UniqTag: Content-Derived Unique and Stable Identifiers for Gene Annotation

<div><p>When working on an ongoing genome sequencing and assembly project, it is rather inconvenient when gene identifiers change from one build of the assembly to the next. The gene labelling system described here, UniqTag, addresses this common challenge. UniqTag assigns a unique identifier to each gene that is a representative <i>k</i>-mer, a string of length <i>k</i>, selected from the sequence of that gene. Unlike serial numbers, these identifiers are stable between different assemblies and annotations of the same data without requiring that previous annotations be lifted over by sequence alignment. We assign UniqTag identifiers to ten builds of the Ensembl human genome spanning eight years to demonstrate this stability. The implementation of UniqTag in Ruby and an R package are available at <a href="https://github.com/sjackman/uniqtag" target="_blank">https://github.com/sjackman/uniqtag</a> sjackman/uniqtag. The R package is also available from CRAN: install.packages ("uniqtag"). Supplementary material and code to reproduce it is available at <a href="https://github.com/sjackman/uniqtag-paper" target="_blank">https://github.com/sjackman/uniqtag-paper</a>.</p></div

The number of common UniqTag identifiers between build 75 of the Ensembl human genome and nine other builds, the number of common gene and protein identifiers between builds, and the number of genes with peptide sequences that are identical between builds.

<p>The number of common UniqTag identifiers between build 75 of the Ensembl human genome and nine other builds, the number of common gene and protein identifiers between builds, and the number of genes with peptide sequences that are identical between builds.</p

Oleic Acid Metabolism <i>via</i> a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus <i>Grosmannia clavigera</i>

<div><p>The bark beetle-associated fungus <i>Grosmannia clavigera</i> participates in the large-scale destruction of pine forests. In the tree, it must tolerate saturating levels of toxic conifer defense chemicals (e.g. monoterpenes). The fungus can metabolize some of these compounds through the ß-oxidation pathway and use them as a source of carbon. It also uses carbon from pine triglycerides, where oleic acid is the most common fatty acid. High levels of free fatty acids, however, are toxic and can cause additional stress during host colonization. Fatty acids induce expression of neighboring genes encoding a cytochrome P450 (CYP630B18) and its redox partner, cytochrome P450 reductase (CPR2). The aim of this work was to study the function of this novel P450 system. Using LC/MS, we biochemically characterized CYP630 as a highly specific oleic acid ω-hydroxylase. We explain oleic acid specificity using protein interaction modeling. Our results underscore the importance of ω-oxidation when the main ß-oxidation pathway may be overwhelmed by other substrates such as host terpenoid compounds. Because this CYP-CPR gene cluster is evolutionarily conserved, our work has implications for metabolism studies in other fungi.</p></div

Time course of the root mean square (RMS) changes of the heme moiety in <i>Gs</i>CYP630B18 with oleic acid (left) and stearic acid (right) as a substrate.

<p>The protein was constrained for the first 100 ps.</p

Enzyme activity of <i>Gs</i>CPR1 and <i>Gs</i>CPR2.

<p>EU—one enzyme unit of CPR reduces 1.0 μmol oxidized cytochrome c per minute in the presence of 100 mM NADPH at pH 7.8 and 25°C.</p><p>m—membrane-bound</p><p>Positive control—rabbit liver CPR</p><p>Negative control—membrane fraction of yeast strain expressing empty plasmid pYEDP60U</p><p>Enzyme activity of <i>Gs</i>CPR1 and <i>Gs</i>CPR2.</p

Calculated kinetic parameters for <i>Gs</i>CPR1 and <i>Gs</i>CPR2.

<p>Calculated kinetic parameters for <i>Gs</i>CPR1 and <i>Gs</i>CPR2.</p

Schematic representation of selective conservation of the CYP630-CPR2 gene cluster in Pezizomycotina.

<p>The presence of the gene cluster found in seven Pezizomycotina classes is given as a fraction of the species that the gene cluster was identified in relative to all the species of a given class whose genomes were searched. Homologs of either gene were not identified outside of Pezizomycotina. The relative orientation of both genes is given (< >—divergent; > <—convergent, << or >>—co-oriented) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref047" target="_blank">47</a>]. The phylogeny was modeled after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref048" target="_blank">48</a>].</p

Oleic acid oxidation by <i>Gs</i>CYP630B18 in combination with <i>Gs</i>CPR1 or <i>Gs</i>CPR2.

<p>Extracted ion chromatograms (297.2–297.4 m/z, run in negative-ion ESI-MS) for oleic acid conversion by GsCYP630B18 in combination with GsCPR1 or GsCPR2 with the highest peak (m/z 297.3) identified as 18-hydroxyoleic acid. Darkened lines indicate where the peaks were integrated for relative ion intensity comparison. Samples GsCYP630 with GsCPR1 (top left panel) and GsCYP630 with GsCPR2 (top right panel) are shown in red. Controls (empty E. coli membrane fractions with GsCPR1or GsCPR2) are shown in black. Significant differences for the major product peak between controls and samples were indicated by P-value (P < 0.05, n = 3). Additional data for each analysis are shown below the LC/MS traces as m/z value, retention time, fold change as ratio of mean intensities between samples and controls, P-value, and peak intensity as average feature intensity within sample/control class.</p

Standard plots for determining ferricyanide reduction kinetics catalyzed by <i>Gs</i>CPR1 or <i>Gs</i>CPR2 in the presence of NADPH.

<p>The CPR-catalyzed reduction of ferricyanide in concentrations between 2 μM and 500 μM at 420 nm in the presence of saturating 100 mM NADPH in 100 mM potassium phosphate (pH 7.6). Different concentrations of ferricyanide are represented in shades of grey and dashed lines. Decreasing absorbance at 420 nm, quantifying the reduction of ferricyanide to ferrocyanide, was plotted by normalizing all points relative to the point zero baseline.</p

Changes in transcript abundance of genes involved in the <i>Gs</i>CYP630B18 putative fatty acid oxidation pathway following growth on monoterpenes, triglycerides or oleic acid as the sole carbon sources.

<p>RNA-Seq data for selected genes from mycelia grown for 10 days on YNB minimal medium with a mixture of monoterpenes (YNB+MT) (monoterpenes: (+)-limonene, (+)-3-carene, racemic α-pinene and (−)-β-pinene at a ratio of 5:3:1:1), for 5 days with triglycerides (YNB+TG), or oleic acid (YNB+OA) as the sole carbon source, relative to controls grown on mannose [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref067" target="_blank">67</a>].</p><p>*<i>P-</i>values are not significant.</p><p>LpL—lipoprotein lipase, GK—glycerol kinase, ACSL—long-chain fatty acyl-CoA synthetase, FATPs—fatty acid transporter proteins, ACLs—acyl-CoA coenzymes, CT—carnitine acetyl transferase, FOX2—multifunctional β-oxidation enzyme, CYP630B18—cytochrome P450, CPR2—cytochrome P450 reductase 2.</p><p>Changes in transcript abundance of genes involved in the <i>Gs</i>CYP630B18 putative fatty acid oxidation pathway following growth on monoterpenes, triglycerides or oleic acid as the sole carbon sources.</p