eprimer3.txt

emboss的linux版本的源代码

                                 eprimer3 



Function

   Picks PCR primers and hybridization oligos

Description

   eprimer3 is an interface to the 'primer3' program from the Whitehead
   Institute.

   The Whitehead program must be set up and on the path in order for
   eprimer3 to find and run it.

   Primer3 picks primers for PCR reactions, considering as criteria:

     * oligonucleotide melting temperature, size, GC content, and
       primer-dimer possibilities,
     * PCR product size,
     * positional constraints within the source sequence, and
     * miscellaneous other constraints.

   All of these criteria are user-specifiable as constraints.

   eprimer3 can also pick hybridisation oligos that are internal to the
   product.

  ADVICE FOR PICKING PRIMERS

   We suggest referring to: Wojciech Rychlik, "Selection of Primers for
   Polymerase Chain Reaction" in BA White, Ed., "Methods in Molecular
   Biology, Vol. 15: PCR Protocols: Current Methods and Applications",
   1993, pp 31-40, Humana Press, Totowa NJ

    Cautions

   Some of the most important issues in primer picking can be addressed
   only before using eprimer3. These are sequence quality (including
   making sure the sequence is not vector and not chimeric) and avoiding
   repetitive elements.

   Techniques for avoiding problems include a thorough understanding of
   possible vector contaminants and cloning artifacts coupled with
   database searches using blast, fasta, or other similarity searching
   program to screen for vector contaminants and possible repeats.
   Repbase (J. Jurka, A.F.A. Smit, C. Pethiyagoda, and others, 1995-1996,
   ftp://ncbi.nlm.nih.gov/repository/repbase) is an excellent source of
   repeat sequences and pointers to the literature. eprimer3 now allows
   you to screen candidate oligos against a Mispriming Library (or a
   Mishyb Library in the case of internal oligos).

   Sequence quality can be controlled by manual trace viewing and quality
   clipping or automatic quality clipping programs. Low- quality bases
   should be changed to N's or can be made part of Excluded Regions. The
   beginning of a sequencing read is often problematic because of primer
   peaks, and the end of the read often contains many low-quality or even
   meaningless called bases. Therefore when picking primers from
   single-pass sequence it is often best to use the INCLUDED_REGION
   parameter to ensure that eprimer3 chooses primers in the high quality
   region of the read.

   In addition, eprimer3 takes as input a Sequence Quality list for use
   with those base calling programs

   (e.g. Phred, Bass/Grace, Trout) that output this information.

    What to do if eprimer3 cannot find a primers?

   Try relaxing various parameters, including the self-complementarity
   parameters and max and min oligo melting temperatures. For example,
   for very A-T-rich regions you might have to increase maximum primer
   size or decrease minimum melting temperature. It is usually unwise to
   reduce the minimum primer size if your template is complex (e.g. a
   mammalian genome), since small primers are more likely to be
   non-specific. Make sure that there are adequate stretches of non-Ns in
   the regions in which you wish to pick primers. If necessary you can
   also allow an N in your primer and use an oligo mixture containing all
   four bases at that position.

   Try setting the '-explain' option.

Usage

   Here is a sample session with eprimer3


% eprimer3 tembl:hsfau1 hsfau.eprimer3 -explain 
Picks PCR primers and hybridization oligos

   Go to the input files for this example
   Go to the output files for this example

Command line arguments

   Standard (Mandatory) qualifiers:
  [-sequence]          seqall     The sequence from which to choose primers.
                                  The sequence must be presented 5' to 3'
  [-outfile]           outfile    [*.eprimer3] Whitehead primer3_core program
                                  output file

   Additional (Optional) qualifiers (* if not always prompted):
   -[no]primer         toggle     [Y] Tell EPrimer3 to pick primer(s)
*  -task               menu       [1] Tell EPrimer3 what task to perform.
                                  Legal values are 1: 'Pick PCR primers', 2:
                                  'Pick forward primer only', 3: 'Pick reverse
                                  primer only', 4: 'No primers needed'.
                                  (Values: 1 (Pick PCR primers); 2 (Pick
                                  forward primer only); 3 (Pick reverse primer
                                  only); 4 (No primers needed))
   -hybridprobe        toggle     [N] An 'internal oligo' is intended to be
                                  used as a hybridization probe (hyb probe) to
                                  detect the PCR product after amplification.
*  -oligomishyblibraryfile infile     Similar to MISPRIMING-LIBRARY, except tha
t
                                  the event we seek to avoid is hybridization
                                  of the internal oligo to sequences in this
                                  library rather than priming from them.
                                  The file must be in (a slightly restricted)
                                  FASTA format (W. B. Pearson and D.J. Lipman,
                                  PNAS 85:8 pp 2444-2448 [1988]); we briefly
                                  discuss the organization of this file below.
                                  If this parameter is specified then
                                  EPrimer3 locally aligns each candidate oligo
                                  against each library sequence and rejects
                                  those primers for which the local alignment
                                  score times a specified weight (see below)
                                  exceeds INTERNAL-OLIGO-MAX-MISHYB. (The
                                  maximum value of the weight is arbitrarily
                                  set to 12.0.)
                                  Each sequence entry in the FASTA-format file
                                  must begin with an 'id line' that starts
                                  with '>'. The contents of the id line is
                                  'slightly restricted' in that EPrimer3
                                  parses everything after any optional
                                  asterisk ('*') as a floating point number to
                                  use as the weight mentioned above. If the
                                  id line contains no asterisk then the weight
                                  defaults to 1.0. The alignment scoring
                                  system used is the same as for calculating
                                  complementarity among oligos (e.g.
                                  SELF-ANY). The remainder of an entry
                                  contains the sequence as lines following the
                                  id line up until a line starting with '>'
                                  or the end of the file. Whitespace and
                                  newlines are ignored. Characters 'A', 'T',
                                  'G', 'C', 'a', 't', 'g', 'c' are retained
                                  and any other character is converted to 'N'
                                  (with the consequence that any IUB / IUPAC
                                  codes for ambiguous bases are converted to
                                  'N'). There are no restrictions on line
                                  length.
                                  An empty value for this parameter indicates
                                  that no library should be used.
   -numreturn          integer    [5] The maximum number of primer pairs to
                                  return. Primer pairs returned are sorted by
                                  their 'quality', in other words by the value
                                  of the objective function (where a lower
                                  number indicates a better primer pair).
                                  Caution: setting this parameter to a large
                                  value will increase running time. (Integer 0
                                  or more)
   -includedregion     range      [(full sequence)] A sub-region of the given
                                  sequence in which to pick primers. For
                                  example, often the first dozen or so bases
                                  of a sequence are vector, and should be
                                  excluded from consideration. The value for
                                  this parameter has the form
                                  (start),(end)
                                  where (start) is the index of the first base
                                  to consider, and (end) is the last in the
                                  primer-picking region.
   -target             range      [(full sequence)] If one or more Targets is
                                  specified then a legal primer pair must
                                  flank at least one of them. A Target might
                                  be a simple sequence repeat site (for
                                  example a CA repeat) or a single-base-pair
                                  polymorphism. The value should be a
                                  space-separated list of
                                  (start),(end)
                                  pairs where (start) is the index of the
                                  first base of a Target, and (end) is the
                                  last
                                  E.g. 50,51 requires primers to surround the
                                  2 bases at positions 50 and 51.
   -excludedregion     range      [(full sequence)] Primer oligos may not
                                  overlap any region specified in this tag.
                                  The associated value must be a
                                  space-separated list of
                                  (start),(end)
                                  pairs where (start) is the index of the
                                  first base of the excluded region, and and
                                  (end) is the last. This tag is useful for
                                  tasks such as excluding regions of low
                                  sequence quality or for excluding regions
                                  containing repetitive elements such as ALUs
                                  or LINEs.
                                  E.g. 401,407 68,70 forbids selection of
                                  primers in the 7 bases starting at 401 and
                                  the 3 bases at 68.
   -forwardinput       string     The sequence of a forward primer to check
                                  and around which to design reverse primers
                                  and optional internal oligos. Must be a
                                  substring of SEQUENCE. (Any string is
                                  accepted)
   -reverseinput       string     The sequence of a reverse primer to check
                                  and around which to design forward primers
                                  and optional internal oligos. Must be a
                                  substring of the reverse strand of SEQUENCE.
                                  (Any string is accepted)
*  -gcclamp            integer    [0] Require the specified number of
                                  consecutive Gs and Cs at the 3' end of both
                                  the forward and reverse primer. (This
                                  parameter has no effect on the internal
                                  oligo if one is requested.) (Integer 0 or
                                  more)
*  -osize              integer    [20] Optimum length (in bases) of a primer
                                  oligo. EPrimer3 will attempt to pick primers
                                  close to this length. (Integer 0 or more)
*  -minsize            integer    [18] Minimum acceptable length of a primer.
                                  Must be greater than 0 and less than or
                                  equal to MAX-SIZE. (Integer 1 or more)
*  -maxsize            integer    [27] Maximum acceptable length (in bases) of
                                  a primer. Currently this parameter cannot
                                  be larger than 35. This limit is governed by
                                  the maximum oligo size for which EPrimer3's
                                  melting-temperature is valid. (Integer up
                                  to 35)
*  -otm                float      [60.0] Optimum melting temperature(Celsius)
                                  for a primer oligo. EPrimer3 will try to
                                  pick primers with melting temperatures are
                                  close to this temperature. The oligo melting
                                  temperature formula in EPrimer3 is that
                                  given in Rychlik, Spencer and Rhoads,
                                  Nucleic Acids Research, vol 18, num 12, pp
                                  6409-6412 and Breslauer, Frank, Bloeker and
                                  Marky, Proc. Natl. Acad. Sci. USA, vol 83,
                                  pp 3746-3750. Please refer to the former
                                  paper for background discussion. (Any
                                  numeric value)
*  -mintm              float      [57.0] Minimum acceptable melting
                                  temperature(Celsius) for a primer oligo.
                                  (Any numeric value)
*  -maxtm              float      [63.0] Maximum acceptable melting
                                  temperature(Celsius) for a primer oligo.