BioInformatics & BestKeeeper Software & Gene Quantification Homepage

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What is Bioinformatics? In the last few decades, advances in molecular biology and the equipment available for research in this field have allowed the increasingly rapid sequencing of large portions of the genomes of several species. In fact, to date, several bacterial genomes, as well as those of some simple eukaryotes (e.g., Saccharomyces cerevisiae, or baker's yeast) and more complex eukaryotes (C. elegans and Drosophila) have been sequenced in full. The Human Genome Project, designed to sequence all 24 of the human chromosomes, is also progressing and a rough draft was completed in the spring of 2000. http://www.library.csi.cuny.edu/~davis/molbiol/lecture_notes/bioinformatics_genomics/bioinformaticsIntro.html
Types of data		Analysis and Interpretation of Data

The various types of data: Many different types of data are collected and stored in databases to facilitate retrieval. Depicted here are amino acid sequences, protein domain cartoons, different renderings of three-dimensional structures, and protein hydrophobicity data. Databases consisting of data derived experimentally such as nucleotide sequences and three-dimensional structures are known as primary databases. Those data that are derived from the analysis or treatment of primary data such as secondary structures, hydrophobicity plots, and domains are stored in secondary databases. A protein database consisting of the conceptual translation of nucleotide sequences would also be considered a secondary database.		The analysis and interpretation of various data types: Illustrated here are various ways in which individual entries in sequence and structure databases can be compiled to reveal patterns and trends in biology. For example, sequence families or neighborhoods can be defined and annotated based on the similarity of each sequence to other members of the family. Common sequence features in sequence families can be identified in multiple alignments. These motifs may provide clues to the biochemical function of members of the family. Clustering of sequences into trees that reflect the degree of similarity between each sequence and all of the others in the family reveals evolutionary relationships. Finally, identification of homologs to each gene in well-characterized metabolic pathways provides information about the prevalence of that pathway in other organisms.
http://www.ncbi.nlm.nih.gov/Education/Bioinformatics/datatypes.html		http://www.ncbi.nlm.nih.gov/Education/Bioinformatics/dataanal.html

PCR Bioinformatics and Web based Primer Design

http://www.herts.ac.uk/natsci/STC/bio/pcrbio.html

There have been a number of key developments in molecular biology techniques however one that has had the most impact in recent years has been the polymerase chain reaction or PCR. One of the reason for the adoption of the PCR is the elegant simplicity of the reaction and relative ease of the practical manipulation steps. Frequently this is one of the first techniques used when analyzing DNA, it has opened up the analysis of cellular and molecular processes to those outside the field of molecular biology. There have been a number of key developments in molecular biology techniques however one that has had the most impact in recent years has been the polymerase chain reaction or PCR. One of the reason for the adoption of the PCR is the elegant simplicity of the reaction and relative ease of the practical manipulation steps. Frequently this is one of the first techniques used when analyzing DNA, it has opened up the analysis of cellular and molecular processes to those outside the field of molecular biology.

qPCR-DAMS:  a Database Tool to Analyze, Manage, and Store Both Relative and
Absolute Quantitative Real-Time PCR data.

Jin N, He K, Liu L.
Physiol Genomics. 2006

Physiological Sciences, Oklahoma State University, Stillwater, OK, USA.

Quantitative real-time PCR is an important high throughput method in biomedical sciences. However, existing software has limitations in handling both relative and absolute quantification. We designed qPCR-DAMS (Quantitative PCR Data Analysis and Management System), a database tool based on Access 2003, to deal with such shortcomings by the addition of integrated mathematical procedures. qPCR-DAMA allows a user choose among four methods for data processing within a single software package: (I) Ratio relative quantification, (II) Absolute level, (III) Normalized absolute expression, and (IV) Ratio absolute quantification. qPCR-DAMS also provides a tool for multiple reference gene normalization. qPCR-DAMS has three quality control steps and a data display system to monitor data variation. In summary, qPCR-DAMS is a handy tool for real-time PCR users. Availability: This software is free for academic use and downloadable http://download.gene-quantification.info/

FastPCR
© 1998-2005 v.3.6 (for Windows)

PCR primer design, DNA and protein tools, repeats and own database searches
http://www.biocenter.helsinki.fi/bi/Programs/fastpcr.htm

FastPCR is a free software for Microsoft Windows and is based on a new approach in the design of PCR primers for standard and long PCRs, inverse PCR, direct amino acid sequence degenerate PCR, multiplex PCR and in silico PCR; for sequence alignments, clustering and any kind repeat sequence searching.

At this moment the program is only for OS Microsoft Windows, but C# .Net Linux and Mac program versions are currently under preparation.

FastPCR Software can simultaneously work with multiple nucleic acid or protein sequences (up to 1,000,000). The multiplex PCR primers design and "in silico" PCR are also supported. The FastPCR program is an ideal software for personal databases homology searches which are similar to the basic local alignment search tool (BLAST) algorithm (a segment-to-segment alignment principle similar to DIALIGN). The program includes various bioinformatics tools and supports the clustering of sequences. A new repeats search theory was developed and applied to the program, which makes the accomplishment of all DNA repeat types searches fast and powerful.

FastPCR software has several specific, ready-to-use templates for many PCR and sequencing applications:

Standard, inverse and long PCR - Locates optimal primers for PCR, hybridisation, or sequencing.

Multiplex PCR primers design - fast primers design with a cross-dimers test for high sensitive multiplex PCRs.

Design group-specific PCR primers.

Degenerate PCR: primers are designed directly on an amino acid sequence.

In Silico PCR - prediction of probable PCR products and the mismatche primer location search.

Primers Secondary structures - self-dimers and cross-dimers primer analyses; primer alignment and melting temperatures calculation.

False priming - primers checking for multiple annealing sites using sequence alignment algorythms.

Primer quality - a unique way for PCR efficiency determination.

Comprehensive primer report - comprehensive pairs and individual primers analysis.

The software supports several file formats: FASTA, text and Excel files.

Tools:

Primer tests and dimer detection;

Powerful Repeats Search: Invert, Direct, Simple and others;

Clustering Sequences;

Make complement, reverse complement and inverted stand;

Search the sequence with universal degenerated code with alignment;

Extract the sequence from selected sites;

Protein/DNA translation;

Calculation the annealing temperature of PCR in case unknowns PCR product.

Database tools;

Restriction analysis.

Each application document contains customisable search settings, based on the latest published primer selection criteria for those applications.

Bioinformatics analysis of alternative splicing

Christopher Lee & Qi Wang

Briefings in Bioinformatics Volume: 6 Number: 1 Page: 23 -- 33

Over the past few years, the analysis of alternative splicing using bioinformatics has emerged as an important new field, and has significantly changed our view of genome function. One exciting front has been the analysis of microarray data to measure alternative splicing genome-wide. Pioneering studies of both human and mouse data have produced algorithms for discerning evidence of alternative splicing and clustering genes and samples by their alternative splicing patterns. Moreover, these data indicate the presence of alternative splice forms in up to 80 per cent of human genes. Comparative genomics studies in both mammals and insects have demonstrated that alternative splicing can in some cases be predicted directly from comparisons of genome sequences, based on heightened sequence conservation and exon length. Such studies have also provided new insights into the connection between alternative splicing and a variety of evolutionary processes such as Alu-based exonisation, exon creation and loss. A number of groups have used a combination of bioinformatics, comparative genomics and experimental validation to identify new motifs for splice regulatory factors, analyse the balance of factors that regulate alternative splicing, and propose a new mechanism for regulation based on the interaction of alternative splicing and nonsense-mediated decay. Bioinformatics studies of the functional impact of alternative splicing have revealed a wide range of regulatory mechanisms, from NAGNAG sites that add a single amino acid; to short peptide segments that can play surprisingly complex roles in switching protein conformation and function (as in the Piccolo C2A domain); to events that entirely remove a specific protein interaction domain or membrane anchoring domain. Common to many bioinformatics studies is a new emphasis on graph representations of alternative splicing structures, which have many advantages for analysis.

Comparison of different melting temperature calculation methods for short DNA sequences.

Alejandro Panjkovich & Francisco Melo

Bioinformatics (21,6): 711 -- 722

Motivation: The overall performance of several molecular biology techniques involving DNA/DNA hybridization depends on the accurate prediction of the experimental value of a critical parameter: the melting temperature Tm. Till date, many computer software programs based on different methods and/or parameterizations are available for the theoretical estimation of the experimental Tm value of any given short oligonucleotide sequence. However, in most cases, large and significant differences in the estimations of Tm were obtained while using different methods. Thus, it is difficult to decide which Tm value is the accurate one. In addition, it seems that most people who use these methods are unaware about the limitations, which are well described in the literature but not stated properly or restricted the inputs of most of the web servers and standalone software programs that implement them.

Results: A quantitative comparison on the similarities and differences among some of the published DNA/DNA Tm calculation methods is reported. The comparison was carried out for a large set of short oligonucleotide sequences ranging from 16 to 30 nt long, which span the whole range of CG-content. The results showed that significant differences were observed in all the methods, which in some cases depend on the oligonucleotide length and CG-content in a non-trivial manner. Based on these results, the regions of consensus and disagreement for the methods in the oligonucleotide feature space were reported. Owing to the lack of sufficient experimental data, a fair and complete assessment of accuracy for the different methods is not yet possible. Inspite of this limitation, a consensus Tm with minimal error probability was calculated by averaging the values obtained from two or more methods that exhibit similar behavior to each particular combination of oligonucleotide length and CG-content class. Using a total of 348 DNA sequences in the size range between 16mer and 30mer, for which the experimental Tm data are available, we demonstrated that the consensus Tm is a robust and accurate measure. It is expected that the results of this work would be constituted as a useful set of guidelines to be followed for the successful experimental implementation of various molecular biology techniques, such as quantitative PCR, multiplex PCR and the design of optimal DNA microarrays.

Availability: A binary software distribution to calculate the consensus Tm described in this work for thousands of oligonucleotides simultaneously for the LINUX operating system is freely available upon request to the authors or from our website http://protein.bio.puc.cl/melting-temperatures.html

Supplementary information: The large set of oligonucleotides, the detailed results of the comparative and accuracy benchmarks, and hundreds of comparative graphs generated during this work are available at our website http://protein.bio.puc.cl/melting-temperatures.html

A data-driven clustering method for time course gene expression data.

Ma P, Castillo-Davis CI, Zhong W, Liu JS.
Nucleic Acids Res. 2006 Mar 1;34(4):1261-9. Print 2006.
Department of Statistics, Harvard University, Cambridge, MA 02138, USA.

Gene expression over time is, biologically, a continuous process and can thus be represented by a continuous function, i.e. a curve. Individual genes often share similar expression patterns (functional forms). However, the shape of each function, the number of such functions, and the genes that share similar functional forms are typically unknown. Here we introduce an approach that allows direct discovery of related patterns of gene expression and their underlying functions (curves) from data without a priori specification of either cluster number or functional form. Smoothing spline clustering (SSC) models natural properties of gene expression over time, taking into account natural differences in gene expression within a cluster of similarly expressed genes, the effects of experimental measurement error, and missing data. Furthermore, SSC provides a visual summary of each cluster's gene expression function and goodness-of-fit by way of a 'mean curve' construct and its associated confidence bands. We apply this method to gene expression data over the life-cycle of Drosophila melanogaster and Caenorhabditis elegans to discover 17 and 16 unique patterns of gene expression in each species, respectively. New and previously described expression patterns in both species are discovered, the majority of which are biologically meaningful and exhibit statistically significant gene function enrichment. Software and source code implementing the algorithm, SSClust, is freely available http://genemerge.bioteam.net/SSClust.html

Distribution-insensitive cluster analysis in SAS on real-time PCR gene
expression data of steadily expressed genes.

Tichopad A, Pecen L, Pfaffl MW.

Comput Methods Programs Biomed. 2006 Apr;82(1):44-50. Epub 2006

Cluster analysis is a tool often employed in the micro-array techniques but used less in the real-time PCR. Herein we present core SAS code that instead of the Euclidian distances takes correlation coefficient as a dissimilarity measure. The dissimilarity measure is made robust using a rank-order correlation coefficient rather than a parametric one. There is no need for an overall probability adjustment like in scoring methods based on repeated pair-wise comparisons. The rank-order correlation matrix gives a good base for the clustering procedure of gene expression data obtained by real-time RT-PCR as it disregards the different expression levels. Associated with each cluster is a linear combination of the variables in the cluster, which is the first principal component. Large set of variables can then be replaced by the set of cluster components with little loss of information. In this way, distinct clusters containing unregulated housekeeping genes along with other steadily expressed genes can be disclosed and utilized for standardization purposes. Simulated data in parallel with the data from a biological experiment were taken to validate the SAS macro. For both cases, good intuitive results were obtained.

Real-time RT-PCR: Neue Ansätze zur exakten mRNA Quantifizierung

BioSpektrum 1/2004 (in German)

Die molekularen Technologien Genomics, Transcriptomics und Proteomics erobern immer mehr die klassischen Forschungsgebiete der Biowissenschaften. Die enorme Flut an gewonnenen Daten und Ergebnissen ist von überproportionalem Nutzen in der molekularen Diagnostik und Physiologie sowie die „Functional Genomics“. Immer neue ausgeklügelte Methoden und Anwendungen sind daher nötig um komplexe physiologische Vorgänge zu beschreiben. Da wir uns erst an Anfang dieser molekularen Ära befinden, ist es notwendig diese Techniken zu optimieren und komplett zu verstehen. Eine dieser technisch ausgefeilten Methoden zur zuverlässigen und exakten Quantifizierung spezifischer mRNA, stellt die real-time RT-PCR dar. Dieser Artikel beschreibt im Wesentlichen die effizienzkorrigierte relative Quantifizierung, die Normalisierung der Expressionsergebnisse anhand eines nicht regulierten „Housekeeping Gens“, die Berechnung der real-time PCR Effizienz sowie die Verrechnung und statistische Auswertung der Expressionsergebnisse. Alle beschriebenen Themenkomplexe können im Detail auf der korrespondierenden Internetseite in internationalen publizierten Originalarbeiten nachgeschlagen werden.

Analysis and Interpretation of Data

A web server for performing electronic PCR

Sequence Mapping by Electronic PCR

Genome Research
Vol. 7, No. 5, pp. 541-550, May 1997

Configuration Options

Analysis and Interpretation of Data

A web server for performing electronic PCR

Sequence Mapping by Electronic PCR

Genome Research Vol. 7, No. 5, pp. 541-550, May 1997

Configuration Options

Genome Research
Vol. 7, No. 5, pp. 541-550, May 1997