This module uses the
vector_clip program to identify and mark the
sequencing vector (those used to produce templates for sequencing, eg m13mp18
or puc18). To achieve this task it needs to know information about the vector
including the cut site position and the position of the primer site relative
to the cut site.See section Defining the Positions of Cloning and Primer Sites for Vector_Clip..
- Option: Use Vector-primer file
Vector_clip may be told to search through a series of vectors and primers held
within an external file. Alternatively we can request that it looks only at
one specific, known, vector. This question is to determine which of the two
mutually exclusive methods to use. In general it is still important for the
Experiment File to contain primer and template data. The Vector-primer module can
be used to add the primer and sequencing vector information to the Experiment File
but not the template name.
- Option: Vector-primer filename.
This is only used if the "Use Vector-primer file" question was answered with
"Yes". Each input sequence will be compared against each vector-primer pair to
find the best match. This provides a simple way of comparing against multiple
vectors or comparing against both forward and reverse primers of a single
vector. For further details on creating this vector-primer file, seesection Vector_Primer file format..
- Option: Select vector-primer subset
This is used in conjuction with the vector-primer filename to indicate which
of the vector-primer pairs listed in this file should be used. Initially this
is set to all vector-primer pairs, but efficiency will be greatly increased if
just the required subset is selected. (Internally pregap4 will then temporarily
produce a new vector-primer filename each time
one, containing just the selected items.) To select more than one
vector-primer pair use the standard listbox mouse bindings: single left click
to pick an item; click and drag to select a range; and control left click to
toggle a single item. The selected list will be saved to the pregap4
configuration file whenever all the parameters for this module are saved.
- Option: Max primer to cut-site length
This parameter is only used when a vector-primer file is defined. The sequence
stored in the vector-primer file may be considerably longer than we expect to
see at the start of the sequences being analysed. By defining the maximum
length of sequence we expect to see,
vector_clip may be more sensitive
and slightly faster.
- Option: Vector file name
This, and the following two options, are only used if the "Use Vector-primer
file" question was answered with "No". The vector file name should be the
name of a file containing just the vector bases or white space, in a plain
- Option: Cut site
The cut site specified as a base count from the start of the vector file.
- Option: Primer site
The primer site specified as a base offset from the cut site. e.g. for m13mp18
forward primers the value is 41. If, instead of the usual single value,
are specified separated by a slash, then this gives the values for the
universal forward and reverse primers (for example "
41/-24"). Only use
this format if the
PR (primer type) experiment file line type is known
AND will be specified in the experiment file. If the PR record
is not specified in the
experiment file, the primer site position will be set to zero, and the vector
clipping is unlikely to work correctly.
(PR values do not have to be known if they
can be derived using naming schemes such as those used
by the Sanger Centre). If the
primer site indicates a custom primer sequence then the primer site is taken
to be 0.
- Option: Percentage minimum 5' match
- Option: Percentage minimum 3' match
Both ends of the sequence are checked using a dynamic programming algorithm to
find the optimal alignment. An end is marked as vector if the
percentage match is at least as high as this supplied parameter.
- Option: Default 5' position
This specifies the value to use for marking the 5' sequencing vector if none
is detected. Specifying this as -1 will cause the absolute value given for the
primer site (which is specified as relative to the cut site).
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Last generated on 22 October 2002.