Step1: Plan the RMA search                                            BACK

Decide the goals of unique peptide extraction and prepare the interested family sequences (amino acid or DNA sequences).

Step2: Enter the your family sequences                           BACK

The RMA system accepts input sequences in two ways; importing sequence files or pasting FASTA sequence data on text editor directly.

Importing Sequence Files:

  1. Select "File --> Open files --> Import files" from the menu bar of RMA system. The file format can be either FASTA sequence files or plain text file with amino acid/DNA data.
  2. Select sequence type (protein or DNA).
  3. Click "Add" button, select appropriate directory path and sequence files.
  4. Clict "OK" button to continue.
  5. You can delete any one of imported sequences or remove all of them by clicking "Delete One Item" or "Clear" button.
  6. For this version, the number of importing family sequences are limited to 500 temporarily.
  7. Click "Cancel" to give up importing files.

Paste FASTA Sequence Format

  1. Select "File --> Open files --> Paste FASTA sequences" from the menu bar of RMA system.
  2. Select sequence type (protein or DNA).
  3. Open your FASTA formatted sequences in a text editor or browers's window as plain text.
  4. Use your mouse to highlight the entire sequence.
  5. Copy and paste the first FASTA formatted sequence into the RMA sequence editor.
  6. Click "Enter" to change into a new line and copy/paste the second FASTA formatted sequence continually.
  7. Click "OK" button to continue.
  8. You should now see your FASTA sequences in the main window.

Step3: Select the favorite searching tools                          BACK

The RMA system provides automatic/semi-automatic reinforced merging technique and traditional brute force searching techniques for extracting unique peptide motifs.

Automatic Reinforced Merging Technique (Automatic RMA)

  1. Select "Tools --> RMA Method --> Automatic" from menu bar of RMA system.
  2. Select sequence type (protein or DNA).
  3. The running status of RMA is displayed at the bottom of the main screen.
  4. Click "Description" button to view the presentation of searched results.
  5. You can double click on any searched unique peptide motif to see its position and related information.
  6. You can select one of the source files from "Sources" list box to quickly view the searched results.

Semi-Automatic Reinforced Merging Technique
(Semi-Automatic RMA)

There are three major phases in semi-automatic RMA systems, including Grouping, Searching, and Merging phases.

Grouping Phase
It is provided for users to select one of the BLOSUM/PAM substitution matrices for clustering 20 amino acids into several groups with respect to threshold values. The clustering algorithms are based on a hierarchical methodology (agglomerative algorithms) which produces clusters of amino acids with similar characteristics. The algorithm stops when it meets the threshold value setting. (ps. In this version, this function are not available temporarily)

Searching Phase
is provided for users to extract primary unique peptide motifs from each sequence. RMA system requires the parameters of the length of primary segment and the tolerant number in mismatching situation. Based on these settings, the module performs modified Boyer Moore algorithms to filter out all possible candidate patterns and their representative percentage (uniqueness level) respectively. The system provides Primary Length Analysis which is an optional analytic tool. Users can skip this function and set the parameters manually. If users are not sure about the length parameter for primary segment, they can perform this function to obtain suggesting settings. However, it requires evaluation time depending on quantities of imported files.

  1. Click "Primary Length Analysis" button to analyze the imported family sequences automatically. The length of primary segment is suggested to set a larger value for sequences with higher similarity.
  2. Select sequence type (protein or DNA).
  3. Click "Search" button to extract primary unique segments.
  4. The resulting table on right hand side shows all primary segments and their related information, including position and uniqueness.

Merging Phase
provides reinforced merging processes to the obtain predicted unique peptide. Users are able to specify the minimum length of final merged segment with local primary segments satisfied the assigned uniqueness level. There are four different method to merge the primary segments, including "merge", "trim merge", "strict merge", and "strict trim merge". Any possible partial segment of the merged substring will satisfy the assigned representative percentage requirement.

  1. Enter the lowest limit value for the length of merged unique patterns. The default values for amino acid and DNA sequences are 8 and 15 respectively.
  2. Enter the lowest limit percentage for the uniqueness level of primary segments.
  3. The default values for amino acid and DNA sequences are 8 and 15 respectively.
  4. Click one of the merging operation buttons to execute the final merging operations.
  5. The system will collect, organize, and show the final results on main window.

Traditional Brute Force Technique                                      BACK

  1. Select "Tools --> Brute Force Method --> from menu bar of RMA system. ( a dialogue window will pop-up automatically)
  2. Enter the length parameter for unique peptide motifs.
  3. Click "Search" button to extract the unique peptide motifs from imported sequences.
  4. Searched results and related information are shown in right table box.
  5. You can click on the "Show on Sequences" button to obtain the exact positon of searched unique peptide motifs.

Step 4. Specify the appropriate search parameters              BACK

There is no parameter settings required for "Automatic" RMA systems. However, for "Semi-Automatic" programs, there are three-phase parameters required for executing RMA manually.

  1. In "Grouping Phase"(optional), users can select traditional BLOSUM substitution matrices from supported files which are employed for grouping 20 amino acids. This process provides substitution information for uniquess representation.
  2. In "Searcing Phase", users have to enter text boxes of "length lower bound" and "length upper bound" for searching primary segments. The number of upper bound has to be greater or equal to lower bound. (These paremeters can be obtained automatical by performing "Primary Length Analysis" function. The parameter of "reserved sites" represents the tolearnce features of primary segment, which should be less than or equal to the "length lower bound" setting.
    A rule of thumb, the length of primary segment can be set as "3" for protein sequences, and "7" for DNA sequences.
  3. In "Merging Phase", select an approiate "Pattern Length" for final merged unique peptide motifs. Generally speaking, "8" is suggested for protein sequences and "15" for DNA sequences. Therefore, the extracted unique peptide motifs possess at least "8" and "15" respectively.
  4. The "Uniqueness(%)" parameter represents the unique level of serched primary segments. A primary segment with a uniqueness level of 100% possesses the highest uniqe feature(i.e. the lowest tolerant characteristics), and 0% represents the least unique feature ( i.e. the highest tolerant characteristics).

Step 5. Execute the program                                        BACK

After all parameters are specified, users have to select one of RMA merging operators, including "MERGE", "TRIM MERGE", "STRICT MERGE", and "STRICT TRIM MERGE". BY clicking one of the merging buttons, the RMA will provide extracted unique peptide motifs on main screen. Each merging operator employs different colors to represent the searched segments, and they are described in RMA output.