Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

Generating 1200bp tiled Amplicons for SARS-CoV2 Whole Genome Sequencing v2

This procedure provides instructions for how to generate amplicons across the entire SARS-CoV-2 genome to be used for downstream whole genome sequencing applications, including Illumina MiSeq/NextSeq or Oxford Nanopore MinION sequencing platforms. The steps involved in this protocol were derived from version 3 of Freed et al protocol nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon)V.3 available at https://dx.doi.org/10.17504/protocols.io.bgggjttw

Spatial and Seasonal Structure of Bacterial Communities Within Alpine Vineyards: Trentino as a Case Study

Bacteria have a fundamental role in determining the fitness of grapevine, the composition of grapes and the features of wines but at present, little information is available. In this work, the bacteria colonizing the different portions of grapevine (bark, leaves and grapes) were explored in the vineyards of the Alpine region of Trentino, considering the impact of different environmental and agronomical variables. The vineyards included in the work were selected based on their different geographical positions (altitude) and grapevine training systems in order to explore the whole variability of the grapevine ecosystem. Moreover, the surface amount of copper was measured on grapes and leaves during the vegetative growth. Bacterial analysis, performed using plate counts and Illumina MiSeq, revealed an increase in the concentration of grape bacteria proportional to the progress of the ripening stage. Conversely, the peak of bacterial concentration onto leaf and bark samples occurred in August, probably due to the more favourable environmental conditions. In bark samples, the bacterial microbiota reached the 7 log CFU/cm2, while 6 log UFC/g were measured in grape samples. A remarkable biodiversity was observed, with 13 phyla, 35 classes, 55 orders, 78 families and 95 genera of bacteria present. The presence of some taxa (Alphaproteobacteria, Desulfovibrionaceae, Clostriadiales, Oscillospira, Lachnospiraceae and Bacteroidales) was ubiquitous in all vineyards, but differences in terms of relative abundance were observed according to the vegetative stage, altitude of the vineyard and training system. Bacteria having oenological implication (Lactobacillus, Pediococcus and Oenococcus) were detected in grape samples collected in August, in low abundance. The data revealed a complex bacterial ecosystem inside the vineyard that, while maintaining common traits, evolves according to environmental and agronomical inputs. This study contributes to define the role of bacteria in the complex balance established in each vineyard between human actions and agricultural environment, known as terroir.

Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed.

Variants of the HNF4A and HNF1A genes in patients with impaired glucose metabolism and dyslipidemia

Maturity onset diabetes of the young is a dominantly inherited form of monogenic diabetes, diagnosed mainly before the age of 35 years. Mutations in the HNF1A and HNF4A genes are associated with diabetes mellitus of the HNF1A-MODY and HNF4A-MODY subtypes, respectively. These two forms of MODY are characterized by dyslipidemia in addition to impaired glucose metabolism due to the altered function HNF1A and HNF4A proteins. The aim of this study was a genetic analysis of young patients with the MODY phenotype and dyslipidemia with a burdened family history. Material and methods. The probands underwent targeted DNA sequencing using the Illumina MiSeq NGS System. The target panel included the coding regions and splicing sites of MODY-associated genes: HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, KCNJ11, ABCC8, and APPL1. Results. A heterozygous single nucleotide deletion NM_000457.4: c.153del (3’rule) was found in proband P1 in the HNF4A gene. In proband P2, single nucleotide deletion NM_000545.8: c.335del (3 ‘rule) in the HNF1A gene was detected in a heterozygous state. Both variants are located in the coding parts of the genes, led to a shift in the reading frame and have not been described in the literature and databases earlier. Conclusions. Taking into account the phenotypic features of probands, we assume that the variants NM_000545.8: c.335del (rule 3) in the HNF1A gene and NM_000457.4: c.153del (rule 3) of the HNF4A gene are associated with different MODY subtypes in these individuals. After verification of MODY-HNF1A and MODY-HNF4A diagnosis, it is necessary to monitor the lipid profile parameters (total cholesterol, low and high density lipoprotein cholesterol, triglycerides) and prescribe appropriate drug therapy.

The Influence of Residue Mixing on the Decomposition of Pepper Root Residues

(1) Background: Residue degradation plays a very important role in terrestrial ecosystems and residue mixing is the main factor affecting the degradation rates. However, in the agricultural systems, the effect of residue mixing on the degradation of pepper residues and the microbial community in pepper root residues is not clear. (2) Methods: In this study, we added different residues into soil by using double-layered nylon litterbags in culture bottles. The treatments including pepper root (P: Capsicum annuum L.), soybean [S: Glycine max (L.) Merr.] and maize (M: Zea mays L.) residue, as well as mixtures of soybean + pepper (SP), maize + pepper (MP), maize + soybean + pepper (MSP) mixtures. Litterbags were harvested after 7, 14, 28, and 56 days, respectively. Mass loss and nitrogen and phosphorus contents in pepper residue were quantified and bacterial and fungal community levels in pepper residues were analyzed using quantitative PCR and high throughput amplicon sequencing; (3) Results: The study showed that the mass loss and fungal community abundance of pepper root residue in mixtures were higher than P, except day 7. The phosphorus contents in MSP-P and MP-P were significantly lower than that for P at day 28 and day 56. Illumina MiSeq sequencing showed that the presence of maize residue significantly altered the microbial community composition of pepper root pepper. Day 56. (4) Conclusions: Our results suggest that residue mixing changed the microbial community abundance in pepper residue and promoted the degradation of pepper residues compared to pepper residue decomposition alone, especially for mixtures with soybean.

Wheat Production Alters Soil Microbial Profiles and Enhances Beneficial Microbes in Double-Cropping Soybean

Plant-parasitic nematodes represent a substantial constraint on global food security by reducing the yield potential of all major crops. The soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is widely distributed across important soybean production areas of the U.S., being the major soybean yield-limiting factor, especially in the Midwestern U.S. Double cropped (DC) soybean is commonly planted following winter wheat. We previously reported double-cropping soybean fields with reduced SCN counts compared to fallow at both R1 growth stage (beginning of flowering) (−31.8%) and after soybean harvest (−32.7%). To test if higher counts of beneficial and SCN antagonistic microorganisms could be correlated with the suppression of SCN in fields previously planted with wheat, three field locations with noted SCN suppression were selected for a metagenomics study. Ten subplots were selected (5 wheat and 5 fallow pre-soybean) from each location. A total of 90 soil samples were selected: 3 fields ×2 treatments × 3 timepoints × 5 replications. Three DNA markers targeted distinct microbial groups: bacteria (16S V4-V5), fungi (ITS2), and Fusarium (tef1). Amplicons were sequenced using an Illumina MiSeq platform (300 bp paired-end). Sequencing datasets were processed in R using the DADA2 pipeline. Fungal populations were affected by location in all sampling periods and differed significantly between DC and fallow plots at soybean planting and after harvest (P < 0.001). Several enriched fungal and bacterial taxa in wheat plots, including Mortierella, Exophiala, Conocybe, Rhizobacter spp., and others, were previously reported to parasitize SCN and other plant-parasitic nematodes, suggesting a potential role of beneficial microbes in suppression of SCN in soybean fields double-cropped with wheat.

Heterogeneity of Soil Bacterial and Bacteriophage Communities in Three Rice Agroecosystems and Potential Impacts On Nutrient Cycling

Background: As genetic entities infecting and replicating only in bacteria, bacteriophages can regulate the community structure and functions of their host bacteria, but they are often overlooked because of their relatively low abundance. The ecological roles of bacteriophages in aquatic and forest environments have been widely explored, but those in agroecosystems remains limited. Here, we used metagenomic sequencing to analyze the diversity and interactions of bacteriophages and their host bacteria in soils from three typical rice agroecosystems in China: double cropping in Guangzhou, southern China, rice–wheat rotation cropping in Nanjing, eastern China and early maturing single cropping in Jiamusi, northeastern China. Bacteriophages were isolated and their functions on soil nitrogen cycling and effect on soil bacterial community structure were verified in pot inoculation experiments and Illumina MiSeq sequencing.Results: Soil bacterial and viral diversity and functions varied among the three agroecosystems. Genes detected in communities from the three agroecosystems were associated with typical functions; soil bacteria in Jiamusi were significantly enriched in genes related to carbohydrate metabolism, in Nanjing with xenobiotics biodegradation and metabolism, and in Guangzhou with virulence factors and scarce in secondary metabolite biosynthesis, which might lead to a significant occurrence of rice bacterial diseases. In the three ecosystems, 368 species of virus were detected. Notably, over-represented auxiliary carbohydrate-active enzyme (CAZyme) genes were identified in the viruses, which might assist host bacteria in metabolizing carbon, and 67.43% of these genes were present in Jiamusi. In bacteriophage isolation and inoculation experiments, Enterobacter bacteriophage-NJ reduced the nitrogen fixation capacity of soil by lysing N-fixing host bacteria and changed the soil bacterial diversity and community structure.Conclusions: Our results showed that diversity and function of paddy soil bacteria and viruses varied in the three agroecosystems. Soil bacteriophages can affect nutrient cycling by expressing auxiliary metabolic genes (AMGs) and lysing the host bacteria that are involved in biogeochemical cycles. These findings form a basis for better understanding bacterial and bacteriophage diversity in different rice agroecosystems, laying a solid foundation for further studies of soil microbial communities that support ecofriendly production of healthy rice.

The Depth-Depended Fungal Diversity and Non-depth-Depended Aroma Profiles of Pit Mud for Strong-Flavor Baijiu

Microorganisms in pit mud are the essential factor determining the style of strong flavor Baijiu. The spatial distribution characteristics of fungal communities and aroma in the pit mud for strong flavor Baijiu from Xinjiang, China, were investigated using Illumina MiSeq high-throughput sequencing and electronic nose technology. A total of 138 fungal genera affiliated with 10 fungal phyla were identified from 27 pit mud samples; of these, Saccharomycopsis, Aspergillus, and Apiotrichum were the core fungal communities, and Aspergillus and Apiotrichum were the hubs that maintain the structural stability of fungal communities in pit mud. The fungal richness and diversity, as well as aroma of pit mud, showed no significant spatial heterogeneity, but divergences in pit mud at different depths were mainly in pH, total acid, and high abundance fungi. Moisture, NH4+, and lactate were the main physicochemical factors involved in the maintenance of fungal stability and quality in pit mud, whereas pH had only a weak effect on fungi in pit mud. In addition, the fungal communities of pit mud were not significantly associated with the aroma. The results of this study provide a foundation for exploring the functional microorganisms and dissecting the brewing mechanism of strong flavor Baijiu in Xinjiang, and also contributes to the improvement of pit mud quality by bioaugmentation and controlling environmental physicochemical factors.

Diversity of bacterium communities in saline-alkali soil in arid regions of Northwest China

Background The saline-alkali soil area accounts for over 1/4-1/5 of the land area in Gansu Province of China, which are mainly distributed in the north of Hexi corridor and Jingtai basin. The unique ecological environment contains unique and diverse microbial resources. The investigation of microbial diversity in saline environment is vital to comprehend the biological mechanisms of saline adaption, develop and utilize microbial resources. Results The Illumina MiSeq sequencing method was practiced to investigate the bacterial diversity and composition in the 5 subtypes and 13 genera of saline-alkali soil in Gansu Province, China. The results from this study show that Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Gemmatimonadetes were the dominant bacterial groups in 13 saline soil. Proteobacteria had the greatest abundance in sulfate-type meadow solonchaks and orthic solonchaks, chloride-type orthic solonchaks and bog solonchaks, sulfate-chloride-type, chloride-sulfate-type, and sulfate-type dry solonchaks. Halobacteria was the dominant bacterial class in soil samples except for sulfate-type meadow solonchaks and orthic solonchaks, chloride-type orthic solonchaks and bog solonchaks. The richness estimators of Ace and Chao 1 and the diversity indices of Shannon and Simpson revealed the least diversity in bacterial community in sulfate-chloride-type orthic solonchaks. Conclusions The sulfate anion was the most important driving force for bacterial composition (17.7%), and the second most influencing factor was pH value (11.7%).