Five input types are provided, including 1. gene name(s) for DBTSS, 2. NCBI nucleotide accession#(s), 3. fasta sequence(s) paste, 4. primer pair information for PCR product and 5. fasta file input. They are introduced as follows:

1. gene name(s) for DBTSS

User can input gene name(s) to search transcriptional start sites from DBTSS (http://dbtss.hgc.jp/) and two organisms are selectable, they are human and mouse, respectively. (Fig. 1)

Fig. 1. Gene name(s) for DBTSS input box.

When the text field of “Gene Names” is inputted correctly and “Query” button is clicked (Fig. 1) (for instance “TEKT4”), the system will query Gene ID information via NCBI database. (Fig. 2)

Fig. 2. Query Gene ID information via NCBI database.

And then, the system will search query transcriptional start sites information via DBTSS database. (Fig. 3)

Fig. 3. Query transcriptional start sites information via DBTSS database.

When the query is finished, related information is shown as Fig. 4. This information contains “Organism”, “Gene Name”, “Gene ID”, “Official Full Name”, and promoter sequence. And now user can set the CpG island parameters and clicked the “CpG Island Search” button to search CpG islands in the promoter sequence. (Fig. 4)

Fig. 4. Promoter sequence are shown and CpG island parameters set after query transcriptional start sites information via DBTSS database.

After the “CpG Island Search” button is clicked (Fig. 4), the system will search CpG islands by the program “CpG Island Searcher”. (Fig. 5)

Fig. 5. Query nucleotide information via NCBI database.

For a result of CpG island search, all CpG islands in the promoter sequence will be output (this case only one CpG island is found). And then user can select which CpG island to analyze methylation RFLP (Restriction Fragment Length Polymorphism). (Fig. 6)

Fig. 6. The result for CpG island search.

There are three options in every CpGIsland #, they are “CpG Mapping”, “CpG Distribution”, and “CpG Island Seq”, respectively. And the CpG island information is described including start position, end position, %GC, ObsCpG/ExpCpG, and length. (Fig. 6)

CpG Mapping” shows the CpG island and CpG position in the promoter sequence with color sequence and color bar. Black color font and represents nucleotide bases, green color font and represents CpG island, and pink color font and represents CpG position. (Fig. 7)

Fig. 7. “CpG Mapping” shows the CpG island and CpG position in the promoter sequence with color sequence and color bar.

“CpG Distribution” shows the CpG island and CpG position in the promoter sequence with color bar. represents nucleotide bases, represents CpG island, and represents CpG position. (Fig. 8)

Fig. 8. “CpG Distribution” shows the CpG island and CpG position in the promoter sequence with color bar.

“CpG Island Seq” shows the CpG island and CpG position in the promoter sequence with color sequence. Black color font represents nucleotide bases, green color font represents CpG island, and pink color font represents CpG position. (Fig. 9)

Fig. 9. “CpG Island Seq” shows the CpG island and CpG position in the promoter sequence with color sequence.

Here, we selected CpG island 1 to analyze methylation RFLP. Click the “Methy-RFLP analysis” button of CpG island 1, and then wait the process of methylation RFLP analysis. (Fig. 10)

Fig. 10. Analyze methylation RFLP.

Finally, available RFLP for methylation enzymes of selecting CpG island are provided and shown as Fig. 11 (partly enzymes are shown). Enzymes are classed with commercial and non-commercial, and the recognition sequences contain two types that are IUPAC and Non-IUPAC.

At the right top, four options are available for displaying additional information; they are “Sequence”, and “Visualization”, “CTCFBS Search”, “Bisulfite T Stretch”, respectively. (Fig. 11)

The bisulfite primers had integrated in Methyl-Typing that shown in Fig. 11 "Primer information". The bisulfite sequencing primers (BSP) were designed in Methyl-Typing according to the criteria of Methyl Primer Express R Software v1.0 user manual (www.appliedbiosystems.com/methylprimerexpress). Using the BSP primers for PCR amplification, it is able to be digested with the mined COBRA enzymes to perform COBRA analysis.

Fig. 11. Available RFLP for methylation enzymes of selecting CpG island are provided.

Result of clicking the “sequence” box shows the original orbisulfite-treated/methylated sequenceswith CpG islands and mined restriction enzymes are colored in green and red, respectively. The bisulfite-treated/un-methylated sequence is also provided for comparison . All the recognition sequences are designed in CpG island. (Fig. 12)

Fig. 12. Result of clicking the “sequence” box.

A convenient function is employed to find enzyme recognition sequences position. Cclick a recognition sequence of enzyme and all the corresponding enzyme recognition sequences will be marked as red color font and the selected enzyme line will also be marked as shallow yellow color (Fig. 13).

Fig. 13. Click a recognition sequence of enzyme and the corresponding enzyme recognition sequences are marked as red color font and the selected line is marked as shallow yellow color.

“Visualization” shows the CpG island and enzyme recognition sequences with color bar. The red color bar represents enzyme recognition sequences, green color bar represents CpG island, and blue color font represents nucleotide bases. Two visualization are shown, one is visualization for all enzymes in CpG island and the other is visualization for a selected enzymes in CpG island. (Fig. 14)

Fig. 14. “Visualization” shows the CpG island and enzyme recognition sequences with color bar and all the recognition sequences are designed in CpG island.

When user selects a recognition sequence, all the corresponding recognition sequences will be marked by red bar and the selected line will also be marked as shallow yellow color (Fig. 15).

Fig. 15. Select a recognition sequence, the corresponding recognition sequences are marked by a red bar and the selected line will be marked as shallow yellow color.

“CTCFBS Search” uses CTCFBSDB (http://insulatordb.utmem.edu/help.php) to search insulators for the input sequence. When user click the “CTCFBS Search” button (Fig. 16), and a new page will be popped up. The input sequence will be loaded automatically and E-value is defaulted by 10.0. (Fig. 16)

Fig. 16. CTCFBS search input box.

User can clicked the “CTCFBS Search” button and the result will be output at an original page. (Fig. 17)

Fig. 17. The result for CTCFBS search.

“Bisulfite T Stretch” provides a search for repeat ‘T’ bases after bisulfite process. The corresponding sequence will be marked by red color and the default length is 10 bps. And user can adjust by the option where select a value is 9, 8, 7, 6, or 5. (Fig. 18)

Fig. 18. Repeat ‘T’ bases search after bisulfite process.

Result for clicking the “BSP (Bisulfite sequencing primer)” box in the followign Fig. 19. The primers F (forward) and R (reverse) are visualized and their sequences were provided. The positions for the primer location and COBRA enzymes were shown as well as the product size for PCR digestion with COBRA enzymes.

Fig. 19. Result for clicking the “BSP (Bisulfite sequencing primer)” box.

 

2. NCBI nucleotide accession#(s)

NCBI nucleotide accession# input is availability in the system for providing various sequences. (Fig. 20)

Fig. 20. NCBI nucleotide accession# input box.

When the text field of “Nucleotide Accession#” is input correctly and “Query” button is clicked (Fig. 20), the system will query nucleotide information via NCBI database. (Fig. 21)

Fig. 21. Query nucleotide information via NCBI database.

When the query is finished, related information is shown as Fig. 22. This information contains “LOCUS”, “DEFINITION”, “ACCESSION”, “VERSION”, and nucleotide sequence. And now user can set the CpG island parameters and clicked the “CpG Island Search” button to search CpG islands in the nucleotide sequence (Fig. 22). And then the other steps are the same as above description.

Fig. 22. Nucleotide sequence are shown and CpG island parameters set after query nucleotide information via NCBI database.

 

The following steps are the same as above description after the “CpG Island Search” button are clicked.

 

3. fasta sequence(s) paste

A fasta format sequence can be type or paste to the sequence input box, and multiple fasta format sequences are available. GpG Island Searcher parameters are defaulted as %GC=55%, ObsCpG/ExpCpG=0.60, and Length=200bp for search the input sequence CpG islands. (Fig. 23)

Fig. 23. Fasta sequence paste input box.

When the “Analysis” button is clicked (Fig. 23), and then the sequences in input box will be processed and shown as fasta format. Every fasta sequence follows a “CpG Island Search” button and user can select they desired sequence to search CpG island (Fig. 24). And then the other steps are the same as above description.

Fig. 24. Every fasta sequence follows a “CpG Island Search” button and user can select they desired sequence to search CpG island.

The following steps are the same as above description after the “CpG Island Search” button are clicked.

 

4. primer pair information for PCR product

Fig. 25. Primer pair information input box.

When the primer pair is inputted correctly and related parameters is set, click the

“Query” button (Fig. 25), the system will query PCR product information via UCSC In-Silico PCR. (Fig. 26)

Fig. 26. Query PCR product information via UCSC database.

And then, the PCR product sequence will output and display as a fasta sequence. (Fig. 27)

Fig. 27. The PCR product sequence is fasta type.

The following steps are the same as above description after the “CpG Island Search” button are clicked.

 

5. fasta file input

A fasta format sequence or multiple fasta format sequences can be saved to a file in local computer. And user can by the “browse…” botton to select its absolute path. GpG Island Searcher parameters are defaulted as %GC=55%, ObsCpG/ExpCpG=0.60, and Length=200bp for search the input sequence CpG islands (Fig. 28). When “Put In” button is clicked, the input file will be processed and shown as fasta format (same as Fig. 24). And then the other steps are the same as above description after the “CpG Island Search” button is clicked.

Fig. 28. Fasta file input box.

The following steps are the same as above description after the “CpG Island Search” button are clicked.