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

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

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Butt, Kariithi, Volkening, Taylor, Leyson, Pantin-Jackwood, Suarez, Stanton and Afonso.)

Introduction: Japanese 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., Methods: RT-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., Results: Our 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., Conclusion: Biosurveillance 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Crispell, Williams, Zielinski, Iwami, Homas and Thomas.)

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 ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (© 2022 The Authors. Published by Elsevier Inc.)

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. [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., (© 2022 Society of Chemical Industry.)

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.

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., (© 2022 John Wiley & Sons Ltd.)

Background: Qiweibaizhu 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 Methods: Eighteen 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., Results: The 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., Conclusion: The 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Zhou, Xiao, Peng and Tan.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Conventional culture-based techniques are largely inadequate in elucidating the microbiota contained in an environment, due to low recovery within a complex bacterial community. This limitation has been mitigated by the use of next-generation sequencing (NGS)-based approaches thereby facilitating the identification and classification of both culturable and uncultivable microorganisms. Amplicon targeted NGS methods, such as 16S ribosomal RNA (16S rRNA) and shotgun metagenomics, are increasingly being applied in various settings such as in food production environments to decipher the microbial consortium therein. Even though multiple food matrices/food production environments have been studied, the low-moisture environment associated with bakery food production remains to be investigated. To address this knowledge gap, in this study, we investigated the microbiome associated with two bakery production sites (designated as A and B) located in Ireland using 16S rRNA-amplicon-based sequencing. Amplicons corresponding to a hypervariable region contained within the 16S rRNA gene were amplified from DNA samples purified from environmental swabs and ingredients collected at both sites at various stages (preparation, production, postproduction, and storage) across the bakery production chain, over three seasons (winter, spring, and summer). These amplicons were sequenced, and data were analyzed using the mothur pipeline and visualized using MicrobiomeAnalyst and a series of R packages. The top seven bacterial phyla identified at both sites were composed of Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Patescibacteria, and Verrucomicrobia. In addition, the phyla Tenericutes (Mycoplasmatota) and Acidobacteria were observed only in samples taken at site B. Different bacterial compositions were identified at each stage of production. These same bacteria were also found to be present in the final processed food suggesting the influence of the environment on the food matrix. This study is the first demonstration of the utility of 16S rRNA amplicon-based sequencing to describe the microbiota associated with bakery processing environments.

Background: Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent's PGM, Pacific Biosciences' RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Results: Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. Conclusions: All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support. [ABSTRACT FROM AUTHOR]

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

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

The 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. [ABSTRACT FROM AUTHOR]

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. [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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Simple Summary: Decomposition is a complex process that involves several factors, such as temperature, pH, humidity, and microbes. Microbes play a significant role in the carcass decomposition process. Microcosm burial set-ups were prepared and poultry carcasses were decomposed for 60 days in unsterilized and sterilized soil, incubated under aerobic or anaerobic conditions. The moisture content, pH, alpha and beta diversity were affected by the soil microbial community and oxygen availability during the decomposition of poultry carcasses. The bacterial taxa composition was also altered during the poultry carcass decomposition. These changes suggested that the soil with an intact microbial community and oxygen availability influenced the bacterial community structure during the decomposition of poultry carcasses. The results of this study provided information on the different bacterial species which might be associated with the decomposition of poultry carcasses. The impact of soil with an intact microbial community and oxygen availability on moisture content, soil pH, and bacterial communities during decomposition of poultry carcasses was investigated. Poultry carcasses were decomposed in soil with or without a microbial community, under aerobic or anaerobic conditions. The samples collected in each microcosm burial set-up were analyzed by targeted 16S rRNA amplicon sequencing and Amplicon sequence variants (ASV) method. Our results showed that moisture was high in the burial set-ups under anaerobic conditions and pH was high in the burial set-ups under aerobic conditions. Meanwhile, the Chao1 and Shannon index significantly differed between the different burial set-ups and across different time points. In addition, bacterial taxa composition during the early period of decomposition differed from that of the late period. A total of 23 phyla, 901 genera, and 1992 species were identified. Firmicutes was the most dominant phyla in all burial set-ups throughout the decomposition. At day 60, Pseudogracilibacillus was dominant in the burial set-ups under aerobic conditions, while Lentibacillus dominated in the burial set-ups under anaerobic conditions. This study demonstrated that the soil microbial community and availability of oxygen significantly affected the changes in moisture content, pH, and bacterial composition during the decomposition process. [ABSTRACT FROM AUTHOR]

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

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2021 The Society for Applied Microbiology.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)