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Nathan Blow, Ph.D. and Patrick C. H. Lo, Ph.D.
BioTechniques, Vol. 56, No. 2, February 2014, p. 53
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In“TUIT” classification

When it comes to classifying bacterial communities in metagenomics, 16S ribosomal RNA (rRNA) sequencing remains one of the preferred methodologies. When used with databases featuring well-characterized sequences from different organisms, 16S rRNA sequencing can be a quick and accurate means of classifying species within a sample. But what about those organisms that lack a reference sequence within one of these databases? How can these be classified quickly and accurately? In these instances, researchers can perform BLAST searches against large nucleotide databases to determine possible sequence identity or locate closely related species. Still, these efforts take time and require significant user knowledge. In an effort to enhance the information from 16S rRNA sequencing studies, Valery Shestopalov and colleagues report a new, open-source program for taxonomic classification based on 16S rRNA in the current issue of BioTechniques. The authors’ new tool, which they have termed TUIT (taxonomic unit identification tool), was designed to meet four challenges: (i) the algorithm should deal effectively with useless hits that give no taxonomic information; (ii) the algorithm should attempt to identify the deepest taxonomic rank possible; (iii) the tool should have flexible settings for similarity criteria based on user preferences; and (iv) the tool should be easy to use for both the expert and non-expert alike. TUIT is a tool that combines the capability of a BLAST search with taxonomic information to provide end users a more detailed classification of a given sequence. In a test using a simulated dataset, TUIT was able to classify DNA sequences with high specificity and analyze sequences as short as 125 base pairs. Although developed for 16S rRNA classification, TUIT is not restricted to classification of 16S rRNA sequences. In the end, this new algorithm should prove to be a welcome addition to the metagenomics toolkit—further assisting researchers as they attempt to identify the thousands of inhabitants of communities in samples as diverse as soil and air.

See “TUIT, a BLAST-based tool for the taxonomic classification of nucleotide sequences

Just the FACS about mitotic cells

Researchers studying how gene regulation is affected by the drastic changes in chromatin structure and function that occur during mitosis utilize methods such as chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin conformation capture (3C). In order for these studies to accurately represent the state of chromatin during mitosis, it is critical to isolate substantial quantities of pure mitotic cells uncontaminated by interphase cells. Mitotic cells represent only a small fraction of asynchronously growing cells in culture, but their numbers can be substantially increased through the use of different drugs to induce mitotic arrest. However, it is typically very difficult to obtain complete mitotic arrest. A more effective alternative is to use intracellular FACS to isolate mitotic cells. For ChIP experiments, cells are fixed, permeabilized, and then incubated with an antibody against histone H3 phosphorylated at serine 10 (H3S10ph). Unfortunately, as documented by G. Blobel and colleagues at the University of Pennsylvania (Philadelphia, PA) in this month's issue, the original commercial H3S10ph antibody used for this method has been discontinued, and subsequent batches of antibody performed poorly when scaled up for FACS purification of large numbers of mitotic cells. In an effort to identify an acceptable alternative, the authors tested three commercially available antibodies against other mitosis-specific markers: (i) anti-phospho-Ser/ Thr-Pro, mitotic protein monoclonal #2 (MPM2); (ii) anti-histone H3 (pS28) (H3S28ph); and (iii) anti-HpTGEKP motif (HpTGEKP). These were first tested by staining small numbers of cells from a suspension and an adherent cell line with increasing antibody concentrations. Adequate separation of brightly stained mitotic cells with 4N DNA content from weakly stained 4N cells in S/G2 phase was achieved with all concentrations of the MPM2 antibody. In fact, the lowest concentration of MPM2 was more effective in mitotic cell isolation than any other antibody at the highest concentration tested, indicating that MPM2 was the most effective for small-scale staining of mitotic cells, followed by H3S10ph. These two antibodies were then compared for staining the large number of cells (2 × 108) typically used for ChIP or 3C experiments, and it was evident that MPM2 clearly resolved the mitotic cells while H3S10ph did not. In the end, the authors conclude that MPM2 is a scalable and cost-effective antibody for FACS sorting of mitotic cells.

See “Comparative analysis of mitosis-specific antibodies for bulk purification of mitotic populations by fluorescence-activated cell sorting