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1. How to extract promoter sequences from DOOR2 database (DOOR2)

Firstly, a user can click on the logo "DOOR2" on the homepage, then the user would be redirected to the page with 2,072 species

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Take E. coli K12 as an example. Type "NC_000913" in the search bar at the upper right-hand corner, then the species of interest is shown in the following page.

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Click on "NC_000913(C)", then two tabs (i.e., "All operons" and "Submit a gene list") used to extract operons or genes will be generated. For tab "All operons", say the first 15 operons, select all of them using check box, then click on the button "Get promoters".

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The corresponding promoters are shown in the following page. If no further modifications are needed, click on the button "De-novo motif finding".

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The user would be linked to the default submission page with their promoters pasted in the corresponding text area. Finally, the user can go ahead to input control sequences and set parameters or submit job directly .

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For the tab "Submit a gene list", the user can paste/upload a gene list with respect to the current species, then click on the button "Get Promoters" as mentioned above to obtain the corresponding promoters for further analyses.

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2. How to extract promoter sequences from Database of Eukaryotic Genes (Eukaryotic species)

A user can click on the logo "Database of Eukaryotic Genes" on the homepage, then the user would be redirected to the following page composed of three groups (i.e., plant, human and mouse).

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Take plant as an example. Click on the logo "Plant", then the basic information of species included in plant will be shown as follows.

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The user can click on "Brachypodium distachyon v3.1" if they are interested in this species, then detailed information of genes included in this species would be generated. Concretely, there are two tabs (i.e., "All genes" and "Submit a gene list") used to obtain the promoters of genes of interest. For tab "All genes", choose proper promoter length, then select genes of interest (e.g., the first 15 genes) using the check box, finally click on the button "Get promoters".

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The corresponding promoters are shown in the following page. If no further modifications are needed, click on the button "De-novo motif finding".

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The user would be linked to the default submission page with their promoters pasted in the corresponding text area. Finally, the user can go ahead to input control sequences and set parameters or submit job directly.

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For the tab "Submit a gene list", the user can paste/upload a gene list with respect to the current species and choose proper promoter length, then click on the button "Get Promoters" as mentioned above to obtain the corresponding promoters for further analyses.

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3. How to submit a job for De-novo motif finding (Motif Finding)

Totally, there are four steps to complete a motif finding job submission.
Step 1: Input query sequences
The user has three ways to upload the query sequences in the FASTA format:
(i) paste the query sequences into the text area or upload a local file; or
(ii) let DOOR2 prepare the promoter sequences, if they focus on bacteria; or
(iii) let Database of Eukaryotic Genes prepare the promoter sequences, if they focus on eukaryotes.
Step 2: Include control sequences (optional)
It is optional and allows the user to include a set of background sequences as a control. The format and submission requirement of background sequences is the same as the query sequences.
Step 3: Set algorithm parameters (optional)
Set the minimal and maximal motif length, as well as the number of output motifs.
Step 4: Submit job
Before submitting the job, the user can leave their email, which will be contacted when the job is done. To protect DMINDA 2.0 from spam and abuse, a single click on "I'm not a robot" is required.

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4. How to interpret the results page of De-novo motif finding

An example result page (jobid: 20160822151106f) is generated using the sample sequences provided by DMINDA2:

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There are six columns for each predicted motif shown in a result table, more details can be found as follows:
(i) Motif logo: A motif web logo is listed as default and more details can be found after clicking on "motif logo". E.g., click on the logo of Motif-19, then a new page with detailed information regarding this motif will be generated. In this page, we first map the predicted motifs to the query sequences, by which users can easily get the relative positions of the motifs to corresponding down-stream genes.

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Also some detailed information of this motif is provided, e.g. PWM, PSSM, consensus and so on (see relevant explanation in FAQ 6-8).

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(ii) Length: The motif length, which can be sorted in practice.
(iii) Pvalue: The P-value of this motif, see more details in FAQ 14 and formula 6 in FAQ 15.
(iv) Enrichment score: The Z-score of this motif, which only appears when a background sequence or comparative genomics strategy is used, see details in FAQ 14.
(v) Number: Number of motif instances for this motif, which can be sorted in practice.
(vi) Motifs: Details of each motif instance, including start and end positions in corresponding query sequences. Specifically, the motif instances in orange are the most conserved and those in yellow are identified by our P-value framework, see details in FAQ 13 and 15.
Besides the above six kinds of information, we can do some follow-up analyses for any selected motifs in this results page. For example, we select the first ten motifs in this page, such as:

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(i) Motif scan: Scan for all motif instances of a query motif in given genomic sequences based on a global P-value.
(ii) Motif compare: Compare the similarity between identified motifs, and cluster similar motifs into subgroups.
(iii) Co-occurrence analysis: Identify co-occurring motifs in provided regulatory sequences which may regulates the same genes.
(iv) Format conversion: Transfer current motif format to MEME and Uniprobe formats. Hence, the user can easily perform further motif analysis using other servers.
Also the input sequences and all results are ready for downloading, indicated in the upper right-hand corner.


5. How to submit a job for motif scanning (Motif Scan)

Similar to the de-novo motif finding, there are three steps for submitting a motif scanning job:

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Step 1: Input query motifs
(i) select motif format, refer to FAQ 5;
(ii) enter motifs with selected format in (i), see more details in FAQ 15-16.
Step 2: Input query sequence
(i) paste query sequences into text area or upload a local file; or
(ii) let DOOR2 prepare the promoter sequences, if they focus on bacteria; or
(iii) let Database of Eukaryotic Genes prepare the promoter sequences, if they focus on eukaryotes.
Step 3: Submit job
Before submitting the job, the user can leave their email, which will be contacted when the job is done. To protect DMINDA 2.0 from spam and abuse, a single click on "I'm not a robot" is required.


6. How to interpret the results page of motif scanning

An example results page (jobid: 20160824125537s) composed of query motifs and identified motif instances would be generated immediately after the job is submitted. A more detailed explanation of the results could be found in Tutorial 4.

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7. How to submit a job for motif comparison and clustering (Motif Compare)

There are four steps to submit a job for motif comparison and clustering:

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Step 1: Input query motifs
(i) select motif format, refer to FAQ 5;
(ii) enter motifs with selected format in (i), see more details in FAQ 15-16.
Step 2: Include host DNA sequences (optional)
paste the query sequences into the text area or upload a local file.
Step 3: Set parameters (optional)
set the similarity thresholds in the clustering algorithm.
Step 4: Submit job
Before submitting the job, the user can leave their email, which will be contacted when the job is done. To protect DMINDA 2.0 from spam and abuse, a single click on "I'm not a robot" is required.


8. How to interpret the results page of motif comparison and clustering

An example results page (jobid: 20160826151133c) would be generated immediately after the job is submitted, and detailed explanation of the results are shown below.
(i) Paired Similarity: A table containing the similarity score between each pair of submitted motifs. All of the three columns can be sorted in practice. Hence, the user can easily get the answer to "which pair of motifs has the highest similarity score" and "for a particular motif, which motif is the most similar". Furthermore, the user can copy and print the results or download the results in CSV, Excel or PDF format.

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In addition, the corresponding motif logos would be generated, if the user clicks on the similarity score.
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(ii) Similarity Matrix: Where the user can get the overall view of the similarities between any pair of motifs. In addition, we color each element on the scale of corresponding similarity score. The darker the color is, the more similar the corresponding two motifs are. The server provides a sort operation for each column to check the most similar motifs for the target motif. Furthermore, the user can copy and print the results or download the results in CSV, Excel or PDF format.

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(iii) Cluster Tree: The cluster result of submitted motifs.

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9. How to perform motif occurrence analysis and interpret the results page

This function is only available in the results page of motif finding and motif scanning, which is served as a follow-up analysis. An example results page is shown as follows.

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Concretely, the co-occurrence P-values of each pair of motifs are shown in the third column, which are calculated based on the hyper-geometric distribution. Other related parameters in the hyper-geometric distribution are shown in the last four columns. In this example results page, one can make a conclusion that motif-2 and motif-19 prefer to occur together, hence may regulate downstream genes together with high probability, as shown in tab "motif mapping".

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10. How to perform de-novo motif finding by MP3 (Motif MP3)

There are two steps to submit a job for motif finding by phylogenetic footprinting framework (MP3).

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The user has two ways to upload the query sequences in the FASTA format:
Step 1: Input promoter sequences of interest in two ways
(i) let DOOR2 prepare the promoter sequences, if they focus on bacteria; or
(ii) paste the query sequences into the text area or upload a local file.
Step 2: Submit job
Before submitting the job, the user can leave their email, which will be contacted when the job is done. To protect DMINDA 2.0 from spam and abuse, a single click on "I'm not a robot" is required.


11. How to interpret the results page of De-novo motif finding by MP3

An example results page (jobid: 2016090820854m) would be generated immediately after the job is submitted.

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Concretely, the motif mapping section lists a curve representing the voting scores along with several candidate binding regions. The right peak successfully covered several (here five) TF binding sites located at the upstream regions of the target gene. The corresponding motif profiles for the given query sequences are listed below with detailed information, as mentioned in Tutorial 4.


12. How to perform regulon finding (Regulon Finding)

A user can click on the logo "Regulon Prediction" on the homepage, then the user would be redirected to the submission page. Totally, there are two steps to complete a regulon prediction job.

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Step 1: Input species name and operon/gene IDs of interest
(i) let DOOR2 prepare the input data; or
(ii) paste/upload the input data manually.
Step 2: Submit job
Before submitting the job, the user can leave their email, which will be contacted when the job is done. To protect DMINDA 2.0 from spam and abuse, a single click on "I'm not a robot" is required.


13. How to interpret the results page of regulon prediction

An example results page (jobid: 20161228201738g) would be generated immediately after the job is submitted.

The results page lists the predicted regulons with their included operons. Furthermore, the user can click on any regulons or operons for more details.
(i) For regulons, we provide the details about their members and their regulatory region sequences:


and detailed information with respect to the genes included in the regulon,


as well as the details of the operons.




For the operons included in the predicted regulons, we provide extra information about their predicted motifs, including motif logo, width, P-value, motif instances and location as follows.


14. How to visualize the regulon network

Based on the predicted regulons as shown in (jobid: 20161228201738g), the user can select regulons of interest and click on the button "Get network" to obtain their network visualization.

The user will be redirected to a new webpage with a Cytoscape-like visualization for selected regulons. It is noteworthy that the user should allow pop-ups from http://bmbl.sdstate.edu in their browser if the visualization webpage can't be generated. As shown in the following image, different styles and layouts are available to change the shape of network, and the elements in network can be dragged manually. Furthermore, the network can be filtered by selecting parameters in the section "Filter". Corresponding properties will be shown in the section "Properties", if the element in network is selected. The user can change the color, size, opacity, shape, width and other parameters of the nodes and edges readily. The refined network can be saved in multiple formats, like XGMML, GraphML, pdf, png, etc.

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Concretely, each rounded rectangle represents an operon, and the corresponding motifs are indicated by circles. After the motif is selected, the detailed information will be shown in the section "Properties" and the corresponding regulon will be highlighted with red color in network as shown above. Similarly, the basic information of the operons will be shown after they are selected.

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The width of the edges in the network represent the similarity between two connected motifs, the user can check the similarity between two motifs by clicking on the edge of interest.

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