LAMP primer designing software PrimerExplorer

	[1]Introduction (operating environment)
	[2]Operation of each screen (designing regular primers/designing loop primers)
	[3]Appendix
	[4]A Guide to LAMP primer designing (Manual) ：in preparation

2. Operation of each screen (designing regular primers)

to designing loop primers

1. Enter the target sequence information

Upload the target sequence file for primer generation

Following formats can be used

Plain text format(sequence only)
FASTA format
GenBank format
Multiple alignment file format
Target sequence save file format

Click [Browse] button to choose the target sequence file on your computer.
The target sequence file saved from the Primer design page can also be used.
Following options are available for the parameter setting.

Automatic Judgment
Based on the GC content of the target sequence, the parameter value is automatically selected from 3 types of parameter settings. When GC content is ≤ 45% or ≥ 60%, the parameter for AT rich sequence or GC rich sequences is selected, respectively. When GC content is between 45% and 60%, the standard parameter value is chosen.
Normal
The standard parameter value is present as the default setting.
User Assignment
Click [Browse] button and choose user's parameter file saved in user's personal computer, which is to be used as the default condition. The parameter file saved from the Detail settings page can also be used. .

For basic designing, select target sequence file and push [Primer Design] button. For the primer design with detail settings, select the target sequence file as well as the parameter file, and push [Primer Design] button.

2. Primer design

Basic procedure for the primer design is as follows:

Click [Generate] button to generate the primer candidates.
Click [Display] button to display the primer list.

2-1. Basic designing page
The explanations of items on the page are listed below.

Target sequence display
	Sequences are usually displayed in capital letters.
	If the sequences in Multiple alignment file was found absent in some parts, the sequence base before and after these parts will be shown in small letter.
	About Consensus information, ‘-’ indicates mutation and ‘.’ indicates absence of sequence base.
【Supplementary Explanation】 At the time of choice operation of the base sequence, there is the phenomenon "If it is going to choose some letters with a mouse, a selection area spreads out automatically" by the limitation of the browser in Ineternet Explorer. Please choose one letter with the cursor key while pushing the Shift key in the case of one letter selection in InternetExplorer.
Message area
The status of the designing process is shown.
(1)	[Generate]	Generate the primer sets. Maximum 5 sets will be shown through the basic screening of the generated primers.
(2)	[Display]	List the generated primer sets in a new window.
(3)	[Detail Settings]	Shift to Detail Settings page.
(4)	Set Mutation	Select the mutation sites on the target sequence, and primers are generated so as not to include the selected mutation sites. Highlight the mutation sequence and then click [Mut/Cons] button. When the sequence is selected as mutation, the symbol "*" under the sequence is turned into "-". By clicking on [Clear] button, mutation set is cleared.
(5)	Fixed Primer	Fix the primer positions (F1c, F2, F3, B1c, B2 or B3) and design the primer sets with the fixed primers. Selected primer regions are underlined with the name of the fixed primer.
(6)	[Save Target]	The target sequence on the screen and their attached information can be saved as file. This file containing the target sequence can be used to re-design the primers.
(7)	Design Option	Conditions for designing common primers/ specific primers will be automatically set.
	Default	Design regular primers.
	Common	Design common primers.
	Specific	Design specific primers.

2-2. Detail Settings page
The explanations of items on the page are listed below.

Target sequence display
Sequences are usually displayed in capital letters.
If the sequences in Multiple alignment file was found absent in some parts, the sequence base before and after these parts will be shown in small letter.
About Consensus information, ‘-’ indicates mutation and ‘.’ indicates absence of sequence base.
Message area
The status of the designing process is shown.
(1)	Select Range	Appoint the target sequence range (target range).
	Ignore range	Do not specify the target sequence range.
	Within F2-B2	Target region is within F2-B2 region (including F2 and B2).
	Between F1c-B1c	Target region is within F1c-B1c region but does not include F1c or B1c region.
	Targeting Range	Specify the beginning and end positions.
(2)	[Generate]	Generate the primer sets.
(3)	[Display]	List the generated primer sets in a new window.
(4)	Show Page	Select the page number for the display of primer list in a new window.
(5)	Sorting Rule	Define the sorting pattern for the generated primer sets.
	Dimer	Based on the dimer forming potential (in order of lower to higher).
	F2 5'position	Listed according to the sequence position of F2 5' end position from 5' end of the target sequence.
	F2-B2 range	Based on the length between F2 and B2 in the order of shortest to longest
	Mutation(S) [ascending order]	Based on the number of the primers containing the mutation site (s) in ascending order
	Mutation(S) [descending order]	Based on the number of the primers containing the mutation site (s) in descending order
	5'/3' edge stability	According to the end stability in the order of lower to higher
	Random	In a random order
	None	Follows the order of primer generation.
	Easy	For primer design, the same method as the Basic designing is applied and the generated primers will be shown. (No limit for the number of primers) The first batch of primers generated by the Sorting rule [None] is grouped for each 10 base starting from 5' end of F2. The primer sets representing the groups with the largest number of generated primers will be shown. (Therefore, the primers shown on the screen are not the total number of generated primers.)
(6)	[Basic Designing]	Shift to Basic designing page
(7)	Set Mutation	Select the mutation sites on the target sequence, and primers are generated so as not to include the selected mutation sites. Highlight the mutation sequence and then click [Mut/Cons] button. When the sequence is selected as mutation, the symbol "*" under the sequence is turned into "-". By clicking on [Clear] button, mutation set is cleared.
(8)	Fixed Primer	Fix the primer positions (F1c, F2, F3, B1c, B2 or B3) and design the primer sets with the fixed primers. Selected primer regions are underlined with the name of the fixed primer.
(9)	[Save Target]	The target sequence on the screen and their attached information can be saved as file. This file containing the target sequence can be used to re-design the primers.
(10)	Design Option	Conditions for designing common primers/ specific primers will be automatically set.
	Default	Design regular primers. When specify, all defaults are cleared, and those check-boxes initially unavailable for making changes become modifiable.
	Common	Design common primers. When specify, applicable settings of Mutation/Consensus will be set automatically. The check-boxes under F1c inner and B1c inner will be filled, but users can make changes to these check-boxes.
	Specific	Design specific primers. When specify, applicable settings of Mutation/Consensus will be set automatically. All check-boxes will be filled and will not be available for modification. When primers are generated and shown on the screen, only the primer sets containing mutations at F1c 5'term, B1c 5'term, F2 3'term and B2 3'term are shown.
(11)	Parameter Condition	Select the parameter condition
	Normal	Standard parameter condition
	AT rich	Parameter condition for AT rich sequence
	GC rich	Parameter condition for GC rich sequence
	Custom	User designed parameter. This item is available only when the user selects "User Assignment".
(12)	[Save Parameter]	The current parameter settings can be saved as files, so as to be used in other primer designing later on.
(13)	[Reset Parameter]	Reset parameter value to the default condition.
Specify the parameter setting by inputting the appropriate value or clicking on spin button（"▲""▼"） beside the number entry field to change the input value.
(14)	Length	Minimum and Maximum primer length.
(15)	Tm	Minimum and Maximum primer Tm value.
(16)	GC rate(%)	Acceptable value for GC rate.
(17)	dG threshold [Kcal/mol]	dG threshold
	5'stability	Its 5' stability value (dG) is below this value.
	3'stability	Its 3' stability value (dG) is below this value.
	Dimer check	The primer's dimer forming energy (dG) (stability between the combined primers) is more than this value.
(18)	Distances	Minimum and Maximum length between primers (primer position)
	(F2-B2)	F2-B2 (including both primer length), amplified region.
	Loop(F1c-F2)	F1c (B1c) -F2 (B2) (including F2 (B2)), loop length.
	F2-F3	F2 (B2) -F3 (B3) (exclude the primer itself)
	F1c-B1c	F1c - B1c (exclude the primer itself)
(19)	Limitations	Maximum number of combinations when primer set is generated.
	F1c/B1c	The number of F1c (B1c) candidates, which can combine with each F2 (B2) when generating FIP (BIP).
	F2/B2	The number of F2 (B2) candidates, which can combine with each F1c (B1c) when generating FIP (BIP).
	F3/B3	The number of F3/B3 (outer primer) candidates, which can combine with one FIP/BIP set.
	Sets	Number of the finally generated primer sets.
(20)	Mutation/Consensus	When this item is selected, software will allow mutant to exist in 3' or 5'end. (In the default condition, primer sets are generated so as not to include any mutation sites).

Caution
The target sequence must be less than 2,000bp in length. When more than 2,000bp of the target sequence is uploaded, the primer designing can not be performed and the following page will appear, in which the message " Target sequence length is too long! Please input the sequence less than 2,000bp" is shown. When you push the OK button, it transits to the screen in front.

3. Generated primer list

Up to 100 generated primer sets will be listed in each page. Each row stands for one primer set, and each primer fragment's location refers to the target sequence above directly.
After pushing [Confirm] button, the primer sets selected will be shown in a new window for Detail information of the selected primer sets. When push [Save List] button, the primers listed on the screen will be saved as an Excel file.

4. Detail information of the selected primer sets.

The primer's information below is shown.

The following information is shown with each primer fragment.

Name
Position of 5' end on the target sequence
Position of 3' end on the target sequence
Length
Tm value
Stability of 5' end
Stability of 3' end
GC rate
Primer sequence

Click [Primer Information] to save the primer information as a file in your PC. This information can be used for designing the loop primer.
Click[Save] to save the primer information in the screen display layout as a Excel file in your PC

	[1]Introduction (operating environment)
	[2]Operation of each screen (designing regular primers/designing loop primers)
	[3]Appendix
	[4]A Guide to LAMP primer designing (Manual)：in preparation