4. Outputs for SNP genotyping information

4.1. The query results for the interactive genes

Back to Fig. 23 for the FMO3 interactive genes (the same as the Fig. 30), users can directly to get the PharmGKB information or the RFLP analysis for designate gene by clicking the

or images.

Fig. 30. The query results for interactive genes, tagSNPs, PharmGKB, and RFLP restriction enzyme availability with hyperlinks. Caution: Tag SNP selection candidates from different operation times in HapMap may show different representative tagSNPs due to the greedy algorithm built, i.e., they will randomly select one tagSNP from one LD block. Some tag SNPs may or may not find in next test. Likely, Drug-SNPing has the same performance. (The figure is the same as the Fig. 23)

4.2. NCBI SNP selection and HapMap tagSNPs

In order to get alternative NCBI SNP selection and tagSNPs of HapMap, users can click the big image “STRING interaction” (indicated by an arrow line 1 in Fig. 30). The visualization for STRING interaction will be shown as Fig. 31. On the top of Fig. 31, two selection parameters, such as 1) NCBI SNPs (indicated by arrow line 1) and 2) HapMap tagSNPs (indicated by arrow line 2), are optional for the visual image to get related SNPs information.

Fig. 31. The visualization for STRING interaction. The meanings of these arrow lines are explained later. Except the “color balls” (protein or gene name) are linked to our designed SNP genotyping path, the relationships (combined association score) between these color balls are online retrieved from the STRING [2].

4.2.1. NCBI SNP selection (clicking arrow line 1 in Fig. 31)

The pulldown windows in the top of Fig. 31 are adjustable for function class, SNP class, and heterozygosity, which are online retrieved from dbSNP in NCBI, as shown in Fig. 32.

Fig. 32. The adjustable function for NCBI SNP selection in Drug-SNPing. The numbers for SNPs listed after “submit” are also adjustable.

As shown in Fig. 31, the default settings for the function class, SNP class, and heterozygosity are in “all” status. For TaqMan probes, the Drug-SNPing also provides four choices, such as 1) SNP500Cancer, 2) ABI (from dbSNP in NCBI), 3) SNP500Cancer + ABI (the TaqMan probes exist in both SNP500 Cancer and ABI resources), and 5) Non-limit [They are indicated by a, b, c, and d in Fig. 31]. The examples for TaqMan probes of NCBI SNPs are shown as follows.

4.2.1.1. Under the TaqMan setting for “SNP500Cancer”

When the radio button of “NCBI SNP” is selected, users can click the ‘FMO3’ gene (arrow line 3 in Fig. 31) under the TaqMan setting for “SNP500Cancer [6]” (a in Fig. 31 or Fig. 33).

Fig. 33. The setting parameters for NCBI SNPs: TaqMan probe with SNP500Cancer.

And then, the waiting page for “Drug SNPs Query” will be shown as Fig. 34.

Fig. 34. The waiting page for “Drug SNPs Query”.

After waiting for a moment, the query results for NCBI SNPs of the ‘FMO3’ gene will be shown as Fig. 35. Here only 5 SNPs are shown due to limited space; total numbers for SNPs are 37 as indicated by arrow line in the right side. Users can click the “Next” button to see the other pages for the remaining SNP information.

Fig. 35. The query results for NCBI SNPs of the ‘FMO3’ gene under the TaqMan setting for SNP500Cancer.

Through the page (Fig. 35), users can select desired SNPs to analyze RFLP for getting available restriction enzymes by clicking the “Analyze sequence” button in Fig. 35 (We will describe later in 4.5. Representative results for SNP genotyping information).

4.2.1.2. Under the TaqMan setting for “ABI”

When the radio button of “NCBI SNP” is selected, users can click the ‘FMO3’ gene (arrow line 3 in Fig. 31) under the TaqMan setting for “ABI” (b in Fig. 31 and Fig. 36).

Fig. 36. The setting parameters for NCBI SNPs: TaqMan probe with “ABI”.

After waiting for a moment, the query results for NCBI SNPs of the ‘FMO3’ gene will be shown as Fig. 37. Here only 4 SNPs are shown; total numbers for SNPs are 105 as indicated by arrow line in the right side. Users can click the “Next” button to see the other pages for the remaining SNP information.

Fig. 37. The query results for NCBI SNPs of the ‘FMO3’ gene under the TaqMan setting for ABI. (Here, we did not shown all SNPs in this image)

Through the page (Fig. 37), users can select desired SNPs to analyze RFLP for getting available restriction enzymes by clicking the “Analyze sequence” button in Fig. 37 (We will describe later in 4.5. Representative results for SNP genotyping information).

4.2.1.3. Under the TaqMan setting for “SNP500Cancer+ABI”

When the radio button of “NCBI SNP” is selected, users can click the ‘FMO3’ gene (arrow line 3 in Fig. 31) under the TaqMan setting for “SNP500Cancer+ABI” (c in Fig. 31 and Fig. 38).

Fig. 38. The setting parameters for NCBI SNPs: TaqMan probe with SNP500+ABI.

After waiting, the query results for NCBI SNPs of the ‘FMO3’ gene will be shown as Fig. 39. Here only 5 SNPs are shown; total numbers for SNPs are 18 as indicated by arrow line in the right side.

Fig. 39. The query results for NCBI SNPs of the ‘FMO3’ gene under the TaqMan setting for SNP500Cancer+ABI. (Here, we did not shown all SNPs (18) in this image).

Through this page (Fig. 39), users can select desired SNPs to analyze RFLP for getting available restriction enzymes by clicking the “Analyze sequence” button in Fig. 39 (We will describe later in 4.5. Representative results for SNP genotyping information).

4.2.1.4. Under the TaqMan setting for “Non-limit”

When the radio button of “NCBI SNP” is selected, users can click the ‘FMO3’ gene (arrow line 3 in Fig. 31) under the TaqMan setting for “Non-limit” (d in Fig. 31 and Fig. 40).

Fig. 40. The setting parameters for NCBI SNPs: TaqMan probe- Non-limit.

After waiting for a moment, the query results for NCBI SNPs of the ‘FMO3’ gene will be shown as Fig. 41. Here only 5 SNPs are shown; total numbers for SNPs are 789 as indicated by arrow line in the right side. Users can click the “Next” button to see the other pages for the remaining SNP information.

Fig. 41. The query results for NCBI SNPs of the ‘FMO3’ gene under the TaqMan setting for Non-limit.

Through the page (Fig. 41), users can select desired SNPs to analyze RFLP for getting available restriction enzymes by clicking the “Analyze sequence” button in Fig. 41 (We will describe later in 4.5. Representative results for SNP genotyping information).

4.2.2. HapMap tagSNPs (clicking arrow line 2 in Fig. 42)

Alternatively, when the radio button of “HapMap tagSNPs” is selected (arrow line 2 in Fig. 42, similar to Fig. 31), uses can click the ‘FMO3’ gene (arrow line 3 in Fig. 42).

Fig. 42. The visualization for STRING interactions. The meanings of these arrow lines are explained later. Except the “color balls” (protein or gene name) are linked to our designed SNP genotyping path, the relationships (combined association score) between these color balls are online retrieved from the STRING [2]. (This figure is similar to Fig. 31)

The waiting page for “Drug tagSNPs Query” will be shown as Fig. 43. Then, the waiting page for “SNP Info Query” will be shown as Fig. 44.

Fig. 43. The waiting page for “Drug tagSNPs Query”.

Fig. 44. The waiting page for “SNP Info Query”.

After waiting for a moment, the query results for HapMap tagSNPs of the ‘FMO3’ gene will be shown as Fig. 45. Caution: Tag SNP selection candidates from different operation times in HapMap may show different representative tagSNPs due to the greedy algorithm built, i.e., they will randomly select one tagSNP from one LD block. Some tag SNPs may or may not find in next test. Likely, Drug-SNPing has the same performance.

Fig. 45. The query results for HapMap tagSNPs of the ‘FMO3’ gene. Caution: Tag SNP selection candidates from different operation times in HapMap may show different representative tagSNPs due to the greedy algorithm built, i.e., they will randomly select one tagSNP from one LD block. Some tag SNPs may or may not find in next test. Likely, Drug-SNPing has the same performance.

Through the page (Fig. 45), users can select desired SNPs to analyze RFLP for getting available restriction enzymes.

4.3. PharmGKB information

For instance, when select the image (indicated by an arrow line 2 in Fig. 46) which is located on the gene name “CYP2C19”, the page of PharmGKB will be linked shown as Fig. 47. All kinds of information for “CYP2C19” are available, such as Clinical PGx, PGx Research, Overview, VIP, Pathways, Is Related To, Publications, and Download/LinkOuts.

Fig. 46. The query results for interactive genes, tagSNPs, PharmGKB, and RFLP restriction enzyme availability with hyperlinks. Caution: Tag SNP selection candidates from different operation times in HapMap may show different representative tagSNPs due to the greedy algorithm built, i.e., they will randomly select one tagSNP from one LD block. Some tag SNPs may or may not find in next test. Likely, Drug-SNPing has the same performance. (The figure is the same as the Fig. 23 and Fig. 30)

Fig. 47. The PharmGKB information is available including Clinical PGx, PGx Research, Overview, VIP, Pathways, Is Related To, Publications, and Download/LinkOuts by clicking the

image which is located on the gene name “CYP2C19” indicated by an arrow line 2 in Fig. 46.

4.4. TagSNP genotyping information

For instance, when select the first

image (indicated by an arrow line 3 in Fig. 46) which is located on same row of the gene name “FMO3”, the waiting page for “SNP Info Query” will be shown as Fig. 48.

Fig. 48. The waiting page for “SNP Info Query”.

After waiting for a moment, the query results for HapMap tagSNPs of the ‘FMO3’ gene will be shown as Fig. 49. (Total tagSNP numbers are 6)

Fig. 49. The query results for HapMap tagSNPs of image selected in the ‘FMO3’ gene. Caution: Tag SNP selection candidates from different operation times in HapMap may show different representative tagSNPs due to the greedy algorithm built, i.e., they will randomly select one tagSNP from one LD block. Some tag SNPs may or may not find in next test. Likely, Drug-SNPing has the same performance.

From the output results of Fig. 35, Fig. 37, Fig. 39, Fig. 41, Fig. 45 and Fig. 49, users can select desired SNPs to analyze RFLP. For example, users can click “All selected” button (indicated by an arrow line in Fig. 49) to select all checkboxes (as shown in Fig. 50). Then, users can click the button “Analyze sequence” (indicated by an arrow line in Fig. 50), the waiting page for “SNP-RFLP” will be shown as Fig. 51.

Fig. 50. The query results for HapMap tagSNPs of image selected in the ‘FMO3’ gene with the “All selected” button is clicked.

Fig. 51. The waiting page for “SNP-RFLP”.

4.5. Representative results for SNP genotyping information

After waiting for a moment, the query results for SNP-RFLP of selected SNPs will be shown as Fig. 52.

Fig. 52. The results for “SNP-RFLP” of the ‘FMO3’ gene after the desired SNPs are analyzed.

4.5.1. SNP fasta information

Clicking the arrow line 1 in Fig. 52, the result for SNP fasta sequence is provided as shown in Fig. 53 for other SNP genotyping methods which are not implemented in the system. User can use the button “Output to File” (indicated by arrow line in Fig. 53) to export the sequence to their designated file.

Fig. 53. SNP fasta sequence is provided for other SNP genotyping methods which are not implemented in the system. This is the result of clicking the arrow line 1 in Fig. 52.

4.5.2. TaqMan probe information

Clicking the arrow line 2 in Fig. 52, the result for TaqMan probe information is provided as shown in Fig. 54. Some SNPs included in the SNP500Cancer do not provide the primer and TaqMan probe sequences (not shown in the example).

Fig. 54. The “TaqMan probe” results for “SNP-RFLP” of the ‘FMO3’ gene. This is the result for clicking arrow line 2 in Fig. 52. Arrow line 1 indicates ABI product number for TaqMan probe provided in the dbSNP in NCBI. Arrow line 2 indicates hyperlink button to ABI for TaqMan probe information. rs2066534 is the selected SNP for demonstration.

4.5.3. Natural primer information

Clicking the arrow line 3 in Fig. 52, the result for information is provided as shown in Fig. 55 to Fig. 57. In Fig. 55, the flanking sequence information for selected SNP and several setting parameters for natural primers of PCR-RFLP genotyping are provided. Clicking the “design” button, the waiting page for natural primer design is shown in Fig. 56. Finally, the information for PCR-RFLP is provided in Fig. 57.

Fig. 55. The sequence information and several setting parameters for natural primers of PCR-RFLP genotyping. These parameters are adjustable in the pull-down windows.

Fig. 56. The waiting page for “Natural primer design”.

Fig. 57. Natural primers and the corresponding restriction enzymes for the selected SNP (rs2066534). (A) Restriction enzymes for the alternative nucleotide of the selected SNP. (B) The information for forward and reverse primers. Its location is shown in green color letters.

4.5.4. Enzyme Information

Clicking the arrow line 4 in Fig. 52, the result for restriction enzyme information is provided as shown in Fig. 58.

Fig. 58. The available restriction enzymes for the sense (+) strand.

In order to get available restriction enzymes for antisense (–) strand, users can click the checkbox on the top in Fig. 58 (as indicated in the arrow line in Fig. 59). Both the available restriction enzymes for sense (+) strand and antisense (–) strand will be shown as Fig. 59.

Fig. 59. Both the available restriction enzymes for sense (+) strand and antisense (–) strand.

4.5.5. Mutagenic primer information

Clicking the arrow line 5 in Fig. 52, the waiting page for SNP flanking sequence query is shown in Fig. 60. This step is essential to retrieve the flanking sequence for selected SNP because the flanking sequence provided in the dbSNP in NCBI is usually short. Using our system, the flanking sequence is extended for the convenient of primer design. Clicking the “design” button in Fig. 61, the waiting page for mutagenic primer design is shown in Fig. 62. Finally, the information for PCR-RFLP is provided in Fig. 63 (no mutagenic primers for rs909530) or Fig. 64 (Mutagenic primers and the corresponding restriction enzymes for rs4147581).

Fig. 60. The waiting page for “SNP flanking sequence query”.

Fig. 61. The sequence information and several setting parameters for mutagenic primers of PCR-RFLP genotyping. These parameters are adjustable in the pull-down windows.

Fig. 62. The waiting page for “Mutagenic primer design”.

Fig. 63. No mutagenic primers for the selected SNP (rs909530). The result shows the sequence information.

Fig. 64. Mutagenic primers and the corresponding restriction enzymes for the selected SNP (rs4147581). (A) Restriction enzymes for the alternative nucleotide of the selected SNP. The artificial changed nucleotide is marked in red color (A→T). (B) The information for forward and reverse primers. Their primer locations are shown in green color letters and the SNP is marked in red color.

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