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Bioinformatics. A Practical Guide to the Analysis of Genes and Proteins - Baxevanis A.D.

Baxevanis A.D. Bioinformatics. A Practical Guide to the Analysis of Genes and Proteins - New York, 2001. - 493 p.
ISBN 0-471-22392-1
Download (direct link): bioinformaticsapractic2001.pdf
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Each marker represents some type of genomic element: a gene, an EST, a polymorphism, a large-insert clone end, or a random genomic stretch. In humans, identifying what a marker represents is relatively straightforward. Simply search for the marker name in GDB or eGenome, and, in most cases, the resulting Web display will provide a summary of what the marker represents, usually along with hyperlinks to relevant functional information. For mice, MGD provides a similar function to GDB. For other organisms, the best source is usually either dbSTS or, if present, Web sites or publications associated with the underlying maps. GenBank and dbSTS are alternatives for finding markers, but, because these repositories are passive (requiring researchers to submit their markers rather than actively collecting markers), many marker sets are not represented. If a marker is known to be expressed, UniGene, LocusLink, and dbEST are excellent sources of additional information. Many genes and some polymorphisms have been independently discovered and developed as markers multiple times, and creating a nonredundant set from a collection of markers is often challenging. GDB, eGenome, MGD, and (for genes) UniGene are good sources to use for finding whether two markers are considered equivalent but even more reliable is a DNA sequence or sequence contig containing both
marker’s primers. BLAST and the related BLAST2 are efficient for quickly determining sequence relatedness (Chapter 8).
Obviously, the most reliable tool for marker ordering is a DNA sequence or sequence contig. For expressed human markers, searching with the marker name in UniGene or Entrez Genomes returns a page stating where (or if) the marker has been mapped in GeneMap ’99 and other maps, a list of mRNA, genomic, and EST sequences, and with Entrez Genomes, a Mapviewer-based graphical depiction of the maps, sequence-ready contigs, and available sequence of the region. Similarly, GDB and eGenome show which DNA sequences contain each displayed marker. For other markers, the sequence from which the marker is derived, or alternatively one of the primer sequences, may be used to perform a BLAST search that can identify completely or nearly homologous sequences. The nonredundant, EST, GSS, and HTGS divisions of GenBank are all potentially relevant sources of matching sequence, depending on the aim of the project. Only long sequences are likely to have worthwhile marker-ordering capabilities. Finished genomic sequence tracts have at least some degree of annotation, and scanning the GenBank record for the large sequence will often yield an annotated list of what markers lie within the sequence and where they are. Keep in mind that such annotations vary considerably in their thoroughness and most are fixed in time; that is, they only recognize markers that were known at the time of the annotation. BLAST, BLAST2, or other sequence-alignment programs are helpful in identification or confirmation of what might lie in a large sequence. Also, the NCBI e-PCR Web interface can be used to identify all markers in dbSTS contained within a given sequence, and this program can be installed locally to query customized marker sets with DNA sequences (Schuler, 1997).
For genomes for which DNA sequencing is complete or is substantially underway, it may be possible to construct local clone or sequence contigs. Among higher organisms, this is currently possible only for the human and mouse genomes. Although individual clone sequences can be found in GenBank, larger sequence contigs —sequence tracts comprising more than one BAC or PAC—are more accessible using the Entrez Genomes Web site (see above). Here, by entering a marker or DNA accession number into the contigs search box, researchers can identify sequence contigs containing that marker or element. This site also provides a graphical view of all other markers contained in that sequence, the base pair position of the markers in the sequence, and, with the Mapviewer utility, graphical representations of clone contigs. This process can also be performed using BLAST or e-PCR, although it is somewhat more laborious.
Once a sequence has been identified for markers in a given region, YAC clone, DNA fingerprinting, and STC data can be used to bridge gaps. For humans and mice, the WICGR YAC data provide a mechanism for identifying YAC clones linking adjacent markers. However, caution should be exercised to rely mainly on doublelinked contigs and/or to experimentally confirm YAC/marker links. Also for human genome regions, the UWHTSC and TIGR Web sites for identifying STCs from DNA sequence or BAC clones are very useful. For example, researchers with a sequence tract can go to the UWHTSC TSC search page, enter their sequence, and find STCs contained in the sequence. Any listed STC represents the end of a BAC clone whose insert contains a portion of the input sequence (Venter et al., 1996). The TIGR search tool is complementary to the UWHTSC search, as the TIGR site requires input of a large-insert clone name, which yields STC sequences. STCs represent large-insert clones that potentially extend a contig or link two adjacent, nonoverlapping contigs.
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