Hot off the presses! May 01 Nature methods

The May 01 issue of the Nature methods is now up on Pubget (About Nature methods): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• E pluribus unum
  - Nature Methods 7(5):331 (2010)
  If the human reference genome is to reflect more of the actual genomic diversity in humans, community participation is needed.
• The author file: Evan Eichler
  - Nature Methods 7(5):333 (2010)
  By mapping the underbelly of human genomes, researchers open new questions.
• QIIME allows analysis of high-throughput community sequencing data
  Caporaso JG Kuczynski J Stombaugh J Bittinger K Bushman FD Costello EK Fierer N Peña AG Goodrich JK Gordon JI Huttley GA Kelley ST Knights D Koenig JE Ley RE Lozupone CA McDonald D Muegge BD Pirrung M Reeder J Sevinsky JR Turnbaugh PJ Walters WA Widmann J Yatsunenko T Zaneveld J Knight R - Nature Methods 7(5):335-336 (2010)
  To the Editor: High-throughput sequencing is revolutionizing microbial ecology studies. Efforts like the Human Microbiome Projects and the US National Ecological Observatory Network are helping us to understand the role of microbial diversity in habitats within our own bodies and throughout the planet.
• Intensity normalization improves color calling in SOLiD sequencing
  - Nature Methods 7(5):336-337 (2010)
  To the Editor: Applied Biosystems' SOLiD system is a commonly used massively parallel DNA sequencing platform for applications from genotyping and structural variation analysis to transcriptome quantification and reconstruction. Like other sequencing technologies, it measures fluorescence intensities from dye-labeled molecules to determine the sequence of DNA fragments.
• Efficient maximum likelihood estimator fitting of histograms
  - Nature Methods 7(5):338-339 (2010)
  To the Editor: Scientists commonly form histograms of counted events from their data, and extract parameters by fitting to a known model. Anytime a scientist counts photons, molecules, cells or data for individuals in histogram bins and fits that to a distribution, he or she is fitting an event-counting histogram.
• QuickPALM: 3D real-time photoactivation nanoscopy image processing in ImageJ
  - Nature Methods 7(5):339-340 (2010)
  To the Editor: Although conventional microscopes have a reso-lution limited by diffraction to about half the wavelength of light, several recent advances have led to microscopy methods that achieve roughly tenfold improvements in resolution. Among them, photoactivated light microscopy (PALM) and stochastic optical resolution microscopy (STORM) have become particularly popular, as they only require relatively simple and affordable modifications to a standard total internal reflection fluorescence (TIRF) microscope and have been extended to three-dimensional (3D) super-resolution and multicolor imaging.
• Encoding the unnatural
  - Nature Methods 7(5):343 (2010)
  Researchers evolved a ribosome that efficiently decodes quadruplet codons, thus opening up the possibility of genetically incorporating multiple unnatural amino acids into proteins.
• Connecting the dots in 3D
  - Nature Methods 7(5):344-345 (2010)
  New software tools help take the pain out of working with huge three-dimensional image datasets and aid in mapping neuronal networks.
• Transcription factor interaction maps
  - Nature Methods 7(5):344-345 (2010)
  A systematic map of pair-wise physical interactions among mammalian transcription factors will enable studies of transcriptional control in development and disease.
• News in brief
  - Nature Methods 7(5):345 (2010)
  Neuroscience Imaging dopamine with MRI Magnetic resonance imaging (MRI) is a powerful, noninvasive technology for studying the brain. But neurotransmitters such as dopamine have not been directly observed by MRI.
• Neurons light the way
  - Nature Methods 7(5):346 (2010)
  Monitoring the activity of neurons in vivo in the freely behaving zebrafish larvae is now possible using bioluminescence, an approach with great potential for unveiling how neuronal networks control behavior.
• Cultural shifts
  - Nature Methods 7(5):348 (2010)
  A study of the genetic variation in 17 human embryonic stem cell lines shows hundreds of changes, some associated with cancer.
• Keeping it in the family
  - Nature Methods 7(5):350 (2010)
  Whole-genome sequencing of DNA from two children with Mendelian disorders and from their healthy parents allows efficient correction of sequencing errors and the identification of causal genes.
• Clever PCR: more genotyping, smaller volumes
  - Nature Methods 7(5):351-356 (2010)
  With microfluidics and multiplexing, researchers can get more information from PCR products in less time and with fewer reagents.
• The economy of photons
  - Nature Methods 7(5):357-359 (2010)
  Many of the fundamental advances in light microscopy over the last half century, beginning with the inception of confocal microscopy by Marvin Minsky in 1955 (ref. 1), have aimed to improve the spatial resolution of light microscopy.
• Super-SILAC for tumors and tissues
  - Nature Methods 7(5):361-362 (2010)
  Over the past decade, advances in technology have enabled protein mass spectrometry to evolve from enabling simple protein identifications and mapping of single post-translational modifications to making possible quantitative, whole-proteome analysis. However, a longstanding problem in the field has been how to accurately compare comprehensive proteomes and selected subsets thereof, such as targeted interactomes and networks or groups of proteins carrying defined modifications.
• Measuring the growth rate of cells, one at a time
  - Nature Methods 7(5):363 (2010)
  Cell mass increase results from all biogenesis processes (production of proteins, lipids, nucleic acids and other molecules). In contrast to specific molecular processes, which can be monitored in single cells using the many fluorescence-based microscopy techniques available, it is difficult to reliably measure the overall mass of single cells and, a fortiori, to determine their growth rate.
• Characterization of missing human genome sequences and copy-number polymorphic insertions
  - Nature Methods 7(5):365-371 (2010)
  The extent of human genomic structural variation suggests that there must be portions of the genome yet to be discovered, annotated and characterized at the sequence level. We present a resource and analysis of 2,363 new insertion sequences corresponding to 720 genomic loci. We found that a substantial fraction of these sequences are either missing, fragmented or misassigned when compared to recent de novo sequence assemblies from short-read next-generation sequence data. We determined that 18–37% of these new insertions are copy-number polymorphic, including loci that show extensive population stratification among Europeans, Asians and Africans. Complete sequencing of 156 of these insertions identified new exons and conserved noncoding sequences not yet represented in the reference genome. We developed a method to accurately genotype these new insertions by mapping next-generation sequencing datasets to the breakpoint, thereby providing a means to characterize copy-! number status for regions previously inaccessible to single-nucleotide polymorphism microarrays.
• Fast, single-molecule localization that achieves theoretically minimum uncertainty
  Smith CS Joseph N Rieger B Lidke KA - Nature Methods 7(5):373-375 (2010)
  We describe an iterative algorithm that converges to the maximum likelihood estimate of the position and intensity of a single fluorophore. Our technique efficiently computes and achieves the Cramér-Rao lower bound, an essential tool for parameter estimation. An implementation of the algorithm on graphics processing unit hardware achieved more than 105 combined fits and Cramér-Rao lower bound calculations per second, enabling real-time data analysis for super-resolution imaging and other applications.
• Optimized localization analysis for single-molecule tracking and super-resolution microscopy
  Mortensen KI Churchman LS Spudich JA Flyvbjerg H - Nature Methods 7(5):377-381 (2010)
  We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.
• Super-SILAC mix for quantitative proteomics of human tumor tissue
  Geiger T Cox J Ostasiewicz P Wisniewski JR Mann M - Nature Methods 7(5):383-385 (2010)
  We describe a method to accurately quantify human tumor proteomes by combining a mixture of five stable-isotope labeling by amino acids in cell culture (SILAC)-labeled cell lines with human carcinoma tissue. This generated hundreds of thousands of isotopically labeled peptides in appropriate amounts to serve as internal standards for mass spectrometry–based analysis. By decoupling the labeling from the measurement, this super-SILAC method broadens the scope of SILAC-based proteomics.
• Using buoyant mass to measure the growth of single cells
  Godin M Delgado FF Son S Grover WH Bryan AK Tzur A Jorgensen P Payer K Grossman AD Kirschner MW Manalis SR - Nature Methods 7(5):387-390 (2010)
  We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the 'instantaneous' growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.
• A streptavidin variant with slower biotin dissociation and increased mechanostability
  Chivers CE Crozat E Chu C Moy VT Sherratt DJ Howarth M - Nature Methods 7(5):391-393 (2010)
  Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.
• In situ detection and genotyping of individual mRNA molecules
  Larsson C Grundberg I Söderberg O Nilsson M - Nature Methods 7(5):395-397 (2010)
  Increasing knowledge about the heterogeneity of mRNA expression within cell populations highlights the need to study transcripts at the level of single cells. We present a method for detection and genotyping of individual transcripts based on padlock probes and in situ target-primed rolling-circle amplification. We detect a somatic point mutation, differentiate between members of a gene family and perform multiplex detection of transcripts in human and mouse cells and tissue.
• High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision
  - Nature Methods 7(5):399-405 (2010)
  Two-photon calcium imaging of neuronal populations enables optical recording of spiking activity in living animals, but standard laser scanners are too slow to accurately determine spike times. Here we report in vivo imaging in mouse neocortex with greatly improved temporal resolution using random-access scanning with acousto-optic deflectors. We obtained fluorescence measurements from 34–91 layer 2/3 neurons at a 180–490 Hz sampling rate. We detected single action potential–evoked calcium transients with signal-to-noise ratios of 2–5 and determined spike times with near-millisecond precision and 5–15 ms confidence intervals. An automated 'peeling' algorithm enabled reconstruction of complex spike trains from fluorescence traces up to 20–30 Hz frequency, uncovering spatiotemporal trial-to-trial variability of sensory responses in barrel cortex and visual cortex. By revealing spike sequences in neuronal populations on a fast time scale, high-speed calcium im! aging will facilitate optical studies of information processing in brain microcircuits.
• Conditional gene expression and RNAi using MEC-8–dependent splicing in C. elegans
  Calixto A Ma C Chalfie M - Nature Methods 7(5):407-411 (2010)
  We describe a method for conditional regulation of gene expression based on the processing of an intron cassette. The RNA processing factor MEC-8 is necessary for the function of the Caenorhabditis elegans touch receptor neurons; mec-8 mutants are touch insensitive. We show here that this insensitivity involves the loss of MEC-8–dependent splicing of mec-2, which encodes a component of the mechanosensory transduction complex. MEC-8 is needed to remove the ninth intron in mec-2 pre-mRNA to form the longest of three mRNAs, mec-2a. Without MEC-8, splicing causes the termination of the transcript. Inclusion of mec-2 intron 9 is sufficient to convey mec-8–dependent regulation on other genes and, in mec-8(u218ts) mutants, resulted in their temperature-dependent expression. Because mec-8 is expressed ubiquitously in embryos and extensively in larvae, this system should produce temperature-sensitive expression for most genes. As an example, we report a strain that exhibits! temperature-dependent RNA interference.