I'm dreaming of a COVID-free Christmas

BioTechniques Editors explore the evolution of point-of-care testing over the past 2 years, inside and outside of the COVID-19 pandemic.

A high-quality fungal genome assembly resolved from a sample accidentally contaminated by multiple taxa

Contamination in sequenced genomes is a relatively common problem and several methods to remove non-target sequences have been devised. Typically, the target and contaminating organisms reside in different kingdoms, simplifying their separation. The authors present the case of a genome for the ascomycete fungus Teratosphaeria eucalypti, contaminated by another ascomycete fungus and a bacterium. Approaching the problem as a low-complexity metagenomics project, the authors used two available software programs, BlobToolKit and anvi'o, to filter the contaminated genome. Both the de novo and reference-assisted approaches yielded a high-quality draft genome assembly for the target fungus. Incorporating reference sequences increased assembly completeness and visualization elucidated previously unknown genome features. The authors suggest that visualization should be routine in any sequencing project, regardless of suspected contamination.

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed ‘MILKSHAKE,’ is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue’s modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

High-yield expression of periplasmic single-chain variable fragments by solid Escherichia coli cultures

High-yield expression of quality antibody fragments is indispensable for research and diagnosis. Most recombinant antibody fragments are expressed in Escherichia coli using liquid cultures; however, their yields and quality are often poor. Here the authors expressed a single-chain variable fragment in E. coli cultivated on the wet surface of a solid support. Compared with a liquid culture, the authors obtained 2.5-times more single-chain variable fragments with membrane-cultivated E. coli. This method has two important advantages: it enables high yields of periplasmic single-chain variable fragments compared with liquid culture and offers simple and rapid expression and extraction.

ncRNAseq: simple modifications to RNA-seq library preparation allow recovery and analysis of mid-sized non-coding RNAs

Despite their abundance, mid-sized RNAs (30–300 nt) have not been extensively studied by high-throughput sequencing, mostly due to selective loss in library preparation. The authors propose simple and inexpensive modifications to the Illumina TruSeq protocol (ncRNAseq), allowing the capture and sequencing of mid-sized non-coding RNAs without detriment to the coverage of coding mRNAs. This protocol is coupled with a two-step alignment: a pre-alignment to a curated non-coding genome, passing only the non-mapping reads to a standard genomic alignment. ncRNAseq correctly assigns the highest read-numbers to established abundant non-coding RNAs and correctly identifies cytosolic and nuclear enrichment of known non-coding RNAs in two cell lines.

Comparison of methods for mapping rhizosphere processes in the context of their surrounding root and soil environments

The rhizosphere embodies a complex biogeochemical zone with enhanced rates of nutrient exchange between plants, soil, and microbial communities. Understanding controls on rhizosphere dynamics is critical to support emerging concepts including rhizosphere engineering and reduced dependence on chemical fertilizers which have direct application to food production, increased biofuel generation, and habitat restoration efforts. Yet, its fine spatial scale and complex interactions between geochemical and microbial processes within complex spatiotemporal gradients make the rhizosphere notoriously difficult to study. Emerging instrumentation and methodologies, however, are providing improved resolution to rhizosphere measurements and helping to address critical knowledge gaps in rhizosphere function, ecology, and establishment. Here, we examine recent advances in analysis techniques and the resulting potential for improved understanding of rhizosphere function.