Programming for the DNA analysis of FCM data on an IBM microcomputer

The analysis of data generated on a flow cytometer (FCM) is often performed on a computer obtained especially for dedicated use with the flow cytometer. This computer component can be expensive and also presents the FCM user with the added burden of mastering specialized programming language or of accepting the secret analytical processes of protected proprietary program routines. We believe that the evolution of more accurate and efficient FCM analyses that have the power to consider complex signal distributions can be assisted by the availability of analysis programs written in languages common to many users. DNA analysis routines written for a relatively inexpensive microcomputer (IBM PC/XT) in Basic and Pascal are described here. The routines can automatically process multiple FCM data files and can provide high-resolution graphic hardcopy. A foreground/background utilization is also described that allows the computer to be available for other uses in the laboratory.     

Computer programs for the analysis and the management of DNA sequences.

A program package is described for the management and the analysis of DNA sequence data. The programs - with the exception of a few Fortran routines - are written in the programming language APL. They are best used interactively although batch processing is possible. The package has been in constant use for about 3 years and contains programs for most of the routine problems presently found in a DNA sequencing laboratory.     

Automated band mapping in electrophoretic gel images using background information

Some popular methods for polymorphism and mutation discovery involve ascertainment of novel bands by the examination of electrophoretic gel images. Although existing strategies for mapping bands work well for specific applications, such as DNA sequencing, these strategies are not well suited for novel band detection. Here, we describe a general strategy for band mapping that uses background banding patterns to facilitate lane calling and size calibration. We have implemented this strategy in GelBuddy, a user-friendly Java-based program for PC and Macintosh computers, which includes several utilities to assist discovery of mutations and polymorphisms. We demonstrate the use of GelBuddy in applications based on single-base mismatch cleavage of heteroduplexed PCR products. Use of software designed to facilitate novel band detection can significantly shorten the time needed for image analysis and data entry in a high-throughput setting. Furthermore, the interactive strategy implemented in GelBuddy has been successfully applied to DNA fingerprinting applications, such as AFLP. GelBuddy promises to make electrophoretic gel analysis a viable alternative to DNA resequencing for discovery of mutations and polymorphisms.     

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.     

Methylation blockage and other improvements to a comprehensive DNA analysis program.

A comprehensive DNA analysis computer program was described in the second special issue of Nucleic Acids Research on the applications of computers to research on nucleic acids by Stone and Potter (1). Criteria used in designing the program were user friendliness, ability to handle large DNA sequences, low storage requirement, migratability to other computers and comprehensive analysis capability. The program has been used extensively in an industrial-research environment. This paper talks about improvements to that program. These improvements include testing for methylation blockage of restriction enzyme recognition sites, homology analysis, RNA folding analysis, integration of a large DNA database (GenBank), a site specific mutagenesis analysis, a protein database and protein searching programs. The original design of the DNA analysis program using a command executive from which any analytical programs can be called, has proven to be extremely versatile in integrating both developed and outside programs to the file management system employed.     

MacSECPROT: Two programs in basic to evaluate protein secondary structure

Two interactive programs in BASIC are described, which provide useful tools to evaluate protein secondary structure. Output is given in two formats: (1) graphics are displayed on screen, which can be printed immediately, and (2) textfiles are saved to disk as permanent records and can be printed with a word-processing program. The programs are fast and easy to use and could be a valuable teaching aid in biochemical and molecular biology courses. Program lists are written in Microsoft® BASIC for the Apple® Macintosh™, but can be adapted to other machines accepting graphic commands.     

Spreadsheet-based program for alignment of overlapping DNA sequences.

Molecular biology laboratories frequently face the challenge of aligning small overlapping DNA sequences derived from a long DNA segment. Here, we present a short program that can be used to adapt Excel spreadsheets as a tool for aligning DNA sequences, regardless of their orientation. The program runs on any Windows or Macintosh operating system computer with Excel 97 or Excel 98. The program is available for use as an Excel file, which can be downloaded from the BioTechniques Web site. Upon execution, the program opens a specially designed customized workbook and is capable of identifying overlapping regions between two sequence fragments and displaying the sequence alignment. It also performs a number of specialized functions such as recognition of restriction enzyme cutting sites and CpG island mapping without costly specialized software.     

Comprehensive restriction enzyme lists to update any DNA sequence computer program

Restriction enzyme lists are presented for the practical working geneticist to update any DNA computer program. These lists combine formerly scattered information and contain all presently known restriction enzymes with a unique recognition sequence, a cut site, or methylation (in)sensitivity. The lists are in the shortest possible form to also be functional with small DNA computer programs, and will produce clear restriction maps without any redundancy or loss of information. The lists discern between commercial and noncommercial enzymes, and prototype enzymes and different isoschizomers are cross-referenced. Differences in general methylation sensitivities and (in)sensitivities against Dam and Dcm methylases of Escherichia coli are indicated. Commercial methylases and intron-encoded endonucleases are included. An address list is presented to contact commercial suppliers. The lists are constantly updated and available in electronic form as pure US ASCII files, and in formats for the DNA computer programs DNA-Strider for Apple Macintosh, and DNAsis for IBM personal computers or compatibles via e-mail from the internet address: netservembl-heidelberg.de by sending only the message help relibrary.     

GELYMAC: a Macintosh application for calculating DNA fragment size from gel electrophoresis migration data

A program called GELYMAC takes data on the distances migratedby DNA fragments in a one-dimensional electro-phoretic gel and,using a cubic-spline best-fit of marker fragment distance migratedversus molecular size, calculates the molecular sizes of thefragments. Written in the Rascal (Realtime Pascal) programminglanguage, the program runs on the Macintosh family of microcomputers.Rapid entry of marker and experimental fragment migration datais afforded using a scroll bar system adjacent to a graphicrepresentation of a gel. Output includes tabular listing ofthe data, graphic cartoons of the gel, and the fragment locationsand molecular sizes for individual gel lanes, and the calibrationcurve used in data computations.     

4P: fast computing of population genetics statistics from large DNA polymorphism panels

Massive DNA sequencing has significantly increased the amount of data available for population genetics and molecular ecology studies. However, the parallel computation of simple statistics within and between populations from large panels of polymorphic sites is not yet available, making the exploratory analyses of a set or subset of data a very laborious task. Here, we present 4P (parallel processing of polymorphism panels), a stand‐alone software program for the rapid computation of genetic variation statistics (including the joint frequency spectrum) from millions of DNA variants in multiple individuals and multiple populations. It handles a standard input file format commonly used to store DNA variation from empirical or simulation experiments. The computational performance of 4P was evaluated using large SNP (single nucleotide polymorphism) datasets from human genomes or obtained by simulations. 4P was faster or much faster than other comparable programs, and the impact of parallel computing using multicore computers or servers was evident. 4P is a useful tool for biologists who need a simple and rapid computer program to run exploratory population genetics analyses in large panels of genomic data. It is also particularly suitable to analyze multiple data sets produced in simulation studies. Unix, Windows, and MacOs versions are provided, as well as the source code for easier pipeline implementations.     

The CCPN data model for NMR spectroscopy: development of a software pipeline

To address data management and data exchange problems in the nuclear magnetic resonance (NMR) community, the Collaborative Computing Project for the NMR community (CCPN) created a "Data Model" that describes all the different types of information needed in an NMR structural study, from molecular structure and NMR parameters to coordinates. This paper describes the development of a set of software applications that use the Data Model and its associated libraries, thus validating the approach. These applications are freely available and provide a pipeline for high-throughput analysis of NMR data. Three programs work directly with the Data Model: CcpNmr Analysis, an entirely new analysis and interactive display program, the CcpNmr FormatConverter, which allows transfer of data from programs commonly used in NMR to and from the Data Model, and the CLOUDS software for automated structure calculation and assignment (Carnegie Mellon University), which was rewritten to interact directly with the Data Model. The ARIA 2.0 software for structure calculation (Institut Pasteur) and the QUEEN program for validation of restraints (University of Nijmegen) were extended to provide conversion of their data to the Data Model. During these developments the Data Model has been thoroughly tested and used, demonstrating that applications can successfully exchange data via the Data Model. The software architecture developed by CCPN is now ready for new developments, such as integration with additional software applications and extensions of the Data Model into other areas of research.     

Replication Dynamics: Biases and Robustness of DNA Fiber Analysis

The factors that govern replication programs are still poorly identified in metazoans, especially in mammalian cells. Thanks to molecular combing, the dynamics of DNA replication can be assessed at the genome-scale level from the cumulative analysis of single DNA fibers. This technique notably enables measurement of replication fork speed and fork asymmetry and that of distances separating either initiation or termination events. The results presented here aim to evaluate requirements critical to accurate measurement of replication parameters by molecular combing. We show that sample size, fiber length and DNA counterstaining are crucial to gain robust information concerning replication dynamics. Our results thus provide a methodological frame to investigate the DNA replication program through molecular combing analyses.     

High-throughput DNA extraction from forest trees

It is difficult to extract pure high-quality DNA from trees, which may not be amenable to advances in extraction methods suitable for other plants. A new commercial high-throughput DNA extraction system, using a silica binding matrix for purification and a multisample mixer mill for tissue disruption, was evaluated for its suitability withEucalyptus spp.,Pinus spp., andAraucaria cunninghamii (hoop pine). DNA suitable for a range of molecular biology applications was successfully extracted from all genera. The method was highly reliable when tested in more than 500 preparations and could be adapted to different tree species with relatively minor modifications.     

DNACompress: fast and effective DNA sequence compression

While achieving the best compression ratios for DNA sequences, our new DNACompress program significantly improves the running time of all previous DNA compression programs.     

MorphoJ: an integrated software package for geometric morphometrics

Increasingly, data on shape are analysed in combination with molecular genetic or ecological information, so that tools for geometric morphometric analysis are required. Morphometric studies most often use the arrangements of morphological landmarks as the data source and extract shape information from them by Procrustes superimposition. The MorphoJ software combines this approach with a wide range of methods for shape analysis in different biological contexts. The program offers an integrated and user-friendly environment for standard multivariate analyses such as principal components, discriminant analysis and multivariate regression as well as specialized applications including phylogenetics, quantitative genetics and analyses of modularity in shape data. MorphoJ is written in Java and versions for the Windows, Macintosh and Unix/Linux platforms are freely available from http://www.flywings.org.uk/MorphoJ_page.htm.     

Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome

This paper describes software (written in Pascal and running on Macintosh computers) allowing localization of unknown DNA fragments from the Escherichia coli chromosome on the restriction map established by Kohara et al. (1987). The program identifies the segment's map position using a restriction pattern analysis obtained with all, or some, of the eight enzymes used by Kohara et al. (1987). Therefore, the sequenced genes available in the EMBL library may be localized on the E. coli chromosome restriction map. This allowed correction of the map (mainly by introducing missing sites in the published maps) at the corresponding positions. Analysis of the data indicates that there is only a very low level of polymorphism, at the nucleotide level, between the E. coli K12 strains used by the various laboratories involved in DNA sequencing. The program is versatile enough to be used with other genomes.     

PRIMO: A Primer Design Program That Applies Base Quality Statistics for Automated Large-Scale DNA Sequencing

PRIMO is a computer program that designs walking primers for large-scale DNA sequencing projects. Oligonucleotide primers are predicted automatically, using quality information associated with each base call, eliminating the need for manually viewing the sequence traces or inspecting contig assemblies to determine appropriate locations for primer design. This allows PRIMO to run in batch mode on an arbitrarily large number of templates. For shotgun sequencing, PRIMO reads assembled sequence contigs with corresponding base quality statistics and automatically designs walking primers as needed to extend and join contigs, or improve their overall quality. In the opposite extreme of single-pass or completely directed sequencing, PRIMO reads the unassembled sequence for each template and designs walking primers for extending each read. If the base-calling software does not provide base quality statistics, PRIMO assigns its own measure of base quality determined by the shapes of individual peaks in the trace data for each template. In this way, PRIMO can be used in the finishing stages of a shotgun sequencing project, in sequencing by directed primer walking, or in some intermediate strategy. The code is written in ANSI C and maintained in two versions: one for the Macintosh and the other for UNIX.