Latest Articles Include:
- The scientist and the smartphone
- Nature methods 7(2):87 (2010)
Mobile computing platforms such as the iPhone are beginning to make inroads into the laboratory—serious prospect or fairy tale?
- The author file
- Nature methods 7(2):89 (2010)
Adapting optics: techniques for seeing stars scale to cells.
- Genome editing with modularly assembled zinc-finger nucleases
- Nature methods 7(2):91 (2010)
To the Editor: In a Correspondence in Nature Methods, some members of the Zinc Finger Consortium reported discouragingly high failure rates for the modular assembly of zinc-finger DNA-binding proteins and concluded that more time-consuming and labor-intensive selection-based methods were "the only publicly available alternatives for academic researchers interested in using ZFN technology". Zinc finger nucleases (ZFNs) are artificial restriction enzymes made by fusing reprogrammable zinc-finger DNA-binding units to the FokI nuclease domain, which efficiently induce, site-specific mutations in higher eukaryotic cells, and thus hold great promise in many fields.
- Reply to "Genome editing with modularly assembled zinc-finger nucleases"
- Nature methods 7(2):91-92 (2010)
Joung et al. reply: The publications cited by Kim et al. describing successful construction of zinc-finger nucleases (ZFNs) by modular assembly only further support our original conclusion that this method has a high failure rate for engineering functional zinc-finger arrays. Two of the three reports cited in their Correspondence provide data that enable calculation of failure rates for modular assembly.
- IntOGen: integration and data mining of multidimensional oncogenomic data
- Nature methods 7(2):92-93 (2010)
To the Editor: The use of high-throughput techniques has come to the fore in modern cancer research. Several projects collate and analyze multiple datasets from cancer gene studies.
- Systems biology: Big surprises in a little package
- Nature methods 7(2):95 (2010)
An in-depth, systems biology approach to analyzing a 'reduced genome' bacterium reveals startling complexity.
- Imaging and visualization: One-shot structure determination
- Nature methods 7(2):96-97 (2010)
By sampling a two-dimensional diffraction pattern on a spherical detector, three-dimensional structure determination of single molecules should be possible from a single measurement.
- Genomics: To each his own
- Nature methods 7(2):96-97 (2010)
De novo assembly of human genome sequences that are not currently included in the reference genome opens the possibility of a human pan-genome.
- News in brief
- Nature methods 7(2):97 (2010)
Gene regulation Mutagenesis screens in human cells The classical mutagenesis approach is a powerful method to determine the genes induced in various biological processes. However, the diploid nature of the human and other mammalian genomes has limited large-scale mutagenesis studies.
- Bioinformatics: Found in translation
- Nature methods 7(2):98 (2010)
Annotation of clinical databases using controlled vocabulary permits cross-species comparisons of phenotypes associated with human disease.
- Imaging and visualization: Taking imaging into the red
- Nature methods 7(2):100 (2010)
Subtle modifications to a red fluorescent protein make it highly effective for intravital imaging.
- Cell biology: Supporting actors
- Nature methods 7(2):102 (2010)
Two GFP-binding peptides, Enhancer and Minimizer, modulate GFP fluorescence and will enable a number of in vivo applications.
- Mind the gaps
- Nature methods 7(2):105-106 (2010)
Amid all the excitement about next-generation sequencing, scientists often neglect to mention the problems that are caused by short read lengths. Genome assemblies produced from short reads are far more fragmented than those produced from long reads, with many more gaps and with relatively poor long-range linking information.
- Advancing neurochemical monitoring
- Nature methods 7(2):106-108 (2010)
Identifying the neural basis of behavior is a core focus of neuroscience. One prominent methodology in this pursuit is monitoring the neurotransmitters that underlie communication between neurons.
- Correcting distorted optics: back to the basics
- Nature methods 7(2):108-110 (2010)
How can optical imperfections that cause image distortion be measured and corrected? This is an important problem in many fields, ranging from imaging very large structures as in astronomy down to the smallest as in high-resolution microscopy. Many often quite complicated ways have been invented to determine exactly how a given optical system differs from perfection.
- Target-enrichment strategies for next-generation sequencing
- Nature methods 7(2):111-118 (2010)
We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.
- Parallel, tag-directed assembly of locally derived short sequence reads
- Nature methods 7(2):119-122 (2010)
We demonstrate subassembly, an in vitro library construction method that extends the utility of short-read sequencing platforms to applications requiring long, accurate reads. A long DNA fragment library is converted to a population of nested sublibraries, and a tag sequence directs grouping of short reads derived from the same long fragment, enabling localized assembly of long fragment sequences. Subassembly may facilitate accurate de novo genome assembly and metagenome sequencing.
- Two-color, two-photon uncaging of glutamate and GABA
Kantevari S Matsuzaki M Kanemoto Y Kasai H Ellis-Davies GC - Nature methods 7(2):123-125 (2010)
We developed a caged GABA (γ-aminobutyric acid), which, when combined with an appropriate caged glutamate, allows bimodal control of neuronal membrane potential with subcellular resolution using optically independent two-photon uncaging of each neurotransmitter. We used two-color, two-photon uncaging to fire and block action potentials from rat hippocampal CA1 neurons in brain slices with 720-nm and 830-nm light, respectively. Our method should be generalizable to other chemical messenger pairs.
- Chronic microsensors for longitudinal, subsecond dopamine detection in behaving animals
Clark JJ Sandberg SG Wanat MJ Gan JO Horne EA Hart AS Akers CA Parker JG Willuhn I Martinez V Evans SB Stella N Phillips PE - Nature methods 7(2):126-129 (2010)
Neurotransmission operates on a millisecond timescale but is changed by normal experience or neuropathology over days to months. Despite the importance of long-term neurotransmitter dynamics, no technique exists to track these changes in a subject from day to day over extended periods of time. Here we describe and characterize a microsensor that can detect the neurotransmitter dopamine with subsecond temporal resolution over months in vivo in rats and mice.
- FRT-seq: amplification-free, strand-specific transcriptome sequencing
- Nature methods 7(2):130-132 (2010)
We report an alternative approach to transcriptome sequencing for the Illumina Genome Analyzer, in which the reverse transcription reaction takes place on the flowcell. No amplification is performed during the library preparation, so PCR biases and duplicates are avoided, and because the template is poly(A)+ RNA rather than cDNA, the resulting sequences are necessarily strand-specific. The method is compatible with paired- or single-end sequencing.
- Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution
Gu H Bock C Mikkelsen TS Jäger N Smith ZD Tomazou E Gnirke A Lander ES Meissner A - Nature methods 7(2):133-136 (2010)
Bisulfite sequencing measures absolute levels of DNA methylation at single-nucleotide resolution, providing a robust platform for molecular diagnostics. We optimized bisulfite sequencing for genome-scale analysis of clinical samples: here we outline how restriction digestion targets bisulfite sequencing to hotspots of epigenetic regulation and describe a statistical method for assessing significance of altered DNA methylation patterns. Thirty nanograms of DNA was sufficient for genome-scale analysis and our protocol worked well on formalin-fixed, paraffin-embedded samples.
- Bright cyan fluorescent protein variants identified by fluorescence lifetime screening
- Nature methods 7(2):137-139 (2010)
Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.
- Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues
Ji N Milkie DE Betzig E - Nature methods 7(2):141-147 (2010)
Biological specimens are rife with optical inhomogeneities that seriously degrade imaging performance under all but the most ideal conditions. Measuring and then correcting for these inhomogeneities is the province of adaptive optics. Here we introduce an approach to adaptive optics in microscopy wherein the rear pupil of an objective lens is segmented into subregions, and light is directed individually to each subregion to measure, by image shift, the deflection faced by each group of rays as they emerge from the objective and travel through the specimen toward the focus. Applying our method to two-photon microscopy, we could recover near-diffraction–limited performance from a variety of biological and nonbiological samples exhibiting aberrations large or small and smoothly varying or abruptly changing. In particular, results from fixed mouse cortical slices illustrate our ability to improve signal and resolution to depths of 400 μm.
- Systems analysis of EGF receptor signaling dynamics with microwestern arrays
- Nature methods 7(2):148-155 (2010)
We describe microwestern arrays, which enable quantitative, sensitive and high-throughput assessment of protein abundance and modifications after electrophoretic separation of microarrayed cell lysates. This method allowed us to measure 91 phosphosites on 67 proteins at six time points after stimulation with five epidermal growth factor (EGF) concentrations in A431 human carcinoma cells. We inferred the connectivities among 15 phosphorylation sites in 10 receptor tyrosine kinases (RTKs) and two sites from Src kinase using Bayesian network modeling and two mutual information-based methods; the three inference methods yielded substantial agreement on the network topology. These results imply multiple distinct RTK coactivation mechanisms and support the notion that small amounts of experimental data collected from phenotypically diverse network states may enable network inference.
- Mass spectrometry for biologists
- Nature methods 7(2):157-161 (2010)
Mass spectrometry–based proteomics is still rapidly expanding, not just in terms of the methods and instruments but also the biological questions.