtainable with the other approaches. We conclude that a tiered approach constitutes a strategy for obtaining the maximum amount of information on aquaculture microbial communities and can inform basic research on community evolution dynamics. For specific and/or applied questions, single-method approaches are more practical and cost-effective and could lead to better farm management practices.

Abstract Background Intratumoral microbial communities have been recently discovered to exist in a variety of cancers and have been found to be intricately involved in tumour progression. Therefore, investigating the profiles and functions of intratumoral microbial distribution in hepatocellular carcinoma (HCC) is imperative. Methods To verify the presence of microorganisms in HCC, we performed fluorescence in situ hybridization (FISH) using HCC tissues and conducted MiSeq using 99 HCC and paracancerous tissues to identify the key microorganisms and changes in metabolic pathways affecting HCC progression through a variety of bioinformatics methods. Results Microbial diversity was significantly higher in HCC tissues than in adjacent tissues. The abundances of microorganisms such as Enterobacteriaceae, Fusobacterium and Neisseria were significantly increased in HCC tissues, while the abundances of certain antitumour bacteria such as Pseudomonas were decreased. Processes such as fatty acid and lipid synthesis were significantly enhanced in the microbiota in HCC tissues, which may be a key factor through which intratumoral microbes influence tumour progression. There were considerable differences in the microbes and their functions within tumour tissue collected from patients with different clinical features. Conclusion We comprehensively evaluated the intratumoral microbial atlas of HCC tissue and preliminarily explored the mechanism of the effects of the microbial community involving changes in lipid metabolism and effects on HCC progression, which lays the foundation for further research in this field.

Indian lilac or neem (Azadirachta indica) is found in tropical and subtropical regions of the Indian subcontinent. Each part of the tree is a source of various phytochemicals. Neem gum is an exudate from mature parts of the plant stem. Biochemically, it has an acidic pH range (5–6) and is composed of monosaccharides, saponins, phenols, and tannins. This study aimed to elucidate the diversity of neem gum-associated microflora through high throughput metagenomics approach using 16S rRNA variable region sequencing. The bacterial community of neem gum was dominated by Firmicutes (~82%), Proteobacteria (~18%), and Actinobacteria (~0.02%). Among the genera, Lactococcus was found to be the most dominant bacterium. The predominance of Lactococcus in neem gum is probably due to its acidic nature, which provides a suitable microenvironment for its proliferation. In addition, Lactococcus and beneficial microorganisms such as Pseudomonas, Burkholderia, Pantoea, Klebsiella, and Methylobacterium were also present in the gum. This study highlights the fact that neem gum can be exploited as a unique source of microorganisms for biotechnological and agricultural applications. [ABSTRACT FROM AUTHOR]

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

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

The emergence of the Coronavirus Disease 2019 (COVID-19) pandemic has fostered the use of high-throughput techniques to sequence the entire severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and track its evolution. The present study proposes a rapid and relatively less expensive sequencing protocol for 384 samples by adapting the use of an Illumina NovaSeq library to an Illumina MiSeq flow cell instrument. The SARS-CoV-2 genome sequences obtained with Illumina NovaSeq and those obtained using MiSeq instruments were compared with the objective to validate the new, modified protocol. A total of 356 (94.6%) samples yielded interpretable sequences using the modified Illumina COVIDSeq protocol, with an average coverage of 91.6%. By comparison, 357 (94.9%) samples yielded interpretable sequences with the standard COVIDSeq protocol, with an average coverage of 95.6%. Our modified COVIDSeq protocol could save 14,155 euros per run and yield results from 384 samples in 53.5 h, compared to four times 55.5 h with the standard Illumina MiSeq protocol. The modified COVIDSeq protocol thus provides high quality results comparable to those obtained with the standard COVIDSeq protocol, four times faster, while saving money. [ABSTRACT FROM AUTHOR]

Background: Microbial communities in recirculating aquaculture systems (RAS) play a role in system success, nutrient cycling, and water quality. Considering the increasing socio-economic role of fish farming, e.g., regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective, especially regarding the growth, development, and welfare of the farmed animal. However, the current data on the composition of microbial communities within RAS is patchy, which is partly attributable to diverging method choices that render comparative analyses challenging. Therefore, there is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems. Results: We compared sequencing approach performances (3 types of 16S short amplicon sequencing, PacBio long-read amplicon sequencing, and amplification-free shotgun metagenomics) in the characterization of microbial communities in two commercial RAS fish farms. Results showed that 16S primer choice and amplicon length affect some values (e.g., diversity measures, number of assigned taxa or distinguishing ASVs) but have no impact on spatio-temporal patterns between sample types, farms and time points. This implies that 16S rRNA approaches are adequate for community studies. The long-read amplicons underperformed regarding the quantitative resolution of spatio-temporal patterns but were suited to identify functional services, e.g., nitrification cycling and the detection of pathogens. Finally, shotgun metagenomics extended the picture to fungi, viruses, and bacteriophages, opening avenues for exploring inter-domain interactions. All sequencing datasets agreed on major prokaryotic players, such as Actinobacteriota, Bacteroidota, Nitrospirota, and Proteobacteria. Conclusion: The different sequencing approaches yielded overlapping and highly complementary results, with each contributing unique data not obtainable with the other approaches. We conclude that a tiered approach constitutes a strategy for obtaining the maximum amount of information on aquaculture microbial communities and can inform basic research on community evolution dynamics. For specific and/or applied questions, single-method approaches are more practical and cost-effective and could lead to better farm management practices. [ABSTRACT FROM AUTHOR]

Background: Intratumoral microbial communities have been recently discovered to exist in a variety of cancers and have been found to be intricately involved in tumour progression. Therefore, investigating the profiles and functions of intratumoral microbial distribution in hepatocellular carcinoma (HCC) is imperative. Methods: To verify the presence of microorganisms in HCC, we performed fluorescence in situ hybridization (FISH) using HCC tissues and conducted MiSeq using 99 HCC and paracancerous tissues to identify the key microorganisms and changes in metabolic pathways affecting HCC progression through a variety of bioinformatics methods. Results: Microbial diversity was significantly higher in HCC tissues than in adjacent tissues. The abundances of microorganisms such as Enterobacteriaceae, Fusobacterium and Neisseria were significantly increased in HCC tissues, while the abundances of certain antitumour bacteria such as Pseudomonas were decreased. Processes such as fatty acid and lipid synthesis were significantly enhanced in the microbiota in HCC tissues, which may be a key factor through which intratumoral microbes influence tumour progression. There were considerable differences in the microbes and their functions within tumour tissue collected from patients with different clinical features. Conclusion: We comprehensively evaluated the intratumoral microbial atlas of HCC tissue and preliminarily explored the mechanism of the effects of the microbial community involving changes in lipid metabolism and effects on HCC progression, which lays the foundation for further research in this field. [ABSTRACT FROM AUTHOR]

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

BACKGROUND: The gut microbiota (GM) plays an important role in human health and is being investigated as a possible target for new therapies. Although there are many studies showing that emodin can improve host health, emodin-GM studies are scarce. Here, the effects of emodin on the GM were investigated in vitro and in vivo. RESULTS: In vitro single bacteria cultivation showed that emodin stimulated the growth of beneficial bacteria Akkermansia, Clostridium, Roseburia, and Ruminococcus but inhibited major gut enterotypes (Bacteroides and Prevotella). Microbial community analysis from a synthetic gut microbiome model through co-culture indicated the consistentGMchange by emodin. Interestingly, emodin stimulated Clostridium and Ruminococcus (which are related to Roseburia and Faecalibacterium) in a mice experiment and induced anti-inflammatory immune cells, which may correlate with its impact on specific gut bacteria. CONCLUSION: Emodin (i) showed similar GM changes in monoculture, co-culture, and in an in vivo mice experiment and (ii) simulated regulatory T-cell immune responses in vivo. This suggest that emodin may be used to modulate the GM and improve health. [ABSTRACT FROM AUTHOR]

Lichens are not only fungal–algal symbiotic associations but also matrices for association with bacteria, and the bacterial diversity linked to lichens has been receiving more attention in studies. This study compares the diversity and possible metabolism of lichen-associated bacteria from saxicolous foliose and fruticose taxa Alectoria, Canoparmelia, Crocodia, Menegazzia, Usnea, and Xanthoparmelia from the Venezuelan Guiana Shield and the South African Highveld Plateau. We used DNA extractions from the lichen thalli to amplify the eukaryotic 18S rRNA gene (rDNA) and the V3–V4 region of the bacterial 16S rDNA, of which amplicons were then Sanger- and MiSeq-sequenced, respectively. The V3–V4 sequences of the associated bacteria were grouped into operational taxonomic units (OTUs) ascribed to twelve bacterial phyla previously found in the rock tripe Umbilicaria lichens. The bacterial OTUs emphasized the uniqueness of each region, while, at the species and higher ranks, the regional microbiomes were shown to be somewhat similar. Nevertheless, regional biomarker OTUs were screened to predict relevant metabolic pathways, which implicated different regional metabolic features.

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION’s versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic.

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

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, 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 (8 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.

Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples (n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples (n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19–34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range−0.82 to−0.98). In field-collected clinical swab samples, NDV (Ct range 12–37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq.

Bactrocera melastomatos Drew & Hancock and Bactrocera umbrosa (Fabricius) are fruit flies of the subfamily Dacinae under the family Tephritidae [1]. B. melastomatos occurs in India (Andaman Island), Thailand, Peninsular Malaysia, Singapore, and Indonesia (Sumatra, Kalimantan, Java) [1] while B. umbrosa is distributed from southern Thailand and Malaysia to New Guinea and New Caledonia [2]. The adult male flies of B. melastomatos are attracted to Cue lure while the adult male flies of B. umbrosa are attracted to methyl eugenol [3]. Fruit flies of Bactrocera melastomatos infest Melastomataceae while those of B. umbrosa infest Moraceae. We compare the diversity of microbiota associated with the wild adult males of these two specialist fruit flies infesting different families of host plants. Targeted 16S rRNA gene (V3-V4 region) was sequenced using the Illumina MiSeq platform. Six bacterial phyla (Actinobacteria, Armatimonadetes, Bacteroidetes, Cyanobacteria/Melainabacteria group, Firmicutes, Proteobacteria) were detected at 97% similarity clustering and 0.001% abundance filtering. Four phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria) were present in all the specimens studied. Proteobacteria was the predominant phylum in both B. melastomatos and B. umbrosa. Enterobacteriaceae was the predominant family in UM B. melastomatos and B. umbrosa, and Orbaceae was the predominant family in Awana B. melastomatos. Klebsiella was the predominant genus in B. umbrosa, Citrobacter in UM B. melastomatos, and Orbus in Awana B. melastomatos. Double Wolbachia infections were present in UM B. melastomatos. In general, the bacterial diversity and richness varied within and between the samples of B. melastomatos and B. umbrosa.

IntroductionJapanese encephalitis virus (JEV) is a mosquito-borne viral pathogen, which is becoming a growing public health concern throughout the Indo-Pacific. Five genotypes of JEV have been identified. Current vaccines are based on genotype III and provide a high degree of protection for four of the five known genotypes.MethodsRT-PCR, Magpix, Twist Biosciences Comprehensive Viral Research Panel (CVRP), and SISPA methods were used to detect JEV from mosquito samples collected in South Korea during 2021. These methods were compared to determine which method would be most effective for biosurveillance in the Indo-Pacific region.ResultsOur data showed that RT-PCR, Twist CVRP, and SISPA methods were all able to detect JEV genotype I, however, the proprietary Magpix panel was only able to detect JEV genotype III. Use of minION sequencing for pathogen detection in arthropod samples will require further method development.ConclusionBiosurveillance of vectorborne pathogens remains an area of concern throughout the Indo-Pacific. RT-PCR was the most cost effective method used in the study, but TWIST CVRP allows for the identification of over 3,100 viral genomes. Further research and comparisons will be conducted to ensure optimal methods are used for large scale biosurveillance.

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

BACKGROUND: As major stress hormones, glucocorticoids can directly or indirectly affect the intestinal microflora, although few studies have focused on changes in the composition of the intestinal microflora. In this study, rabbits were randomly divided into two groups: gavage administration with saline, and the same doses of dexamethasone (1 mg kg−1). After 7 days, the microbial diversity of the jejunum contents was analysed. RESULTS: The gut microflora richness and diversity had no significant difference between the two groups. The proportions of Firmicutes and Bacteroidetes were the most abundant in the jejunum of meat rabbits. Dexamethasone injection led to a change in the structure of the gut microflora composition, and we found that there were six biomarkers with linear discriminant analysis score >4 (Firmicutes, Caproiciproducens, Clostridiales, Clostridia, Psychrobacter, and Psychrobacter faecalis), moreover, the results of this study provide new insight into alleviating the effects of stress on meat rabbits. CONCLUSION: It was concluded that glucocorticoids caused changes in the composition of intestinal microflora. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Increased research attention is being given to bacterial diversity associated with lichens. Rock tripe lichens (Umbilicariaceae) were collected from two distinct Antarctic biological regions, the continental region near the Japanese Antarctic station (Syowa Station) and the maritime Antarctic South Orkney Islands (Signy Island), in order to compare their bacterial floras and potential metabolism. Bulk DNA extracted from the lichen samples was used to amplify the 18S rRNA gene and the V3-V4 region of the 16S rRNA gene, whose amplicons were Sanger- and MiSeq-sequenced, respectively. The fungal and algal partners represented members of the ascomycete genus Umbilicaria and the green algal genus Trebouxia, based on 18S rRNA gene sequences. The V3-V4 sequences were grouped into operational taxonomic units (OTUs), which were assigned to eight bacterial phyla, Acidobacteriota, Actinomyceota, Armatimonadota, Bacteroidota, Cyanobacteria, Deinococcota, Pseudomonadota and the candidate phylum Saccharibacteria (also known as TM7), commonly present in all samples. The OTU floras of the two biological regions were clearly distinct, with regional biomarker genera, such as Mucilaginibacter and Gluconacetobacter, respectively. The OTU-based metabolism analysis predicted higher membrane transport activities in the maritime Antarctic OTUs, probably influenced by the sampling area's warmer maritime climatic setting. [ABSTRACT FROM AUTHOR]

Traditional detection of aquatic invasive species via morphological identification is often time‐consuming and can require a high level of taxonomic expertise, leading to delayed mitigation responses. Environmental DNA (eDNA) detection approaches of multiple species using Illumina‐based sequencing technology have been used to overcome these hindrances, but sample processing is often lengthy. More recently, portable nanopore sequencing technology has become available, which has the potential to make molecular detection of invasive species more widely accessible and substantially decrease sample turnaround times. However, nanopore‐sequenced reads have a much higher error rate than those produced by Illumina platforms, which has so far hindered the adoption of this technology. We provide a detailed laboratory protocol and bioinformatic tools (msi package) to increase the reliability of nanopore sequencing to detect invasive species, and we test its application using invasive bivalves while comparing it with Illumina‐based sequencing. We sampled water from sites with pre‐existing bivalve occurrence and abundance data, and contrasting bivalve communities, in Italy and Portugal. Samples were extracted, amplified, and sequenced by the two platforms. The mean agreement between sequencing methods was 69% and the difference between methods was nonsignificant. The lack of detections of some species at some sites could be explained by their known low abundances. This is the first reported use of MinION to detect aquatic invasive species from eDNA samples. [ABSTRACT FROM AUTHOR]

Abstract 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.

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

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

BackgroundQiweibaizhu powder (QWBZP) has been shown to be effective in treating antibiotic-associated diarrhea (AAD). Previous research has reported that plant polysaccharides can promote the growth of beneficial intestinal bacteria and inhibit the multiplication of pathogenic bacteria, thus effectively treating diarrhea. Here, we investigated the effect of QWBZP crude polysaccharide on the diversity of intestinal mucosal bacteria and their community structure composition in mice with AAD, and the aim of this study was to provide the scientific basis for the efficacy of QWBZP crude polysaccharide on diarrhea.Materials and MethodsEighteen Kunming (KM) mice were randomly divided into the normal (mn) group, the model (mm) group, and the QWBZP crude polysaccharide treatment (ma) group, with six mice in each group. An AAD model was constructed using a mixed antibiotic solution and treated with gavage crude polysaccharide solution of QWBZP. The intestinal mucosa was extracted from the jejunum to the ileum of mice, and the metagenome was extracted and then analyzed using MiSeq sequencing to characterize the intestinal mucosal bacteria in mice.ResultsThe spleen and thymus indices of each group of mice had no significant differences. The Chao1 and ACE indices of the mn and mm groups were similar, the Simpson index was the largest and the Shannon index was the smallest in the mm group, and there was no significant difference in the diversity indices of all three groups. In the PCA and PCoA, the mn and ma group samples were both relatively concentrated with a high similarity of community structure. In contrast, the samples in the mm group were more scattered and farther away from the samples in the mn and ma groups, i.e., the community structure similarity within and between the groups was low. Compared with the mm group, the relative abundance of Proteobacteria, Lactobacillus, and the Firmicutes/Bacteroidetes (F/B) ratio in the ma group was decreased, while that of Enterococcus continued to increase. In the LEfSe analysis, there were significant differences in the characteristic bacteria in the mn, mm, and ma groups.ConclusionThe single crude polysaccharide component is not very effective in treating AAD, so the clinical efficacy of the QWBZP crude polysaccharide is subject to further investigation.

Aims: Yak is a dominant ruminant, well adapted to grazing on pasture year around in the harsh climate of the 3000‐meter‐high Qinghai‐Tibetan Plateau. The complex microbial community that resides within the yak rumen is responsible for fermentation and contributes to its climatic adaptation. This study aimed to characterize the rumen microbiota responses to wide seasonal variations, especially those necessary for survival in the cold seasons. Methods and Results: In the present study, we performed 16s rRNA gene sequencing to investigate the seasonal variations in microbiota composition, diversity and associated volatile fatty acids (VFAs) in yak rumen. The results showed that rumen microbiota were dominated by Bacteroides (72.13%–78.54%) and Firmicutes; the relative abundance of Firmicutes was higher in summer (17.44%) than in winter (10.67%; p < 0.05). The distribution of taxa differed among spring, summer and winter rumen communities (PERMANOVA, p = 0.001), whereas other taxa (e.g., Fibrobacter, Verrucomicrobia, Anaerostipes and Paludibacter), which could potentially help overcome harsh climate conditions were observed in higher abundance during the cold spring and winter seasons. The highest total VFA concentration in the yak rumen was obtained in summer (p < 0.05), followed by spring and winter, and both positive and negative correlations between VFAs and specific genera were revealed. Conclusions: Microbiota in yak rumen appear to be highly responsive to seasonal variations. Considering environmental factors, we suggest that seasonal adaptation by microbial communities in rumen enables their hosts to survive seasonal scarcity and cold stress in the spring and winter. Significance and Impact of Study: The present study furthers our understanding of how microbial adaptation to seasonal variations in nutrient availability and climate may function in high plateau ruminants, providing insights into the tripartite relationship between the environment, host and microbiota. [ABSTRACT FROM AUTHOR]

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

This study analyzed the impact of land use changes on the structure and function of soil fungal communities in the Sanjiang Plain wetland. Soil samples were collected from natural wetlands, paddy fields, and restored wetlands in the Sanjiang Plain, after which the internal transcribed spacer (ITS) of soil fungi was sequenced by Miseq technology. Our results demonstrated that the reclamation of wetlands into paddy fields can significantly reduce the α diversity index of soil fungi (P <0.05), whereas the restoration of wetlands can significantly increase the soil fungus Chao1 index (P <0.05). A total of 16 phyla were identified in three types of land use soil, among which Ascomycota, Basidiomycota, Mortierellomycota, and Rozellomycota were the most dominant (relative abundance > 1%). Mycorrhizal fungi and parasitic fungi are the main functional groups of fungi in natural wetlands and restored wetlands, whereas saprophytes were the main functional groups of paddy fields. Soil pH, total carbon, organic carbon, total nitrogen, and total phosphorus are the main factors affecting the diversity of soil fungal communities. Therefore, our findings indicated that resource development in natural wetlands increases potential ecological risks and reduces ecosystem stability. [ABSTRACT FROM AUTHOR]

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

Abstract Background The guts of blood-sucking insects host a community of bacteria that can shift dramatically in response to biotic and abiotic factors. Identifying the key factors structuring these microbial communities has important ecological and epidemiological implications. Methods We used the yellow fever mosquito, Aedes aegypti, to investigate the impact of mixed blood meals on gut microbiota of vector mosquitoes. Adult females were experimentally fed on sugar or blood from chicken, rabbit or a mixture of chicken and rabbit blood, and their gut microbiota were characterized using 16S rRNA gene amplification and MiSeq sequencing. Results The gut bacterial communities of mosquitoes fed on the three blood meal treatments clustered separately, suggesting that host species identity and mixed blood-feeding are key determinants of gut bacterial community composition in mosquitoes. Mixed blood meal had a synergistic effect on both operational taxonomic unit (OTU) richness and the Shannon diversity index, suggesting that mixed blood-feeding can offset the nutritional deficit of blood meals from certain host species. The microbial communities observed in this study were distinct from those identified from similarly fed Ae. aegypti from our previous study. Conclusions These findings demonstrate that vector host-feeding preferences can influence gut microbial composition and diversity, which could potentially impact pathogen acquisition and transmission by the vector. The results also demonstrate that different microenvironmental conditions within the laboratory may play an important role in structuring the microbial communities of independently reared mosquito colonies.

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

Premise: Foliar fungal endophytes vary in their distributions across landscapes or plant host taxa, indicative of specialized ecologies and host specific adaptations. Accounts of specialization, however, depend on the taxonomic breadth and geographic range of the host plants included in each study. A broad region‐scale study or deep sampling of diverse potential host species still remains relatively rare but is becoming increasingly possible with high‐throughput sequencing. Methods: Amplicon sequencing was used to rapidly identify the fungal endophytic community among six pine (Pinus, Pinaceae) species co‐occurring across northeastern United States and to test for site and host specialization. We focused on the endophytic genus Lophodermium (Rhytismataceae), whose species members are thought to specialize on different pine species, to test if amplicon sequencing could rapidly verify previously implied or discover new patterns of host specificity. Results: While amplicon sequencing could analyze more samples at greater depths and recover greater numbers of unique Lophodermium taxa than when endophyte communities were surveyed with traditional culturing methods, patterns of specialization were not better supported. This may be because amplicon sequencing can indiscriminately capture non‐host specific organisms found incidentally from plant tissues or because we have overestimated host‐specificity in the past with biased culturing techniques. Conclusions: Amplicon sequencing can quickly identify patterns of host specificity by allowing large‐scale surveys but has limitations in quantifying the level of intimacy of these relationships. [ABSTRACT FROM AUTHOR]

Abstract Background The major histocompatibility complex (MHC) is responsible for detecting and addressing foreign pathogens inside the body. While the general structure of MHC genes is relatively well conserved among mammalian species, it is notably different among ruminants due to a chromosomal inversion that splits MHC type II genes into two subregions (IIa, IIb). Recombination rates are reportedly high between these subregions, and a lack of linkage has been documented in domestic ruminants. However, no study has yet examined the degree of linkage between these subregions in a wild ruminant. The white-tailed deer (Odocoileus virginianus), a popular ruminant of the Cervidae family, is habitually plagued by pathogens in its natural environment (e.g. Haemonchus contortus, Elaeophora). Due to the association between MHC haplotypes and disease susceptibility, a deeper understanding of MHC polymorphism and linkage between MHC genes can further aid in this species’ successful management. We sequenced MHC-DRB exon 2 (IIa) and MHC-DOB exon 2 (IIb) on the MiSeq platform from an enclosed white-tailed deer population located in Alabama. Results We identified 12 new MHC-DRB alleles, and resampled 7 alleles, which along with other published alleles brings the total number of documented alleles in white-tailed deer to 30 for MHC-DRB exon 2. The first examination of MHC-DOB in white-tailed deer found significantly less polymorphism (11 alleles), as was expected of a non-classical MHC gene. While MHC-DRB was found to be under positive, diversifying selection, MHC-DOB was found to be under purifying selection for white-tailed deer. We found no significant linkage disequilibrium between MHC-DRB and MHC-DOB, suggesting that these loci are unlikely to be closely linked. Conclusions Overall, this study identified 12 new MHC-DRB exon 2 alleles and characterized a new, non-classical, MHC II gene (MHC-DOB) for white-tailed deer. We also found a lack of significant linkage between these two loci, which supports previous findings of a chromosomal inversion within the MHC type II gene region in ruminants, and suggests that white-tailed deer may have a recombination hotspot between these MHC regions similar to that found for Bos taurus.

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.