The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.
This paper presents the utilization of progressive alignment principle for positional adjustment of a set of genomic signals with different lengths. The new method of multiple alignment of signals based on dynamic time warping is tested for the purpose of evaluating the similarity of different length genes in phylogenetic studies. Two sets of phylogenetic markers were used to demonstrate the effectiveness of the evaluation of intraspecies and interspecies genetic variability. The part of the proposed method is modification of pairwise alignment of two signals by dynamic time warping with using correlation in a sliding window. The correlation based dynamic time warping allows more accurate alignment dependent on local homologies in sequences without the need of scoring matrix or evolutionary models, because mutual similarities of residues are included in the numerical code of signals.
- Klíčová slova
- Correlation, Genomic signal processing, Multiple alignment, Phylogenetic tree, Similarity distance,
- MeSH
- algoritmy MeSH
- bakteriální RNA genetika MeSH
- druhová specificita MeSH
- fylogeneze MeSH
- genom bakteriální * MeSH
- genomika metody MeSH
- počítačové zpracování signálu MeSH
- RNA ribozomální 18S genetika MeSH
- sekvenční seřazení metody MeSH
- výpočetní biologie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální RNA MeSH
- RNA ribozomální 18S MeSH
BACKGROUND: Understanding the architecture and function of RNA molecules requires methods for comparing and analyzing their tertiary and quaternary structures. While structural superposition of short RNAs is achievable in a reasonable time, large structures represent much bigger challenge. Therefore, we have developed a fast and accurate algorithm for RNA pairwise structure superposition called SETTER and implemented it in the SETTER web server. However, though biological relationships can be inferred by a pairwise structure alignment, key features preserved by evolution can be identified only from a multiple structure alignment. Thus, we extended the SETTER algorithm to the alignment of multiple RNA structures and developed the MultiSETTER algorithm. RESULTS: In this paper, we present the updated version of the SETTER web server that implements a user friendly interface to the MultiSETTER algorithm. The server accepts RNA structures either as the list of PDB IDs or as user-defined PDB files. After the superposition is computed, structures are visualized in 3D and several reports and statistics are generated. CONCLUSION: To the best of our knowledge, the MultiSETTER web server is the first publicly available tool for a multiple RNA structure alignment. The MultiSETTER server offers the visual inspection of an alignment in 3D space which may reveal structural and functional relationships not captured by other multiple alignment methods based either on a sequence or on secondary structure motifs.
- MeSH
- algoritmy * MeSH
- internet * MeSH
- konformace nukleové kyseliny * MeSH
- RNA chemie MeSH
- sekvenční analýza RNA metody MeSH
- sekvenční seřazení metody MeSH
- software * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- RNA MeSH
The development of a diagnostic polymerase chain reaction (PCR) or quantitative PCR (qPCR) assay for universal detection of highly variable viral genomes is always a difficult task. The purpose of this chapter is to provide a guideline on how to align, process, and evaluate a huge set of homologous nucleotide sequences in order to reveal the evolutionarily most conserved positions suitable for universal qPCR primer and hybridization probe design. Attention is paid to the quantification and clear graphical visualization of the sequence variability at each position of the alignment. In addition, specific problems related to the processing of the extremely large sequence pool are highlighted. All of these steps are performed using an ordinary desktop computer without the need for extensive mathematical or computational skills.
- MeSH
- DNA primery genetika MeSH
- polymerázová řetězová reakce metody MeSH
- sekvenční seřazení metody MeSH
- teoretické modely MeSH
- virus chřipky A genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
MOTIVATION: Understanding the architecture and function of RNA molecules requires methods for comparing and analyzing their 3D structures. Although a structural alignment of short RNAs is achievable in a reasonable amount of time, large structures represent much bigger challenge. However, the growth of the number of large RNAs deposited in the PDB database calls for the development of fast and accurate methods for analyzing their structures, as well as for rapid similarity searches in databases. RESULTS: In this article a novel algorithm for an RNA structural comparison SETTER (SEcondary sTructure-based TERtiary Structure Similarity Algorithm) is introduced. SETTER uses a pairwise comparison method based on 3D similarity of the so-called generalized secondary structure units. For each pair of structures, SETTER produces a distance score and an indication of its statistical significance. SETTER can be used both for the structural alignments of structures that are already known to be homologous, as well as for 3D structure similarity searches and functional annotation. The algorithm presented is both accurate and fast and does not impose limits on the size of aligned RNA structures. AVAILABILITY: The SETTER program, as well as all datasets, is freely available from http://siret.cz/hoksza/projects/setter/.
- MeSH
- algoritmy * MeSH
- konformace nukleové kyseliny * MeSH
- RNA chemie MeSH
- sekvenční analýza RNA metody MeSH
- sekvenční seřazení metody MeSH
- software * MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- RNA MeSH
RNA interference (RNAi) has become an important tool to study and utilize gene silencing by introducing short interfering RNA (siRNA). In order to predict the most efficient siRNAs, a new software tool, RNA Workbench (RNAWB), has been designed and is freely available (after registration) on http://www.rnaworkbench.com. In addition to the standard selection rules, RNAWB includes the possibility of statistical analyses of the applied selection rules (criteria). The role of RNA secondary structures in the RNA interference process as well as the application of sequence rules are discussed to show the applicability of the software.
- MeSH
- algoritmy * MeSH
- genový targeting metody MeSH
- malá interferující RNA genetika MeSH
- molekulární sekvence - údaje MeSH
- RNA interference * MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza RNA metody MeSH
- sekvenční seřazení metody MeSH
- software * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- malá interferující RNA MeSH
Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous tissue.
- MeSH
- cysteinové endopeptidasy metabolismus MeSH
- encefalitogenní základní proteiny metabolismus MeSH
- genetická transkripce MeSH
- imunohistochemie metody MeSH
- infekce červy třídy Trematoda metabolismus MeSH
- isomerie MeSH
- kathepsin B analýza chemie MeSH
- messenger RNA genetika MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- prekurzory enzymů analýza MeSH
- rekombinantní proteiny analýza MeSH
- RNA helmintů genetika MeSH
- Schistosomatidae chemie genetika MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
- Názvy látek
- asparaginylendopeptidase MeSH Prohlížeč
- cysteinové endopeptidasy MeSH
- encefalitogenní základní proteiny MeSH
- kathepsin B MeSH
- messenger RNA MeSH
- prekurzory enzymů MeSH
- rekombinantní proteiny MeSH
- RNA helmintů MeSH
BACKGROUND: The program InDeVal was originally developed to help researchers find known regions of insertion/deletion activity (with the exception of isolated single-base indels) in newly determined Poaceae trnL-F sequences and compare them with 533 previously determined sequences. It is supplied with input files designed for this purpose. More broadly, the program is applicable for finding specific target regions (referred to as "variable regions") in DNA sequence. A variable region is any specific sequence fragment of interest, such as an indel region, a codon or codons, or sequence coding for a particular RNA secondary structure. RESULTS: InDeVal input is DNA sequence and a template file (sequence flanking each variable region). Additional files contain the variable regions and user-defined messages about the sequence found within them (e.g., taxa sharing each of the different indel patterns).Variable regions are found by determining the position of flanking sequence (referred to as "conserved regions") using the LPAM (Length-Preserving Alignment Method) algorithm. This algorithm was designed for InDeVal and is described here for the first time. InDeVal output is an interactive display of the analyzed sequence, broken into user-defined units. Once the user is satisfied with the organization of the display, the information can be exported to an annotated text file. CONCLUSIONS: InDeVal can find multiple variable regions simultaneously (28 indel regions in the Poaceae trnL-F files) and display user-selected messages specific to the sequence variants found. InDeVal output is designed to facilitate comparison between the analyzed sequence and previously evaluated sequence. The program's sensitivity to different levels of nucleotide and/or length variation in conserved regions can be adjusted. InDeVal is currently available for Windows in Additional file 1 or from http://www.sci.muni.cz/botany/elzdroje/indeval/.
- MeSH
- DNA rostlinná genetika MeSH
- inzerční mutageneze genetika MeSH
- kodon genetika MeSH
- konzervovaná sekvence genetika MeSH
- lipnicovité genetika MeSH
- mutační analýza DNA metody MeSH
- sekvenční delece genetika MeSH
- sekvenční seřazení metody MeSH
- software * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA rostlinná MeSH
- kodon MeSH
Despite the wealth of information on the functional and pharmacological properties of the M2 muscarinic receptor, we know relatively little of structure and regulation of the M2 receptor gene. Here, we describe the organisation of the human M2 gene and its promoters. Four exons are present in the 5' untranslated region of the human M2 mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR-32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2 promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA-box. In addition, we describe another M2 promoter that is specific for rat. We show that GATA-4 transcription factor binds to two sites within the regulatory regions of the M2 gene using reporter gene assays, electromobility shift assays and mutational analysis.
- MeSH
- buněčné linie MeSH
- chromatin metabolismus MeSH
- DNA vazebné proteiny metabolismus MeSH
- exony MeSH
- imunoprecipitace metody MeSH
- klonování DNA metody MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- luciferasy metabolismus MeSH
- messenger RNA genetika MeSH
- molekulární sekvence - údaje MeSH
- mutageneze fyziologie MeSH
- myoblasty MeSH
- myši MeSH
- neuroblastom MeSH
- orgánová specificita MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- promotorové oblasti (genetika) fyziologie MeSH
- protein vázající TATA box metabolismus MeSH
- receptor muskarinový M2 genetika metabolismus MeSH
- regulace genové exprese fyziologie MeSH
- reportérové geny fyziologie MeSH
- retardační test metody MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení metody MeSH
- transkripční faktor GATA4 MeSH
- transkripční faktory metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- chromatin MeSH
- DNA vazebné proteiny MeSH
- luciferasy MeSH
- messenger RNA MeSH
- protein vázající TATA box MeSH
- receptor muskarinový M2 MeSH
- transkripční faktor GATA4 MeSH
- transkripční faktory MeSH
SUMMARY: PRIMEX (PRImer Match EXtractor) can detect oligonucleotide sequences in whole genomes, allowing for mismatches. Using a word lookup table and server functionality, PRIMEX accepts queries from client software and returns matches rapidly. We find it faster and more sensitive than currently available tools. AVAILABILITY: Running applications and source code have been made available at http://bioinformatics.cribi.unipd.it/primex
- MeSH
- algoritmy * MeSH
- genom * MeSH
- oligonukleotidy genetika MeSH
- sekvenční analýza DNA metody MeSH
- sekvenční seřazení metody MeSH
- software * MeSH
- uživatelské rozhraní počítače MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- srovnávací studie MeSH
- validační studie MeSH
- Názvy látek
- oligonukleotidy MeSH
