Nucleic Acids Research MAXAMIZE. A DNA sequencing strategy advisor

The MAXAMIZE advisory system determines from user-provided restriction maps an optimal ' strategy to do nucleotide sequencing by methods involving end-labeled fragments. The maps may be either simple linear restriction maps of fragments or complex circular maps including restriction sites of a vector. The whole system is interactive and is written in the Genetic English language provided by the GENESIS System, a molecular genetics knowledge representation and manipulation package. In addition, MAXAMIZE provides bookkeeping facilities for sequencing and offers advice on how to verify the newly obtained sequence data. Introduction Using the knowledge representation and manipulation tools developed by the MOLGEN project [1], we have developed a system which provides assistance in determining a nucleotide sequence using a given restriction map. The problem is to derive an efficient sequencing strategy which minimizes ttie number of gels run and sequencing reactions performed and maximizes the number of nucleotides read in each step. Starting from the restriction map, the sequencing advisor determines how much sequence information can be obtained considering both the average length the user can sequence from any labeled terminus and whether the fragments can be separated. Having determined the best order of successive digests, the system predicts for each digest the pattern of fragments (on a gel) and advises which of them have to be eluted and sequenced. In addition, the sequencing advisor provides the user with bookkeeping capacities which include the ability to continually update the restriction-map with the newly gathered sequence information. Also, the user is advised: 1. how to verify the experimentally determined sequence 2. how to map RNA ends (5, 3' or both) using the nuclease protection technique [2] 3. which additional enzymes might be good cutter candidates in a region where there are no sites for enzymes already tested. This advisor is most useful for directed sequencing performed using the Maxam and Gilbert method [3] [4]. See [5] for the description of a system which assists in providing bookkeeping for both site specific and random sequencing experiments. © IRL Press limited, 1 Falconborg Court, London W1V 5FG. U.K. 295 Downloaded from https://academic.oup.com/nar/article-abstract/10/1/295/2358573/MAXAMIZE-A-DNA-sequencing-strategy-advisor by guest on 15 September 2017 Nucleic Acids Research Method of Solution The sequencing advisor is written in a subset of Genetic English (Genglish) provided by GENESIS [1]. GENESIS is based on the Unit System [6] [7] [8] [9], a general-purpose knowledge acquisition program written in Interiisp, which runs on the Digital Equipment Corporation DecSystem 10 and 20 series of computers. GENESIS provides the ability to represent store and modify molecular genetic information 3uch as restriction maps, sequences, and restriction enzyme properties, as well as more general types of information like numbers, strings, lists, and tables. The total collection of Information is known as a knowledge base, and may be easily examined, shared, and updated by a variety of users. Furthermore, the system allows manipulation of the knowledge base by using Genglish, the Genetic English language. This language allows a non-programmer molecular biologist to construct sophisticated computational systems that embody domain-specific expertise. For the case of the system discussed in this paper, all developmental work was performed by one of the authors (R.B.), a molecular biologist with essentially no programming experience. Sequencing Experiment Description The scientist describes his problem by providing MAXAMIZE with a restriction map constructed with the GENESIS map editor. The restriction map can describe one of two type3 of DNA structures: either just the DNA of interest, or a vector containing that DNA. In the latter case, the user has to indicate which region is the inserted DNA by marking it as a specific region on the restriction map. MAXAMIZE first establishes the list of restriction enzymes cutting within the inserted DNA fragment, (or, by default, the whole molecule). Editing facilities allow modification of that list by the user, for example, to avoid using a particular enzyme because he had temporarily exhausted his supply. The user is next asked to provide several experimental parameters: • the average number of bases the user is able to read off a gel • the minimum size difference of two fragments which allows purification • the coordinates of the region(s) to be sequenced • the names of the restriction enzymes (from the list discussed above) which are relevant to the sequencing strategy problem. An example of the experiment description phase is presented below. All user responses are shown in underlined letters: we have added comments in italics. stands for carriage-return. ARE YOU FAMILIAR WITH MAXAMIZE? : fl MAXAMIZE w i l l determine an optimal s t ra tegy to sequence using any def ined res t r i c t i on -map . The res t r i c t i on -map 1s described by using the GENESIS map e d i t o r . I f you are prov id ing a vector map which Includes an Inser ted DNA sequence, then you should def ine a region named INSERT on the map.