Monitoring environmental microbiomes: Alignment of microbiology and computational biology competencies within a culturally integrated curriculum and research framework.

We have developed a flexible undergraduate curriculum that leverages the place-based research of environmental microbiomes to increase the number of Indigenous researchers in microbiology, data science and scientific computing. Monitoring Environmental Microbiomes (MEM) provides a curriculum and research framework designed to integrate an Indigenous approach when conducting authentic scientific research and to build interest and confidence at the undergraduate level. MEM has been successfully implemented as a short summer workshop to introduce computing practices in microbiome analysis. Based on self-assessed student knowledge of topics and skills, increased scientific confidence and interest in genomics careers were observed. We propose MEM be incorporated in a scalable course-based research experience for undergraduate institutions, including tribal colleges and universities, community colleges and other minority serving institutions. This coupled curricular and research framework explicitly considers cultural perspectives, access and equity to train a diverse future workforce that is more informed to engage in microbiome research and to translate microbiome science to benefit community and environmental health.

Comparative single-strand conformation polymorphism (SSCP) and microscopy-based analysis of nitrogen cultivation interactive effects on the fungal community of a semiarid steppe soil.

The effects of nitrogen accretion on fungal diversity and community structure in early-seral (cultivated) and native (uncultivated) shortgrass steppe soils were evaluated using single-strand conformation polymorphism (SSCP) and microscopy in a comparative experiment. Selected haplotypes generated from fungal 18S gene fragments were also sequenced for species identification. Microscopy-based analyses showed significantly shorter fungal hyphal lengths in the early-seral control plots in comparison with the native control plots (P<0.0003), independent of nitrogen addition. Although diversity indices did not show significant differences between the plots, SSCP analyses indicated that fungal community structure differed in the native and early-seral control sites. In nitrogen-amended sites, gene sequences from dominant haplotypes indicated a shift to a more common nitrogen-impacted fungal community. While nitrogen amendments appear to be more important than cultivation in influencing these soil fungal communities, hyphal lengths were only decreased due to cultivation. The use of microscopic and molecular techniques, as carried out in this study, provided integrative information concerning fungal community responses to wide spread stresses being imposed globally on terrestrial ecosystems, that is not provided by the individual techniques.

Heteroduplex resolution using T7 endonuclease I in microbial community analyses.

Microbial community analyses using molecular techniques, such as PCR followed by genomic library construction, have been helpful in better understanding microbial communities. This is especially critical in ecological systems where most of the microbes present cannot be cultured using traditional techniques. Unfortunately, there are problems associated with the use of such molecular techniques for the analysis of microbial community structure, primarily from the frequent formation of PCR artifacts. Multitemplate PCR is often subject to errors such as heteroduplex formation that is generated during the amplification of a particular gene from a mixed community of DNA. Based on work in this laboratory, heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I. Here, the 18S rDNA gene of fungi was amplified from soil community DNA and digested with T7 endonuclease I to resolve any heteroduplexes present in the PCR product before cloning. These samples were compared with replicates that did not receive the T7 endonuclease I treatment. Digestion of the amplified community 18S rDNA with 10 U T7 endonuclease I/microgram DNA prior to cloning eliminated heteroduplexes, leaving only the desired clones. Without the T7 endonuclease I treatment, heteroduplexes were produced in approximately 10% of the recombinants screened. The addition of this step may eliminate heteroduplexes from PCR products and ensure that subsequent genomic library construction is not compromised.