High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing

Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold. Here we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.     

Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs

Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ∼8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution.     

Evaluation of methods for sequence analysis of highly repetitive DNA templates.

The DNA Sequencing Research Group (DSRG) of the ABRF conducted a study to assess the ability of DNA sequencing core facilities to successfully sequence a set of well-defined templates containing difficult repeats. The aim of this study was to determine whether repetitive templates could be sequenced accurately by using equipment and chemistries currently utilized in participating sequencing laboratories. The effects of primer and template concentrations, sequencing chemistries, additives, and instrument formats on the ability to successfully sequence repeat elements were examined. The first part of this study was an analysis of the results of 361 chromatograms from participants representing 40 different laboratories who attempted to sequence a panel of difficult-to-sequence templates using their best in-house protocols. The second part of this study was a smaller multi-laboratory evaluation of a single robust protocol with the same panel of templates. This study provides a measure of the potential success of different approaches to sequencing across homopolymer tracts and repetitive elements.     

Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-kappaB activation: TAO2 regulates TAK1 pathways

Osmotic stress activates MAPKs, including JNK and p38, which play important roles in cellular stress responses. Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the MAPK kinase kinase (MAPKKK) family and can activate JNK and p38. TAK1 can also activate IkappaB kinase (IKK) that leads to degradation of IkappaB and subsequent NF-kappaB activation. We found that TAK1 is essential for osmotic stress-induced activation of JNK but is not an exclusive mediator of p38 activation. Furthermore, we found that although TAK1 was highly activated upon osmotic stress, it could not induce degradation of IkappaB or activation of NF-kappaB. These results suggest that TAK1 activity is somehow modulated to function specifically in osmotic stress signaling, leading to the activation of JNK but not of IKK. To elucidate the mechanism underlying this modulation, we screened for potential TAK1-binding proteins. We found that TAO2 (thousand-and-one amino acid kinase 2) associates with TAK1 and can inhibit TAK1-mediated activation of NF-kappaB but not of JNK. We observed that TAO2 can interfere with the interaction between TAK1 and IKK and thus may regulate TAK1 function. TAK1 is activated by many distinct stimuli, including cytokines and stresses, and regulation by TAO2 may be important to activate specific intracellular signaling pathways that are unique to osmotic stress.     

Bioinformatics visualization and integration with open standards: the Bluejay genomic browser

We have created a new Java-based integrated computational environment for the exploration of genomic data, called Bluejay. The system is capable of using almost any XML file related to genomic data. Non-XML data sources can be accessed via a proxy server. Bluejay has several features, which are new to Bioinformatics, including an unlimited semantic zoom capability, coupled with Scalable Vector Graphics (SVG) outputs; an implementation of the XLink standard, which features access to MAGPIE Genecards as well as any BioMOBY service accessible over the Internet; and the integration of gene chip analysis tools with the functional assignments. The system can be used as a signed web applet, Web Start, and a local stand-alone application, with or without connection to the Internet. It is available free of charge and as open source via http://bluejay.ucalgary.ca.     

The virtuous self‐tolerance of virtual memory T cells

“Virtual” memory CD8⁺ T cells are a subset of immune cells produced by homeostatic mechanisms involving response to self‐antigens, raising the possibility that these cells could mediate autoimmunity. New work by Drobek et al demonstrates that virtual memory T cells are indeed favored by stronger T‐cell receptor signals but exhibit minimal autoreactivity while maintaining self‐tolerance.