Metagenomic Information Recovery from Human Stool Samples Is Influenced by Sequencing Depth and Profiling Method.

Sequencing of the 16S rRNA gene (16S) has long been a go-to method for microbiome characterization due to its accessibility and lower cost compared to shotgun metagenomic sequencing (SMS). However, 16S sequencing rarely provides species-level resolution and cannot provide direct assessment of other taxa (e.g., viruses and fungi) or functional gene content. Shallow shotgun metagenomic sequencing (SSMS) has emerged as an approach to bridge the gap between 16S sequencing and deep metagenomic sequencing. SSMS is cost-competitive with 16S sequencing, while also providing species-level resolution and functional gene content insights. In the present study, we evaluated the effects of sequencing depth on marker gene-mapping- and alignment-based annotation of bacteria in healthy human stool samples. The number of identified taxa decreased with lower sequencing depths, particularly with the marker gene-mapping-based approach. Other annotations, including viruses and pathways, also showed a depth-dependent effect on feature recovery. These results refine the understanding of the suitability and shortcomings of SSMS, as well as annotation tools for metagenomic analyses in human stool samples. Results may also translate to other sample types and may open the opportunity to explore the effect of sequencing depth and annotation method.

Revealing the Draft Genome Sequence of Bradyrhizobium sp. Strain USDA 3458, an Effective Symbiotic Diazotroph Isolated from Cowpea (Vigna unguiculata) Genotype IT82E-16.

Pairing plants with plant growth-promoting bacteria is critical to the future of agriculture. Bradyrhizobium sp. strain USDA 3458 isolated from Vigna unguiculata (cowpea) paired with cowpea genotype IT82E-16 represents a novel combination in arid regions. Here, we report the draft genome sequence of strain USDA 3458.

Elucidation of the Genome of Bradyrhizobium sp. Strain USDA 3456, a Historic Agricultural Diazotroph from Cowpea (Vigna unguiculata).

Bradyrhizobium sp. strain USDA 3456 is a historic strain from the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) National Rhizobium Germplasm Collection isolated from Vigna unguiculata (cowpea) in 1966. Strain USDA 3456 has been utilized in global agricultural applications, including improving soil nitrogen fertility. The draft genome sequence here provides a genetic reference of a novel diazotroph.

Rapid establishment of a flowering cline in Medicago polymorpha after invasion of North America.

To establish and spread in a new location, an invasive species must be able to carry out its life cycle in novel environmental conditions. A key trait underlying fitness is the shift from vegetative to reproductive growth through floral development. In this study, we used a common garden experiment and genotyping-by-sequencing to test whether the latitudinal flowering cline of the North American invasive plant Medicago polymorpha was translocated from its European native range through multiple introductions, or whether the cline rapidly established due to evolution following a genetic bottleneck. Analysis of flowering time in 736 common garden plants showed a latitudinal flowering time cline in both the native and invaded ranges where genotypes from lower latitudes flowered earlier. Genotyping-by-sequencing of 9,658 SNPs in 446 individuals revealed two major subpopulations of M. polymorpha in the native range, only one of which is present in the invaded range. Additionally, native range populations have higher genetic diversity than invaded range populations, suggesting that a genetic bottleneck occurred during invasion. All invaded range individuals are closely related to plants collected from native range populations in Portugal and southern Spain, and population assignment tests assigned invaded range individuals to this same narrow source region. Taken together, our results suggest that latitudinal clinal variation in flowering time has rapidly evolved across the invaded range despite a genetic bottleneck following introduction.

Associative nitrogen fixation (ANF) in switchgrass (Panicum virgatum) across a nitrogen input gradient.

Associative N fixation (ANF), the process by which dinitrogen gas is converted to ammonia by bacteria in casual association with plants, has not been well-studied in temperate ecosystems. We examined the ANF potential of switchgrass (Panicum virgatum L.), a North American prairie grass whose productivity is often unresponsive to N fertilizer addition, via separate short-term 15N2 incubations of rhizosphere soils and excised roots four times during the growing season. Measurements occurred along N fertilization gradients at two sites with contrasting soil fertility (Wisconsin, USA Mollisols and Michigan, USA Alfisols). In general, we found that ANF potentials declined with long-term N addition, corresponding with increased soil N availability. Although we hypothesized that ANF potential would track plant N demand through the growing season, the highest root fixation rates occurred after plants senesced, suggesting that root diazotrophs exploit carbon (C) released during senescence, as C is translocated from aboveground tissues to roots for wintertime storage. Measured ANF potentials, coupled with mass balance calculations, suggest that ANF appears to be an important source of N to unfertilized switchgrass, and, by extension, to temperate grasslands in general.

Methodologies for probing the metatranscriptome of grassland soil.

Metatranscriptomics provides an opportunity to identify active microbes and expressed genes in complex soil communities in response to particular conditions. Currently, there are a limited number of soil metatranscriptome studies to provide guidance for using this approach in this challenging matrix. Hence, we evaluated the technical challenges of applying soil metatranscriptomics to a highly diverse, low activity natural system. We used a non-targeted rRNA removal approach, duplex nuclease specific (DSN) normalization, to generate a metatranscriptomic library from field collected soil supporting a perennial grass, Miscanthus x giganteus (a biofuel crop), and evaluated its ability to provide insight into its active community members and their expressed protein-coding genes. We also evaluated various bioinformatics approaches for analyzing our soil metatranscriptome, including annotation of unassembled transcripts, de novo assembly, and aligning reads to known genomes. Further, we evaluated various databases for their ability to provide annotations for our metatranscriptome. Overall, our results emphasize that low activity, highly genetically diverse and relatively stable microbiomes, like soil, requires very deep sequencing to sample the transcriptome beyond the common core functions. We identified several key areas that metatranscriptomic analyses will benefit from including increased rRNA removal, assembly of short read transcripts, and more relevant reference bases while providing a priority set of expressed genes for functional assessment.

Diversity of bacteria at healthy human conjunctiva.

PURPOSE: Ocular surface (OS) microbiota contributes to infectious and autoimmune diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional cultivation techniques. This pilot study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of bacteria. METHODS: Composition of the bacterial community was characterized using deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA. The DNA sequences were classified and the diversity parameters measured using bioinformatics software ESPRIT and MOTHUR and tools available through the Ribosomal Database Project-II (RDP-II). RESULTS: Deep sequencing of conjunctival rDNA from four subjects yielded a total of 115,003 quality DNA reads, corresponding to 221 species-level phylotypes per subject. The combined bacterial community classified into 5 phyla and 59 distinct genera. However, 31% of all DNA reads belonged to unclassified or novel bacteria. The intersubject variability of individual OS microbiomes was very significant. Regardless, 12 genera-Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter, Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas, Streptococcus, Streptophyta, and Methylobacterium-were ubiquitous among the analyzed cohort and represented the putative "core" of conjunctival microbiota. The other 47 genera accounted for <4% of the classified portion of this microbiome. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens. CONCLUSIONS: The first DNA sequencing-based survey of bacterial population at the conjunctiva have revealed an unexpectedly diverse microbial community. All analyzed samples contained ubiquitous (core) genera that included commensal, environmental, and opportunistic pathogenic bacteria.