Research articles

Fluorescence-activated cell sorting of worm embryos promises to replace manual sorting of staged embryos and yields large populations highly enriched in specific developmental stages, allowing high-throughput genomic analysis.

A digital atlas of C. elegans at the post-embryonic L1 stage is presented, along with automated methods for nucleus segmentation and annotation. These resources will enable quantitative analyses of nuclear spatial arrangements as well as high-throughput single-cell analyses in this organism.

This software package provides genome-wide detection of structural variants (insertions, deletions, inversions and inter- and intrachromosomal translocations) from 50-base-pair paired-end reads. The sizes of the detected variants vary from 10 base pairs to 1 megabase pair.

This algorithm for the assignment of phylogenetic groups to fragments generated by metagenomic sequencing projects improves on the currently required 1 kb fragment length for classification. Trained on 539 complete genomes, Phymm can classify reads as short as 100 bp. Combining Phymm with the sequence alignment algorithm BLAST further improves accuracy.

High-throughput analyses of macromolecular shape and oligomeric state at ∼15 Å resolution are possible with a partially automated small angle X-ray scattering (SAXS) pipeline. Though X-ray crystallography provides higher-resolution structural information than SAXS, SAXS analysis is faster and has a higher success rate, which may have implications for how structural genomics research is performed.

Padlock probes, synthesized in large scale on programmable microarrays, capture expressed single-nucleotide polymorphisms for high-throughput sequencing in this method for RNA allelotyping. The approach combines the sensitivity of digital expression measurements with the efficiency of targeted resequencing to quantify allele specific gene expression in various tissues across several individuals.

Using combinations of fluorescently labeled peptide–major histocompatability complex (pMHC) tetramers, T-cell populations with multiple antigen specificities can be monitored in parallel from small samples of human blood. Also in this issue, Newell et al. present a very similar combinatorial encoding method for this purpose.

Fluorescence resonance energy transfer (FRET) between a small-molecule fluorophore donor and a transition metal ion acceptor, a method called 'transition metal ion FRET,' works over shorter distances than the classical FRET approach and can thus be used to monitor very small conformational changes in proteins.

Activation of caged doxycycline or cyanodoxycycline by biologically innocuous doses of UV light allows for precise temporal and spatial control of transgene expression in hippocampal slices, mouse embryos and Xenopus laevis tadpoles.

Although fast temperature jump methods to study protein folding dynamics have long been applied, pressure has been a neglected thermodynamic parameter. A method to generate rapid and large drops in pressure is complementary to fast temperature jump methods and could be useful for direct comparisons to molecular dynamics simulations.

An automated system for tracking large numbers of fruit flies over time and for detecting their behaviors is presented, and should allow high-throughput quantitative studies of fly behavior.

As tissues mature, they undergo shape changes that are the result of individual and collective cell movement triggered by cell-autonomous behavior or external forces. By measuring patterns of strain rates the authors can model these forces and quantify tissue shaping behavior.

Lentiviral vectors that express a fluorescent reporter and a selectable marker in pluripotent cells improve and simplify isolation of induced pluripotent stem cell lines in mouse and human.

An atomic force microscope with a side-view fluorescent imaging path facilitates the direct correlation of mechanical force measurements with observations of changes in cell shape and cytoskeleton rearrangements resulting from the applied forces or during active generation of forces by the cell. The combined instrument could help lead to insights in understanding cell mechanics, contractility and cell-cell adhesion.

Previous whole-transcriptome analysis by RNA-Seq required hundreds of thousands of cells or microgram amounts of RNA. A modification of the cDNA library preparation method now allows unbiased capture of the majority of genes expressed in a single blastomere and oocyte. cDNA sequencing on the SOLiD platform facilitates the quantitative analysis of the transcriptome complexity in a single cell.

piggyBac transposons carrying reprogramming factors are used to reprogram mouse embryonic fibroblasts, with efficiencies equivalent to retroviral transduction, and then removed from the induced pluripotent state cell genome without a trace.

Dense mapping of DNase I cleavage sites across the whole yeast genome by next-generation sequencing reveals a global view of the binding of regulatory proteins to genomic DNA. The high resolution allows the identification of new binding sites for known factors as well as the de novo derivation of factor binding motifs.

The PCR step in the preparation of sequencing libraries for the Illumina Genome Analyzer can introduce coverage bias, especially in very (A+T)-rich genomes. By directly annealing template DNA to adapters with sequences needed for attachment in the flow cell, PCR can be omitted as cluster amplification in the flow cell enriches for fully ligated templates.

An automated system to measure aggression and courtship in pairs of interacting Drosophila is presented and should allow large-scale screens of these behaviors in the future.

Dual-color fluorescence recovery after photobleaching (FRAP) is used to investigate dimerization and higher-order complex formation of receptors at the surface of live cells. A defined fraction of receptors is immobilized with antibodies, and the mobility of the nonimmobilized fraction is measured by FRAP.