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As molecular biology becomes increasingly informational, computer software to manipulate and analyze nucleic acid sequences has evolved from a useful tool to an absolute necessity, researchers say. Foster City, Calif. 94404 (800) 345-5ABI Fax: (415) 572-2743 Products: GENESCAN 672 for microsatellite markers on Macintosh: call for price. DNASTAR Inc. 1228 S. Park St. Madison, Wis. 53715 (608) 258-7420 Fax: (608) 258-7439 Products: Lasergene comprehensive package: $3,995; Lasergene options, including BIO-CORE 1, Map-Draw, and GeneFont (basic molecular analysis system) and MegAlign, Protean, GeneMan, or SeqMan: $945. Genetics Computer Group Inc. (GCG) University Research Park 575 Science Dr., Suite B Madison, Wis. 53711 (608) 231-5200 Fax: (608) 231-5202 Product: GCG Sequence Analysis Software Package: $4,000 (nonprofit organizations) or $12,000 (for-profit organizations) Hitachi Software Engineering America Ltd. 1111 Bayhill Dr., Suite 395 San Bruno, Calif. 94066 (800) 624-6176 Fax: (415) 615-7699 Products: DNASIS 7.0 for PC: $1,700; Hitachi MacDNASIS 2.0 for Macintosh: $1,950 IntelliGenetics Inc. 700 E. El Camino Real Mountain View, Calif. 94040 (415) 962-7300 Fax: (415) 962-7302 Products: GeneWorks sequence-analysis software (Macintosh): $2,000-$3,000, depending on customization; PC/GENE: $2,700-$3,800 Millipore Corp. 80 Ashby Rd. Bedford, Mass. 01730-9125 (617) 275-9200 Fax: (617) 275-5550 Products: Bio Image Short Tandem Repeat Polymorphism Software for microsatellite mapping: call for price; DNA Sequence Assembly Manager Software, assembles contigs: call for price. National Biosciences Inc. 3650 Annapolis La. Plymouth, Minn. 55447-5434 (800) 747-4362 Fax: (800) 369-5118 Products: OLIGO 4.1 Primer Analysis Software for PC or Macintosh: $800 National Center for Biotechnology Information National Library of Medicine 8600 Rockville Pike Building 38A, Room 8N806 Bethesda, Md. 20894-0001 (301) 496-2475 Fax: (301) 480-9241 Products: BLASTX, BLASTN: free; call for information. Oak Ridge National Laboratory Computer Sequence Analysis Group Engineering, Physics and Math Division Oak Ridge, Tenn. 37831 (615) 574-6134 Products: GRAIL/CRM: free; call for information. The first generation of DNA software accelerated sequencing by analyzing images of DNA fragments on electrophoresis gels. Today's programs perform dozens of functions on strings of nucleotides, including cutting, comparing, and detecting information. There is much to choose from. A primary consideration in selecting software is one's hardware. IntelliGenetics Inc. of Mountain View, Calif., for example, offers flexibility: Its GeneWorks sequence-analysis software operates on a Macintosh, while its PC/GENE package runs on IBM- compatible personal computers. The Genetics Computer Group Inc. (GCG) of Madison, Wis., offers the Wisconsin Package, which operates on a VAX/VMS system, from the Maynard, Mass.-based Digital Equipment Corp., and several UNIX systems, from AT&T Bell Laboratories, Murray Hill, N.J. Software from the National Center for Biotechnology Information, a division of the National Library of Medicine in Bethesda, Md., is also UNIX-compatible. Some DNA software packages offer an impressive list of functions to prepare the user for nearly any research contingency, while others are more targeted and specific in the tasks they support. IntelliGenetics' PC/GENE and GeneWorks; MacDNASIS from Hitachi Software Engineering America Ltd. in San Bruno, Calif.; GCG's Wisconsin Package; and others offer an array of functions, including basic editing; overlapping sequences to form contig maps, a method of approaching a gene by aligning overlapping sequences; generating restriction maps; comparing sequences and calculating the percentage of divergence; searching databases; transcribing DNA to RNA and displaying its secondary structure; converting nucleic acid sequences to amino acid sequences; and recognizing patterns, such as protein encoding regions and repeated short sequences. Millipore Corp. of Bedford, Mass., by contrast, offers five more specific software packages used for genome research and mapping, says marketing communications manager Greg Hoff. Each package provides several related functions. The restriction fragment length polymorphism (RFLP) databasing software, for example, allows the user to build databases of RFLP films, and to compare and analyze information in the databases. (An RFLP is a DNA sequence that varies among individuals and is detectable because it alters a restriction enzyme cutting site, changing the sizes of fragments that result from applying the enzyme to DNA.) Software packages targeted to a specific technique can also be comprehensive. OLIGO 4.0 from National Biosciences Inc. of Plymouth, Minn., selects oligonucleotide primers for use in the polymerase chain reaction (PCR) from a researcher's DNA or RNA sequence or from a database. In selecting primers, the program considers such parameters as secondary structure, specificity, and degeneracy, and calculates annealing and melting temperatures. The program also can edit a sequence and translate a single-stranded DNA sequence in all three reading frames (starting with the first, second, or third base). DNASTAR of Madison, Wis., offers " la carte" modules such as MegAlign (for sequence alignment), MapDraw (for restriction analysis), and GeneMan (to access databases). Additionally, the firm offers the comprehensive software package Lasergene, which also is available in a form that supports five simultaneous users. Francis Barany, an associate professor of microbiology at Cornell University Medical College, says that with Lasergene, at the press of a button, he displays maps of the cancer genes with which he is working, altering the maps in seconds by punching in new DNA cutting enzymes. Some programs are friendlier than others. Potential users often shy away from the software available from the National Library of Medicine's National Center for Biotechnology Information, says Warren Gish, a researcher at the facility, because of the effort necessary to master a UNIX workstation, which the software runs on. The center's programs--BLASTN to compare nucleic acid sequences and BLASTX to detect protein encoding gene regions--are "accessed over Internet, a worldwide network that is very popular in academia, and is growing in popularity in the commercial sector. Using a file transfer protocol [FTP], a user can connect to a server [computer] here at the National Center for Biotechnology Information, and copy the software onto a local computer," Gish says. But a biologist who is not a computer aficionado might be better off purchasing software from a commercial vendor, Gish says. "It costs to use company software, but the users have a right to expect that the system be more turnkey, that they have access to someone to call with a problem. Companies offer more hand- holding," Gish says, referring to the training sessions, hotlines, and newsletters that typically accompany purchase of DNA sequence-analysis software. Easily accessible is the GRAIL program from Oak Ridge National Laboratory. "It is simple and friendly, designed for non-computer people," says Edward C. Uberbacher, a member of the computer sequence-analysis group in Oak Ridge's engineering physics and math division, who describes himself as half biologist, half computer scientist. "People send in a [DNA] sequence, and the system sends back a message describing where in the sequence are the coding parts of a gene," he adds, referring to a subcomponent of GRAIL called CRM, which stands for "coding recognition module." Earlier this year, as moviegoers learned about the fatty acid buildup behind adrenoleukodystrophy in the film Lorenzo's Oil, an international team of researchers was using the GRAIL/CRM program to identify the gene that causes this inherited neurological deterioration of late childhood. Following clues provided by affected boys missing genetic material in parts of their X chromosomes, the researchers used the software to identify a likely protein-encoding portion of the candidate gene region. The program then compared the predicted protein's amino acid sequence to those in standard protein databases--and found something unexpected, yet logical (Jean Mosser, et al., Nature, 361:726, 1993). Rather than implicate the very long chain fatty acid CoA (VLCFA-CoA) synthetase gene thought to be the culprit, the computer search identified a peptide transporter protein that ferries VLCFA-CoA synthetase into the peroxisome, the cellular site of fatty acid breakdown. DNA sequence-analysis software must readily adapt to the unknown genetic complexities that are certain to arise from the ongoing genome projects, including surprises in gene structure and function, as well as emerging mapping strategies. Consider the recent discovery of the gene that causes Huntington's disease (Huntington's Disease Collaborative Research Group, Cell, 72:971, 1993). Part of the reason why a decade passed between the discovery of a marker and the final identification of the gene was that its protein product resembles no other. But the gene does have one distinguishing feature--the trinu-cleotide CAG repeated many more times in those who are affected by this dominantly inherited, adult-onset neurological disorder than in others. Huntington's disease joins three other nucleotide repeat disorders recently identified--fragile X syndrome, myotonic dystrophy, and spinal and bulbar muscular atrophy. Most, if not all, sequence-analysis software would not require revamping to handle this unexpected, new form of mutation. "You can put in a specific sequence, and search for it. [The program] stops everywhere it locates the sequence," says Cordell Brown, manager of sales and marketing at Hitachi, explaining how MacDNASIS would detect a nucleotide repeat. A sensitive computer is better at detecting short repeats than a human is. The first maps of the human genome were assembled in the 1980s using RFLP markers. This approach was sketchy, roughly equivalent to filling in the outlines of states on a blank map of the United States. Newer mapping strategies are more like localizing the cities on such a map. One faster and easier mapping route uses as signposts short, repeated DNA sequences (one to five bases). These are called short or simple sequence tandem repeat polymorphisms or, more succinctly, microsatellites. Up to half a billion microsatellite sequences are scattered across the human genome. Mapping genes using microsatellites as guides entails determining the exact number of bases between repeats. This number can differ by as few as one or two bases in different individuals, resulting in very similarly sized electrophoresis gel bands. A computer is much better at discriminating these bands than is the human eye. Millipore's Bio Image Short Tandem Repeat Polymorphism Software, which runs on the company's Bio Image gel/film reader system and SUN Microsystems workstations, increases throughput in detecting genes by five-fold. On a Macintosh, GENESCAN analysis software from Applied Biosystems Inc., Foster City, Calif., eases this new form of mapping. GENESCAN uses fluorescent labels to mark DNA from different individuals (Janet S. Ziegle, et al., Genomics, 14:1026, 1992). The program presents the data either as a simulated gel pattern or as an "electrophoretogram," clearly displaying which alleles are found in which members of a family. In clinical studies, color-coding allows "multiplexing"--looking at more than one gene per family member at a time. "We have determined that a single project with at least 500 individuals would take years to analyze by radioactive methods. But with the GENESCAN software and the ability to multiplex, we can do the same project in a matter of three to four months," said Elizabeth Nanthakumer, director of the DNA analysis facility at the Johns Hopkins University School of Medicine, in an interview that appeared in the Applied Biosystems company newsletter, Biosystems Reporter (November 1991). The DNA sequence-analysis software of the future, say many molecular biologists, will concentrate on identifying more and more motifs (common sequence regions encoding protein with a distinctive three-dimensional structure) to decipher the meaning of gene sequences. And genes in need of analysis are rapidly piling up. At the European Community Symposium on Strategies in cDNA Programs, held in Paris Oct. 12-13, 1992, 70 researchers reported a total of 25,000 partial complementary DNAs (cDNAs). These are DNA sequences that are reverse-transcribed from messenger RNA (mRNA) obtained from specific tissues. The cDNAs therefore reflect genes that are active in a specific cell type. The 25,000 partial cDNAs are thought to represent 10,000 human genes, or about 10 percent of the protein-encoding portion of the human genome. But the need for DNA sequence-analysis software will be compelling even after the genome journey has ended. Concludes Cornell's Francis Barany, "Once the human genome has been sequenced, we're going to be in a position to do many interesting experiments. There will be a time when we have the information and can then answer fundamental questions." Ricki Lewis is a freelance science writer based in Scotia, N.Y. She is the author of a biology text and has just completed a human genetics text.

DNA Sequencing Software Teases Meaning From Genes

May 30, 1993

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