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HHblits is a protein sequence search tool that works by iterative pairwise comparison of profile hidden Markov models. It outperforms existing tools in terms of speed, sensitivity and alignment quality.
Adenosine-to-inosine RNA editing modifies expressed sequences and enhances functional protein diversity. The authors report an in vivo fluorescent reporter that provides a readout of adenosine deaminase RNA-editing activity in Drosophila melanogaster neurons, showing evidence of inter-individual variability in editing activity.
The Splitread algorithm uses a split-read strategy to detect structural variants and small insertions and deletions (indels) in whole-exome and whole-genome sequence datasets at high sensitivity. It maps the breakpoints at single-base-pair resolution, even in low-complexity regions, and can detect novel processed pseudogenes.
In this Analysis, the authors directly experimentally compare microbial opsins used for the control of neural activity. They extract essential principles and key parameters that can help end users with the design and interpretation of optogenetic experiments and guide tool developers in the characterization of future tools.
An efficient haplotype-estimation algorithm that features linear complexity allows the rapid and accurate phasing of diploid genomes from trios, duos and unrelated samples.
The controlled overexpression or knockdown of gene expression in primary organoid cultures of mouse endodermal epithelia is described. This should enable ex vivo studies of mammalian gene function.
Bayesian analysis of fluorophore blinking and bleaching in image data collected from simple xenon arc lamp illumination and high-speed wide-field imaging of standard fluorescent proteins allows localization microscopy in living cells with a 50 nm spatial and a 4 s temporal resolution.
Site-directed seamless modification of bacterial artificial chromosomes is enhanced more than tenfold in efficiency by improving the counterselection step. A set of plasmids and oligonucleotide design software also make this E. coli recombineering approach markedly faster and easier.
Mutations at arbitrarily sampled genomic positions are identified using next-generation sequencing and are used to infer the lineage of DNA damage–prone 'mutator' mouse cells in culture.
The microbial rhodopsin protein, Archaerhodopsin 3, can function as a rapid and highly sensitive genetically encoded voltage indicator in mammalian cells that is capable of detecting single action potentials with a signal-to-noise ratio greater than 10. A mutant lacking proton pumping displays greater sensitivity but a slowed response.
The DNA modification 5-hydroxymethylcytosine has recently been implicated in several biological processes. Enrichment by selective chemical labeling in combination with single-molecule, real-time sequencing provides sensitive detection of this epigenetic mark in genomic DNA at base-pair resolution.
Unique molecular identifiers (UMIs) associate distinct sequences with every DNA or RNA molecule and can be counted after amplification to quantify molecules in the original sample. Using UMIs, the authors obtain a digital karyotype of an individual with Down's syndrome and quantify mRNA in Drosophila melanogaster cells.
Conjugation of triplet-state quenchers to the small organic cyanine fluorophore, Cy5, increases photostability without affecting its spectral characteristics. This allows longer fluorescence imaging with a concomitant reduction in blinking both in vitro and in living cells.
Presented is a study of gene regulation during development using a combination of chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) and directed differentiation of mouse embryonic stem cells inducibly expressing epitope-tagged transcription factors.
An empirical approach for identifying optimal proteotypic peptides and fragmentation patterns from in vitro–synthesized proteins, for targeted proteomics applications, is described.
Molecular engineering allows stoichiometric and co-localized expression of two optogenetic actuators, spaced by a fluorescent protein and an additional transmembrane helix in a single protein fusion. The method provides modular optogenetic tools for bidirectional membrane potential control or synergistic effects on neuronal activity.
A quantitative characterization of the switching properties of 26 organic dyes relates these properties to the quality of localization-based super-resolution images they generate. The data are a useful resource for selecting dyes and point to avenues for future analysis.
A quantitative proteomics approach to characterize protein palmitoylation dynamics on a global scale in cells, as well as to identify enzymes responsible for the regulation of palmitoylation, is described.
An almost-complete, sequence-verified collection of Arabidopsis thaliana root stele transcription factors is reported. The authors use it in the enhanced yeast-one hybrid (eY1H) assay to map gene regulatory interactions in the plant. Also in this issue, Reece-Hoyes et al. describe the eY1H pipeline.
