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Motivation Mutations (or Single Nucleotide Variants) in folded RiboNucleic Acid structures that cause local or global conformational change are riboSNitches. Predicting riboSNitches is challenging, as it requires making two, albeit related, structure predictions. The data most often used to experimentally validate riboSNitch predictions is Selective 2′ Hydroxyl Acylation by Primer Extension, or SHAPE. Experimentally establishing a riboSNitch requires the quantitative comparison of two SHAPE traces: wild-type (WT) and mutant. Historically, SHAPE data was collected on electropherograms and change in structure was evaluated by ‘gel gazing.’ SHAPE data is now routinely collected with next generation sequencing and/or capillary sequencers. We aim to establish a classifier capable of simulating human ‘gazing’ by identifying features of the SHAPE profile that human experts agree ‘looks’ like a riboSNitch. Results We find strong quantitative agreement between experts when RNA scientists ‘gaze’ at SHAPE data and identify riboSNitches. We identify dynamic time warping and seven other features predictive of the human consensus. The classSNitch classifier reported here accurately reproduces human consensus for 167 mutant/WT comparisons with an Area Under the Curve (AUC) above 0.8. When we analyze 2019 mutant traces for 17 different RNAs, we find that features of the WT SHAPE reactivity allow us to improve thermodynamic structure predictions of riboSNitches. This is significant, as accurate RNA structural analysis and prediction is likely to become an important aspect of precision medicine. Availability and Implementation The classSNitch R package is freely available at http://classsnitch.r-forge.r-project.org. Supplementary information Supplementary data are available at Bioinformatics online.

Summary DNA is now routinely used in criminal investigations and court cases, although DNA samples taken at crime scenes are of varying quality and therefore present challenging problems for their interpretation. We present a statistical model for the quantitative peak information obtained from an electropherogram of a forensic DNA sample and illustrate its potential use for the analysis of criminal cases. In contrast with most previously used methods, we directly model the peak height information and incorporate important artefacts that are associated with the production of the electropherogram. Our model has a number of unknown parameters, and we show that these can be estimated by the method of maximum likelihood in the presence of multiple unknown individuals contributing to the sample, and their approximate standard errors calculated; the computations exploit a Bayesian network representation of the model. A case example from a UK trial, as reported in the literature, is used to illustrate the efficacy and use of the model, both in finding likelihood ratios to quantify the strength of evidence, and in the deconvolution of mixtures for finding likely profiles of the individuals contributing to the sample. Our model is readily extended to simultaneous analysis of more than one mixture as illustrated in a case example. We show that the combination of evidence from several samples may give an evidential strength which is close to that of a single-source trace and thus modelling of peak height information provides a potentially very efficient mixture analysis.

The Mouse Multiple Tissue Metabolome Database (MMMDB) provides comprehensive and quantitative metabolomic information for multiple tissues from single mice. Manually curated databases that integrate literature-based individual metabolite information have been available so far. However, data sets on the absolute concentration of a single metabolite integrated from multiple resources are often difficult to be used when different metabolomic studies are compared because the relative balance of the multiple metabolite concentrations in the metabolic pathways as a snapshot of a dynamic system is more important than the absolute concentration of a single metabolite. We developed MMMDB by performing non-targeted analyses of cerebra, cerebella, thymus, spleen, lung, liver, kidney, heart, pancreas, testis and plasma using capillary electrophoresis time-of-flight mass spectrometry and detected 428 non-redundant features from which 219 metabolites were successfully identified. Quantified concentrations of the individual metabolites and the corresponding processed raw data; for example, the electropherograms and mass spectra with their annotations, such as isotope and fragment information, are stored in the database. MMMDB is designed to normalize users' data, which can be submitted online and used to visualize overlaid electropherograms. Thus, MMMDB allows newly measured data to be compared with the other data in the database. MMMDB is available at: http://mmmdb.iab.keio.ac.jp.

Motivation: Determination of the relative copy number of single-nucleotide sequence variants (SNVs) within a DNA sample is a frequent experimental goal. Various methods can be applied to this problem, although hybridization-based approaches tend to suffer from high-setup cost and poor adaptability, while others (such as pyrosequencing) may not be accessible to all laboratories. The potential to extract relative copy number information from standard dye-terminator electropherograms has been little explored, yet this technology is cheap and widely accessible. Since several biologically important loci have paralogous copies that interfere with genotyping, and which may also display copy number variation (CNV), there are many situations in which determination of the relative copy number of SNVs is desirable. Results: We have developed a desktop application, QSVanalyzer, which allows high-throughput quantification of the proportions of DNA sequences containing SNVs. In reconstruction experiments, QSVanalyzer accurately estimated the known relative proportions of SNVs. By analyzing a large panel of genomic DNA samples, we demonstrate the ability of the software to analyze not only common biallelic SNVs, but also SNVs within a locus at which gene conversion between four genomic paralogs operates, and within another that is subject to CNV. Availability and Implementation: QSVanalyzer is freely available at http://dna.leeds.ac.uk/qsv/. It requires the Microsoft .NET framework version 2.0, which can be installed on all Microsoft operating systems from Windows 98 onwards. Contact: msjimc@leeds.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Background: β-Thalassemia is endemic to many regions in Southeast Asia and India, and Methods: Gap-PCR was used to simultaneously amplify the β-globin gene from genomic DNA and to detect the Δ619bp deletion mutation. Multiplex minisequencing was then performed on the amplified β-globin fragment to detect an additional 15 common Southeast Asian and Indian β-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 β-thalassemia and wild-type DNA samples of known genotype. Results: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity). Conclusions: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian β-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of β-thalassemia in endemic regions a step closer to implementation.

Background: Electrophoretic patterns of proteins in serum/plasma are useful in the diagnosis and evaluation of many diseases. Capillary zone electrophoresis (CZE) allows rapid and automated protein separation and produces digital absorbance data, appropriate for mathematical analysis. We previously demonstrated success in detection of monoclonal immunoglobulins in such a system. This study tests new algorithms to produce rapid standardized computer-supported interpretation of the entire electropherogram. Methods: Data from Beckman Paragon CZE 2000 electropherograms were compared with quantitative protein data from >800 routine clinical samples. Algorithms were designed to produce semiquantitative analyses of major proteins and to define different patterns of inflammation based on the electropherogram. Results: The algorithms produced reliable semiquantitative evaluations of prealbumin, albumin, α1-antitrypsin, haptoglobin, and transferrin, but were less accurate for α1-acid glycoprotein. Some genetic variants of albumin and deficiency variants of α1-antitrypsin were easily recognized. Complex clinical traits such as degree and type of inflammation could be evaluated. When used together with previously developed algorithms addressing immunoglobulins, the new algorithms provide relevant clinical interpretation. Selected outputs indicate the need for reflex testing or evaluation by specialists. Conclusions: Automation of both electrophoresis and interpretation can provide a rapid, inexpensive, standardized analysis that can hopefully improve the diagnostic information and clinical outcome for large groups of patients. It also provides objective criteria for clinical interpretations, to be validated or adjusted in future clinical studies.

We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.

A low pH capillary electrophoresis (CE) was used for the measurement of free choline in plant leaves. Choline in the leaf extract was first converted to the benzoyl ester and put into CE. A well-resolved peak in the electropherogram was easily obtained. Involvement of enzymes in a two-step oxidation of choline to glycine betaine was evaluated in different plant species with the same method developed for glycine betaine and betaine aldehyde.

MICROSATELIGHT is a Perl/Tk pipeline with a graphical user interface that facilitates several tasks when scoring microsatellites. It implements new subroutines in R and PERL and takes advantage of features provided by previously developed freeware. MICROSATELIGHT takes raw genotype data and automates the peak identification through PeakScanner. The PeakSelect subroutine assigns peaks to different microsatellite markers according to their multiplex group, fluorochrome type, and size range. After peak selection, binning of alleles can be carried out 1) automatically through AlleloBin or 2) by manual bin definition through Binator. In both cases, several features for quality checking and further binning improvement are provided. The genotype table can then be converted into input files for several population genetics programs through CREATE. Finally, Hardy-Weinberg equilibrium tests and confidence intervals for null allele frequency can be obtained through GENEPOP. MICROSATELIGHT is the only freely available public-domain software that facilitates full multiplex microsatellite scoring, from electropherogram files to user-defined text files to be used with population genetics software. MICROSATELIGHT has been created for the Windows XP operating system and has been successfully tested under Windows 7. It is available at http://sourceforge.net/projects/microsatelight/.

To the Editor We read with interest the article by Panyasai and colleagues1 in which they identified spuriously increased levels of hemoglobin (Hb)A2 by capillary zone electrophoresis (CZE) in 11 patients heterozygous for Hb Hope. We have encountered the same type of false elevation in HbA2 any time there are 2 major peaks in adjoining zones, eg, S/D-Punjab, S/G-Philadelphia, A/Hope, A/Camden, and A/Athens-Georgia. The explanation for this is that in CZE, the baseline is integrated by the tangent method. In the electropherogram in the article by Panyasai et …

An affinity capillary electrophoresis was developed for detecting a point mutation of single-stranded DNA (ssDNA). Poly(ethylene glycol)-oligodeoxyribonucleo-tide block copolymers (PEG-b-ODN) were prepared to use as a novel affinity ligand. Optimization of the analytical conditions, such as the salt concentration of running buffer, the capillary temperature and the base number of the copolymer, gave two distinct peaks for wild-type (WT) and point-mutated ssDNA (MT) on the electropherogram, allowing for the facile discrimination of the single-base difference.

Capillary zone electrophoresis (CZE) is an alternative method for separation of serum proteins (1). Two dedicated and automated multichannel instruments are available, the Paragon 2000 (Beckman-Coulter) and the Capillarys (Sebia). The sensitivity and specificity of Paragon 2000 for detection of monoclonal proteins have been reported to be 95%(2)(3)(4) and 78%(4), respectively. The low reported specificity reflects the frequent occurrence of slight abnormalities in the electropherogram at the anodal part of the γ-globulin fraction (fibrinogen region)(4). Only 2 studies (5)(6) evaluated the Capillarys for detection of monoclonal proteins and reported a high sensitivity, and prospective studies in a routine clinical setting are …

The enhanced resolution of capillary zone electrophoresis (CZE) provides superior sensitivity for the detection of monoclonal (M) proteins in serum samples compared with agarose gel electrophoresis (AGE). This comes at the cost, however, of a lower specificity, calculated as 78% in our hands for the Paragon CZE 2000® with software version 1.602 (Beckman) (1). Abnormalities in the electropherogram that cause confusion are most frequently located in the anodal part of the γ-globulin fraction. Notably, fibrinogen, which remains in incompletely clotted serum samples, migrates at the junction between the β- and γ-fractions. Even when following the strict procedure of keeping serum samples for a minimum of 4 h at room temperature before electrophoresis, we found a suspected fibrinogen or possible M-protein peak in 39 (8.6%) of 454 consecutive routine samples in our laboratory. Recently, a simple method consisting of cold-ethanol precipitation was described for the removal of fibrinogen from serum specimens before agarose protein electrophoresis (2). We evaluated the use of this method in the routine analysis of serum samples by CZE in which a peak at the fibrinogen location could cause confusion with the detection of small M-proteins. Absolute ethanol was added to the specimens at a final concentration of 100 mL/L. …

We describe a computer program system for finding, quantitating, and matching the protein spots resolved on a two-dimensional electropherogram. The programs that locate and quantitate the incorporation of radioactivity into individual spots are totally automatic, as are the programs for matching protein spots between two exposures of the same gel. A semi-automatic method is used to match protein spots between different gels. This procedure is quite fast with the use of a computer-graphic display, which is also helpful in the editing process. A data base is set up and programs have been written to correlate matched protein spots from multi-gel experiments and to efficiently plot out quantitative data from sequences of equivalent spots from many gels or even many multi-gel experiments. The practical use of this system is discussed.

Cellulose acetate electrophoresis of Chironomus utahensis hemolymph demonstrated a six-band pattern for hemoglobins. Three major and three minor bands made up the electropherogram. Slight variations from this general pattern were found in less than 10 percent of the population and may represent ontogenetic changes, genetic variation or slight changes in the electrophoretic conditions. No variations could be correlated with the age of the culture, sex, or site of collection. Cytogenetic observations of the salivary glands revealed three large and one small pair of independent polytene chromosomes. Comparisons of electrophoretic patterns of hemoglobins and chromosome banding patterns with other chironomid species will help to clarify their taxonomic and evolutionary relationships.

Serum from a 71-year-old man with multiple myeloma complicated with renal failure showed a monoclonal IgG lambda. A second spike appearing in the serum protein electropherogram, suggesting a biclonal gammopathy, was found to be due to lambda Bence Jones protein (29.9 g/liter). Lambda Bence Jones protein was also found in smaller concentration (3.8 g/liter) in the urine. Tetramers of Bence Jones protein were not demonstrable by gel filtration and ultracentrifugation, and renal failure was probably the main reason for this very high concentration of Bence Jones protein in the serum.

Agarose-gel electrophoresis of serum of a 72-year-old woman with liver cirrhosis showed virtually no beta-globulins two weeks before the patient's death. There was marked decrease in the concentrations of transferrin, beta-lipoproteins, hemopexin, complement component C3, beta-glycoprotein I, and cholesterol in serum. Absence of a beta-globulin band appears to signify an ominous prognosis.