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Computer tools for Sequencing Projects

*** A wide variety of different computer tools have been created to aid DNA sequencing projects.

*** In this course we will look at just two different software packages: the GCG sequencing project tools, and the Sequencher program (a package devoted exclusively to sequencing).

*** The RCR also offers the MacVector and GeneWorks Packages (for Macintosh computers) both of which have sequencing project tools, I just do not choose to demonstrate them here.

*** In addition to sequence analysis software, computer equipment such as digitizers and gel reading image analysis programs can be used to speed data entry from autoradiographs.

The GCG fragment assembly tools

The GCG Fragment Assembly System is a series of related programs (based on the Staden package) that allow data entry, and assembly of overlapping nucleotide sequence fragments into one continuous sequence (contig). The sequence project tools include:

  • SEQED: a single sequence editor (based on the VMS EDT editor)
  • GELSTART: creates fragment assembly projects, initializes work sessions on existing projects
  • GELENTER: adds individual sequences (reads) to an assembly project, allows input of new sequences from keyboard, digitizer, or import of existing text files
  • GELMERGE: assembles individual sequences into contigs, can automatically remove vector sequences
  • GELASSEMBLE: multiple sequence editor for viewing and editing contigs, allows manual alignment of fragments (even those that won't align with GELMERGE), insertion/deletion of gaps and changing of individual bases
  • GELVIEW: displays contigs as a schematic display of overlapping fragments
  • GELDISASSEMBLE: breaks up contigs into individual sequences within a project


Read the online GCG manual pages for full descriptions of these programs.

Example sequence

Example of a bad sequence


Sequencher

*** Sequencher is a Macintosh program that is entirely dedicated to assisting researchers with DNA sequencing.

*** In addition to the features common to GCG, MacVector, and GeneWorks such as sequence entry and a multiple sequence alignment editor, Sequencher offers specialized tools for working with the output from automated sequencers, especially Applied Biosystems machines.

*** Sequencher can import the electropherograms (graphical representations of the fluorescent intensity of each band in the sequencing gel) directly from the ABI machine. So, rather than just looking at a text file of the sequence data, the quality of the sequence at each base can be assessed - much like an autoradiogram.

*** When multiple sequences are aligned and mismatches are found, the electropherograms can also be aligned and decisions can be made based on the quality of the sequence rather than just which base occurs more often in multiple reads.

*** Sequencher also offers impressive automated tools for removal of vector sequences and low quality sequence data from the ends of reads.

*** Other tools such as translation and restriction mapping are also available.

*** Sequencher can handle large numbers of sequence reads and long contigs provided it is allocated enough RAM (at least 10 MB for contigs over 2000 bp).
Assembly of large numbers of fragments is processor intensive - you will enjoy working with this program more on a high speed Mac.
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Using Computers for Molecular Biology
Stuart M. Brown, Ph.D., RCR, NYU Medical Center
Comments to: browns02@mcrcr.med.nyu.edu