Sequencing Vector Clip

Description

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 vector_clip requires 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 text format.

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, two values 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).