MAPSORT

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Table of Contents
FUNCTION
DESCRIPTION
EXAMPLE
OUTPUT
INPUT FILES
RELATED PROGRAMS
SELECTING ENZYMES
POTENTIAL RESTRICTION SITES
SUBSET, OVERLAP, AND PERFECT SEARCHES
DEFINING PATTERNS
COMMAND-LINE SUMMARY
LOCAL DATA FILES
PARAMETER REFERENCE

FUNCTION

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MapSort finds the coordinates of the restriction enzyme cuts in a DNA sequence and sorts the fragments of the resulting digest by size. MapSort can sort the fragments from single or multiple enzyme digests.

DESCRIPTION

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MapSort is the best way to predict how the fragments of an enzyme digest will look on a gel. You can tell at a glance which enzymes cut a molecule in a given region and whether other fragments of similar size could confound isolation of the fragment of interest. You can concatenate your sequence with its vector before running MapSort to see if a single step isolation is possible and you can examine the pattern of fragments from a multi-enzyme digest. The sequence can be treated as if it were circular or linear. You can see which enzymes cut the sequence only once. Enzymes that cut the sequence, as well as those that do not, are shown at the bottom of the output. The output therefore contains a complete list of all the enzymes considered. You can see the cut sites graphically with MapPlot or PlasmidMap.

EXAMPLE

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Here is a session using MapSort to identify all of the restriction sites and restriction patterns for all the enzyme digests of pbr322:


% mapsort -CIRcular

 (Circular) MAPSORT of what sequence ?  GenBank:SynpBR322

                  Begin (* 1 *) ?
                End (*  4361 *) ?

 Select the enzymes:  Type nothing or "*" to get all enzymes. Type "?"
 for help on which enzymes are available and how to select them.

                                       Enzyme(* * *):

 What should I call the output file (* synpbr322.mapsort *) ?

 Mapping ...........................

%

OUTPUT

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Here is some of the output file synpbr322.mapsort:


 (Circular) MAPSORT of: gb_sy:SynpBR322  Check: 5483  from: 1  to: 4361

J01749 Cloning vector pBR322, complete genome. 6/96
LOCUS       SYNPBR322    4361 bp    DNA   circular  SYN       07-JUN-1996
DEFINITION  Cloning vector pBR322, complete genome.
ACCESSION   J01749 K00005 L08654 M10282 M10283 M10286 M10356 M10784 M10785
            M10786 M33694 V01119
NID         g208958 . . .

 With 216 enzymes: *

                        September 24, 1998 13:39  ..

AatII G_ACGT'C

Cuts at:   4288    4288
   Size:       4361

AccI GT'mk_AC

Cuts at:    652    2245     652
   Size:       1593    2768

///////////////////////////////

XmnI GAAnn'nnTTC

Cuts at:   2033    3965    2033
   Size:       1932    2429

 Enzymes that do cut:

    AatII     AccI   AceIII     AciI   AflIII     AhdI     AluI     AlwI
   Alw26I    AlwNI    ApaBI    ApaLI     ApoI     AvaI    AvaII    BamHI

 ///////////////////////////////////////////////////////////////////////

 Enzymes that do not cut:

    AflII     ApaI     AscI    AvrII     BaeI     BclI    BglII     BmgI
     BplI Bpu1102I    BsaXI    BseRI    BsrGI   BssHII   BstEII    BstXI

 ///////////////////////////////////////////////////////////////////////

INPUT FILES

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MapSort accepts a single nucleotide or protein sequence as input. The function of MapSort depends on whether your input sequence(s) are protein or nucleotide. Programs determine the type of a sequence by the presence of either Type: N or Type: P on the last line of the text heading just above the sequence. If your sequence(s) are not the correct type, see Appendix VI for information on how to change or set the type of a sequence.

RELATED PROGRAMS

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Map maps a DNA sequence and displays both strands of the mapped sequence with restriction enzyme cut points above the sequence and protein translations below. Map can also create a peptide map of an amino acid sequence. MapPlot displays restriction sites graphically. If you don't have a plotter, MapPlot can write a text file that approximates the graph.

MapSort run with either -PLASmid or -FRAGments creates output that can be used by PlasmidMap to make circular restriction maps.

SELECTING ENZYMES

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The program presents you with an enzyme selection prompt that lets you enter enzymes individually or collectively. To get help with selecting enzymes, type a ? at the enzyme prompt. Here is what you see:


Select enzymes:

Type "*" to select all enzymes.
Type "**" to select all enzymes including isoschizomers.
Type individual names like "AluI" to select specific enzymes.
Type "?" to see this message and all available enzymes.
Type "??" to see the available enzymes AND their recognition sites.
Type "?A*" to see what enzymes start with "A."
Type "A*" to select all enzymes starting with "A."
Type parts of names like "Al*" to select all enzymes starting with "AL."
Type "~A*" to unselect all selected enzymes starting with "A."
Type "/*" to see what enzymes you have selected so far.
Type "#" to select no enzymes at all.

Press <Return> after each selection.
Press <Return> and nothing else to end your selections.
Spaces are allowed; upper and lower case are equivalent.

We maintain our enzyme files with a semicolon (;) character in front of all but one member of a family of isoschizomers. (Isoschizomers are restriction endonucleases with the same recognition site.) The isoschizomers beginning with a semicolon are normally not displayed by our mapping programs unless you specifically select them by name or type "**" instead of "*" at the enzyme prompt.

There is more information on enzyme files in Appendix VII.

A command-line expression like -ENZymes=AluI,EcoRII would choose AluI and EcoRII and suppress interactive enzyme selection.

POTENTIAL RESTRICTION SITES

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To assist scientists doing site-directed mutagenesis, this program searches for places in your sequence where a restriction enzyme recognition site occurs with one or more mismatches. Use -MISmatch=1 to identify positions where recognition could occur with one or fewer mismatches.

Use -SILent to find the places in your sequence where a restriction site could be introduced without changing the translation. Read more about using -SILent under the PARAMETER REFERENCE topic below.

SUBSET, OVERLAP, AND PERFECT SEARCHES

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This program normally requires that a sequence pattern be a subset of the enzyme recognition site. If the recognition pattern in the enzyme data file were GCRGC, then the pattern GCAGC in your sequence would be found, since A is within the set of bases defined by R (see Appendix III). If the pattern in the enzyme data file were GCAGC, then a GCRGC in your sequence would not be recognized. If your sequence is very ambiguous, as it might be if it were a backtranslated sequence, then it may be better to use -ALL to do an overlap search. The overlap search would consider an R in your sequence to match an A in the recognition site.

With -PERFect, the program looks for a perfect symbol match between your sequence and the recognition pattern -- GCRGC in the recognition pattern would only match a GCRGC in the sequence.

All searches are case insensitive (upper- or lowercase) for the letters in either the sequence or the enzyme recognition site.

DEFINING PATTERNS

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FindPatterns, Map, MapSort, MapPlot, and Motifs all let you search with ambiguous expressions that match many different sequences. The expressions can include any legal GCG sequence character (see Appendix III). The expressions can also include several non-sequence characters, which are used to specify OR matching, NOT matching, begin and end constraints, and repeat counts. For instance, the expression TAATA(N){20,30}ATG means TAATA, followed by 20 to 30 of any base, followed by ATG. Following is an explanation of the syntax for pattern specification.

Implied Sets and Repeat Counts

Parentheses () enclose one or more symbols that can be repeated some number of times. Braces {} enclose numbers that tell how many times the symbols within the preceding parentheses must be found.

Sometimes, you can leave out part of an expression. If braces appear without preceding parentheses, the numbers in the braces define the number of repeats for the immediately preceding symbol. One or both of the numbers within the braces may be missing. For instance, both the pattern GATG{2,}A and the pattern GATG{2}A mean GAT, followed by G repeated from 2 to 350,000 times, followed by A; the pattern GATG{}A means GAT, followed by G repeated from 0 to 350,000 times, followed by A; the pattern GAT(TG){,2}A means GAT, followed by TG repeated from 0 to 2 times, followed by A; the pattern GAT(TG){2,2}A means GAT, followed by TG repeated exactly 2 times, followed by A. (If the pattern in the parentheses is an OR expression (see below), it cannot be repeated more than 2,000 times.)

OR Matching

If you are searching nucleic acids, the ambiguity symbols defined in Appendix III let you define any combination of G, A, T, or C. If you are searching proteins, you can specify any of several symbol choices by enclosing the different choices in parentheses and separating the choices with commas. For instance, RGF(Q,A)S means RGF followed by either Q or A followed by S. The length of each choice need not be the same, and there can be up to 31 different choices within each set of parentheses. The pattern GAT(TG,T,G){1,4}A means GAT followed by any combination of TG, T, or G from 1 to 4 times followed by A. The sequence GATTGGA matches this pattern. There can be several parentheses in a pattern, but parentheses cannot be nested.

NOT Matching

The pattern GC~CAT means GC, followed by any symbol except C, followed by AT. The pattern GC~(A,T)CC means GC, followed by any symbol except A or T, followed by CC.

Begin and End Constraints

The pattern <GACCAT can only be found if it occurs at the beginning of the sequence range being searched. Likewise, the pattern GACCAT> would only be found if it occurs at the end of the sequence range.

COMMAND-LINE SUMMARY

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All parameters for this program may be added to the command line. Use -CHEck to view the summary below and to specify parameters before the program executes. In the summary below, the capitalized letters in the parameter names are the letters that you must type in order to use the parameter. Square brackets ([ and ]) enclose parameter values that are optional. For more information, see "Using Program Parameters" in Chapter 3, Using Programs in the User's Guide.


Minimal Syntax: % mapsort [-INfile=]GenBank:SynpBR322 -Default

Prompted Parameters:

-BEGin=1 -END=4361           sets the range of interest
-CIRcular                    treats chosen range as circular
-ENZymes=*,[.,.,.]           selects enzymes to be mapped
-OUTfile=synpbr322.mapsort   names the output file

Local Data Files:

-DATa=enzyme.dat          specifies restriction enzymes and recognition sites
-DATa=proenzyme.dat       specifies peptidases and peptide cleavage reagents
-TRANSlate=translate.txt  contains the genetic code

Optional Parameters:

-DIGest         sorts the cuts for all the enzymes together in one digest
-LINear         treats chosen range as linear (default)
-PLAsmid        makes output suitable for display by PLASMIDMAP
-FRAGments      puts "blocks" not "ticks" into the PLASMIDMAP label file
-NOSIZe         suppresses the report of fragment sizes in your output
-MISmatch=1     finds potential sites with one or fewer mismatches.
-SILent         finds translationally silent potential restriction sites
-PERFect        looks only for perfect matches
-ALL            does an "overlapping-set" map
-APPend         appends enzyme file and translation table to your output
-CUTters[=fn]   writes enzyme data file with enzymes that did cut
-NONCUTters[=fn] writes enzyme data file with enzymes that did not cut
-EXCUTters[=fn] writes enzyme data file with enzymes that were excluded
-MINSitelen=6   selects enzymes with 6 (or more) bases in recognition site
-OVErhang=0     selects only blunt-end cutters ("5" for 5', "3" for 3')
-MINCuts=2      shows only enzymes that cut at least 2 times
-MAXCuts=2      shows only enzymes that cut no more than 2 times
-ONCe           shows enzymes that cut only once
-EXCLude=n1,n2  suppresses enzymes that cut between bases n1 and n2

LOCAL DATA FILES

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The files described below supply auxiliary data to this program. The program automatically reads them from a public data directory unless you either 1) have a data file with exactly the same name in your current working directory; or 2) name a file on the command line with an expression like -DATa1=myfile.dat. For more information see Chapter 4, Using Data Files in the User's Guide.

This program reads the public or local version of enzyme.dat to get the enzyme names, recognition sites, cut positions, and overhangs. You can use mapping programs to search for any sequence pattern by adding the pattern to the enzyme data file. If you use the command-line parameter -APPend, this program appends the enzyme data file to the output file. (See Appendix VII for more information about enzyme data files.)

If MapSort finds Type: P on the dividing line in the sequence file, it reads proteolytic cleavage data in the local data file proenzyme.dat.

The translation of codons to amino acids, the identification of potential start codons and stop codons, and the mappings of one-letter to three-letter amino acid codes are all defined in a translation table in the file translate.txt. If the standard genetic code does not apply to your sequence, you can provide a modified version of this file in your working directory or name an alternative file on the command line with an expression like -TRANSlate=mycode.txt. Translation tables are discussed in more detail in Appendix VII. If you use the command-line parameter -APPend and you have provided your own translation scheme, this program appends your translation table to the output file.

PARAMETER REFERENCE

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You can set the parameters listed below from the command line. For more information, see "Using Program Parameters" in Chapter 3, Using Programs in the User's Guide.

-CIRcular

causes MapSort to treat the range of your sequence as if it were circular. If you use this parameter and choose a range from 101 to 200, MapSort acts as if the 100 bases you have chosen were a separate 100-base circular molecule! If you use a whole sequence, MapSort always prompts you to find out if the molecule is circular.

-ENZymes=*[,...]

specifies the restriction enzymes whose recognition sites you want to search. If you search for several different enzymes, separate their names with commas. -ENZymes=* selects all enzymes, -ENZymes=** selects all enzymes, including isoschizomers, and -ENZymes=Al* selects all enzymes whose names start with Al.

-TRANSlate=filename.txt

Usually, translation is based on the translation table in a default or local data file called translate.txt. This parameter allows you to use a translation table in a different file. (See Appendix VII for information about translation tables.)

-DIGest

sorts the cuts and fragments from all of the enzymes you have named together in one digest. You cannot run MapSort with more than 20 enzymes in one digest.

-LINear

is the opposite of -CIRcular. If you have defined a command that runs MapSort with -CIRcular as the default, use the -LINear parameter to make MapSort treat your sequence as linear.

-PLAsmid

causes MapSort to write an output file that can be used to put labeling ticks on the circular restriction map that is drawn by PlasmidMap. See Figure 1 in the Program Manual entry for PlasmidMap.

-FRAGments

causes MapSort to write an output file that can be used to put concentric blocks in the circular restriction map that is drawn by PlasmidMap. See Figure 2 in the Program Manual entry for PlasmidMap.

-NOSIZe

causes the program to suppress the report of fragment sizes in the output file.

-MISmatch=1

causes the program to recognize sites that are like the recognition site but with one or fewer mismatches. If too many mismatches are allowed, the results may not be meaningful. The output from most mapping programs distinguishes between sites with no mismatches and sites with mismatches.

-SILent

shows the places where restriction sites can be introduced (by site-directed mutagenesis) without changing the peptide translation of the sequence. The -SILent parameter assumes that the range you have chosen defines a coding region and reading frame precisely. Sites may be found that have any number of bases changed as long as the changes do not alter the translation. The reading frame is implied by the beginning coordinate you specify. The output from most mapping programs distinguishes between real sites and sites with one or more mismatches. The data file translate.txt defines the genetic code.

-PERFect

sets the program to look for a perfect alphabetic match between the site and the sequence. Ambiguity codes are normally translated so that the site RXY would find sequences like ACT or GAC. With this parameter, the ambiguity codes are not translated so the site RXY would only match the sequence RXY. This parameter is not the same as -MISmatch=0!

-ALL

makes an overlap-set map instead of the usual subset map. If your sequence is very ambiguous (for instance, as a back-translated sequence would be) and you want to see where restriction sites could be, then an overlap-set map is for you. Overlap-set and subset pattern recognition is discussed in more detail in the Program Manual entry for Window.

-APPend

appends the enzyme data file to your output file. If you provided your own translation scheme, that file is also appended.

-CUTters=gamma.cutters

writes out a new enzyme data file containing those selected enzymes that did cut your sequence and were not excluded with any of the -MINCuts, -ONCe, -MAXCuts, and -EXClude parameters. If you do not add a file name to the -CUTters parameter the output file will have the name of your sequence followed by the file name extension .cutters

-NONCUTters=gamma.noncutters

writes out a new enzyme data file containing the selected enzymes that did NOT cut your sequence. If you do not add a file name to this parameter the output file will have the name of your sequence followed by the file name extension .noncutters

-EXCUTters=gamma.excutters

writes out a new enzyme data file containing those enzymes that did cut your sequence but were excluded with any of the -EXClude, -MINCuts, -ONCe, and -MAXCuts parameters. If you do not add a file name to this parameter the output file will have the name of your sequence followed by the file name extension .excutters

The parameters -MINSitelen and -OVErhang restrict the domain of enzymes selected.

-MINSitelen=6

selects only patterns with the specified number or more bases in the recognition site. You can display the sites from any pattern in the enzyme or pattern file that you take the trouble to name individually, but when you use all of the patterns, the program uses all of the patterns whose recognition sites have the specified number or more non-N, non-X bases. -MINSitelen=6 replaces the -SIXbase parameter from earlier versions of the Wisconsin Package.

-OVErhang=0

selects only enzymes that leave blunt ends. Use a 5 with this parameter to search only with enzymes that leave 5' overhangs and a 3 to search only with enzymes that leave a 3' overhang. You can use multiple values, separated by commas. For instance, -OVErhang=5,3 searches with all enzymes that leave either 5' or 3' overhangs. You can display the cuts from any enzyme in the enzyme data file that you take the trouble to name individually, but when you use * (meaning all), the program uses all of the enzymes whose overhangs conform to your choice with this parameter.

The -MINCuts, -MAXCuts, -ONCe, and -EXClude parameters suppress the display of selected enzymes. The list of excluded enzymes in the program output includes both selected enzymes that cut within excluded ranges and selected enzymes that did not cut the right number of times.

-MINCuts=2

excludes enzymes that do not cut at least two times.

-MAXCuts=2

excludes enzymes that cut more than two times.

-ONCe

excludes, from the set of enzymes displayed, those enzymes that cut your sequence more than once (equivalent to setting both mincuts and maxcuts to one).

-EXClude=n1,n2[,n3,n4,...]

excludes enzymes that cut anywhere within one or more ranges of the sequence. If an enzyme is found within an excluded range, then the enzyme is not displayed. The list of excluded enzymes includes enzymes that cut within excluded ranges. The ranges are defined with sets of two numbers. The numbers are separated by commas. Spaces between numbers are not allowed. The numbers must be integers that fall within the sequence beginning and ending points you have chosen. The range may be circular if circular mapping is being done. Exclusion is not done if there are any non-numeric characters in the numbers or numbers out of range or if there is an odd number of integers following the parameter.

Printed: December 9, 1998 16:27 (1162)

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