6 research outputs found
Quality Control Test for Sequence-Phenotype Assignments
<div><p>Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10–20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at <a href="http://bis.ifc.unam.mx/en/software/chispas" target="_blank">http://bis.ifc.unam.mx/en/software/chispas</a>.</p></div
Effect on the sensitivity of critical residues prediction on HokC or the HIV-1 protease by considering ISPAs.
<p>A) Prediction of the known critical residues of HokC is shown for ConSurf (cyan circles) and a random predictor (black circles). B) Prediction of the known critical residues of the HIV-1 protease is shown for ConSurf (cyan circles), POOL (red circles) and a random predictor (black circles). The filled circles correspond to the actual predictions achieved by any of these predictors and the empty circles correspond to the variation on the reliability of these predictions when 20% of the 25 or 46 known critical residues of HokC or the HIV-1 protease are considered ISPAs, respectively. Plots show in the Y-axis the sensitivity values. The image was generated using gnuplot.</p
Quantifying the leakiness of the selection method.
<p>The photo shows a plate where 100 colonies of <i>Escherichia coli</i> cells transformed with a plasmid harboring the wild type <i>hokC</i> gene (pEXT22-frg-<i>hokC</i>; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118288#sec002" target="_blank">Methods</a>) were exposed to IPTG to induce <i>hokC</i> expression. The wild type phenotype corresponds with the absence of cell growth in the presence of IPTG, while a mutant phenotype corresponds with cell growth in the presence of IPTG. In a non-leaky system no mutant phenotype should be observed, yet this image shows the presence of 6 colonies growing in the presence of IPTG. This experiment was repeated 4 times.</p
Sequencing procedure to identify ISPAs.
<p>The figure represents bacterial colonies in plates (big grey circles) that are induced to express (+) or not (-) <i>hokC</i>. Colonies presenting a wild-type and mutant phenotypes are depicted in black and light grey circles, respectively; bars represent the corresponding DNA molecules amplified from these bacterial colonies: black bar represent DNA molecules from cells presenting a wild-type phenotype. During this experimental procedure, it is expected that sequencing errors will be added to the errors introduced by the phenotype assignment; such errors may be reflected in incorrect relationships between DNA sequences and phenotypes. According to our method, if these experimental errors would be accumulated in DNA sequences from a given phenotype at a rate above the experimental errors these should be classified as ISPAs. The DNA sequences obtained by the sequencing procedure are represented at the bottom of the image, indicating in <b>bold</b> the mutated bases and in <i>italics</i> the sequences found in both phenotypes.</p
Effect on the accuracy of a perfect prediction of critical residues in HokC by considering ISPAs.
<p>Prediction of the known critical residues of HokC is shown for a perfect predictor (filled cyan circles) and a random predictor (open black circles). The different circles correspond to the variation on the reliability of these predictions when 20% of the 25 critical residues identified in this and previous studies were considered ISPAs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118288#sec002" target="_blank">Methods</a>). These predictors generate an ordered list of residues and the x-axis indicates the percentage of critical residues taken from the top of these lists. A) Plots in the Y-axis the sensitivity, B) plots in the Y-axis the specificity and C) plots in the Y-axis the Mathews Correlation Coefficient. The image was generated using gnuplot.</p
Spurious mutations in the promoter region of <i>hokC</i>.
<p>Mutations on the promoter region are shown in lower case letters and deletion with dash symbol. DNA sequences isolated from colonies presenting mutant phenotype (Mut XXX) or wild-type phenotype (Wt C08) are shown.</p><p>Spurious mutations in the promoter region of <i>hokC</i>.</p