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Microbiology Resource Announcements (MRA) provides peer-reviewed announcements of scientific resources for the microbial research community. We describe the best practices for writing an announcement that ensures that these publications are truly useful resources. Adhering to these best practices can lead to successful publication without the need for extensive revisions.

As sequencing read length has increased, researchers have quickly adopted longer reads for their experiments. Here, we examine 14 pathogen or host-pathogen differential gene expression data sets to assess whether using longer reads is warranted. A variety of data sets was used to assess what genomic attributes might affect the outcome of differential gene expression analysis including: gene density, operons, gene length, number of introns/exons and intron length. No genome attribute was found to influence the data in principal components analysis, hierarchical clustering with bootstrap support, or regression analyses of pairwise comparisons that were undertaken on the same reads, looking at all combinations of paired and unpaired reads trimmed to 36, 54, 72 and 101 bp. Read pairing had the greatest effect when there was little variation in the samples from different conditions or in their replicates (e.g. little differential gene expression). But overall, 54 and 72 bp reads were typically most similar. Given differences in costs and mapping percentages, we recommend 54 bp reads for organisms with no or few introns and 72 bp reads for all others. In a third of the data sets, read pairing had absolutely no effect, despite paired reads having twice as much data. Therefore, single-end reads seem robust for differential-expression analyses, but in eukaryotes paired-end reads are likely desired to analyse splice variants and should be preferred for data sets that are acquired with the intent to be community resources that might be used in secondary data analyses.

Descriptions of resources, like the genome assemblies reported in Microbiology Resource Announcements, are often frozen at their time of publication, yet they will need to be interpreted in the midst of continually evolving technologies. It is therefore important to ensure that researchers accessing published resources have access to all of the information required to repeat, interpret, and extend these original analyses. Here, we provide a set of suggestions to help make certain that published resources remain useful and repeatable for the foreseeable future.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.

Aspergillus fumigatus is an opportunistic, ubiquitous, saprophytic mould that can cause severe allergic responses in atopic individuals as well as life-threatening infections in immunocompromised patients. A critical step in the establishment of infection is the invasion of airway epithelial cells by the inhaled fungi. Understanding how A. fumigatus senses and responds to airway cells is important to understand the pathogenesis of invasive pulmonary aspergillosis. Here, we analysed the transcriptomes of two commonly used clinical isolates, Af293 and CEA10, during infection of the A549 type II pneumocyte cell line in vitro. We focused our RNA-seq analysis on the core set of genes that are present in the genomes of the two strains. Our results suggest that: (a) A. fumigatus does not mount a conserved transcriptional response to airway epithelial cells in our in vitro model and (b) strain background and time spent in the tissue culture media have a greater impact on the transcriptome than the presence of host cells. Our analyses reveal both common and strain-specific transcriptional programmes that allow for the generation of hypotheses about gene function as it pertains to pathogenesis and the significant phenotypic heterogeneity that is observed among A. fumigatus isolates.

Genomics, transcriptomics, and proteomics have significantly advanced our understanding of obligately host-associated microbes, where interrogation of the biology is often limited by the complexity of the biological system and limited tools. This includes the causative agents of many neglected tropical diseases, including filarial nematodes. Therefore, numerous transcriptomics studies have been undertaken on filarial nematodes. Most of these transcriptomics studies focus on Brugia malayi, which causes lymphatic filariasis and is a laboratory model for human filarial disease. Here, we undertook a meta-analysis of the publicly available B. malayi transcriptomics data enabling the direct cross comparison of samples from almost a dozen studies. This reanalysis highlights the consistency of transcriptomics results across many different studies and experimental designs from across the globe for over a decade of research, across many different generations of a sequencing technology, library preparation protocols, and differential expression tools. Males and microfilariae across samples had similar expression profiles. However, female samples were clustered into two differential expression patterns that were significantly different from one another. Largely, we confirm previous results for all studies reanalyzed including tissue-specific gene expression and anti-Wolbachia doxycycline treatment of microfilaria. However, we did not detect previously reported differential expression upon in vitro or in vivo treatment with ivermectin, albendazole, and DEC, instead identifying a consistent lack of transcriptomic change upon exposure to these anthelminthic drugs. Updated annotation has been provided that denotes poorly supported genes including those overlapping rRNAs. Author summary: Numerous previous studies have measured all of the mRNA in Brugia malayi under a variety of conditions. Here, we collectively analyzed all data from those previous studies to gain new insights, which can be facilitated by the additional comparisons of this type of meta-analysis. Importantly, this meta-analysis demonstrates that worms that are shipped to various locations and handled by different laboratories are largely the same. The exception are adult females, where two profiles are apparent that are vastly different, which could lead to differing results in laboratory experiments. We propose that shipping dates and conditions be provided in manuscripts and that a transcriptome profile be collected in future laboratory studies to benchmark worms. In addition, the inclusion of more worm samples reveals that differences between worm shipments and batches is greater than the difference observed upon treatment with anthelminthic agents (ivermectin, albendazole, and DEC) under the conditions examined in the original studies. During this work, we identified some regions of the genome where genes were misidentified that affect RNA-based analysis and provide an updated annotation file to facilitate their removal in future studies. [ABSTRACT FROM AUTHOR]

Abstract Background Numerous methods exist for enriching bacterial or mammalian mRNA prior to transcriptome experiments. Yet there persists a need for methods to enrich for mRNA in non-mammalian animal systems. For example, insects contain many important and interesting obligate intracellular bacteria, including endosymbionts and vector-borne pathogens. Such obligate intracellular bacteria are difficult to study by traditional methods. Therefore, genomics has greatly increased our understanding of these bacteria. Efficient subtraction methods are needed for removing both bacteria and insect rRNA in these systems to enable transcriptome-based studies. Findings A method is described that efficiently removes >95% of insect rRNA from total RNA samples, as determined by microfluidics and transcriptome sequencing. This subtraction yielded a 6.2-fold increase in mRNA abundance. Such a host rRNA-depletion strategy, in combination with bacterial rRNA depletion, is necessary to analyze transcription of obligate intracellular bacteria. Here, transcripts were identified that arise from a lateral gene transfer of an entire Wolbachia bacterial genome into a Drosophila ananassae chromosome. In this case, an rRNA depletion strategy is preferred over polyA-based enrichment since transcripts arising from bacteria-to-animal lateral gene transfer may not be poly-adenylated. Conclusions This enrichment method yields a significant increase in mRNA abundance when poly-A selection is not suitable. It can be used in combination with bacterial rRNA subtraction to enable experiments to simultaneously measure bacteria and insect mRNA in vector and endosymbiont biology experiments.

Abstract Background Rapid annotation and comparisons of genomes from multiple isolates (pan-genomes) is becoming commonplace due to advances in sequencing technology. Genome annotations can contain inconsistencies and errors that hinder comparative analysis even within a single species. Tools are needed to compare and improve annotation quality across sets of closely related genomes. Results We introduce a new tool, Mugsy-Annotator, that identifies orthologs and evaluates annotation quality in prokaryotic genomes using whole genome multiple alignment. Mugsy-Annotator identifies anomalies in annotated gene structures, including inconsistently located translation initiation sites and disrupted genes due to draft genome sequencing or pseudogenes. An evaluation of species pan-genomes using the tool indicates that such anomalies are common, especially at translation initiation sites. Mugsy-Annotator reports alternate annotations that improve consistency and are candidates for further review. Conclusions Whole genome multiple alignment can be used to efficiently identify orthologs and annotation problem areas in a bacterial pan-genome. Comparisons of annotated gene structures within a species may show more variation than is actually present in the genome, indicating errors in genome annotation. Our new tool Mugsy-Annotator assists re-annotation efforts by highlighting edits that improve annotation consistency.