Genotyping Excel Template Instructions

Choose the correct templates for your data. PharmGKB accepts genotyping data from various kinds of assays. Custom templates are available for PCR, Taqman, RFLP, pyrosequencing, PCR sizing and DHPLC assays. If you used one of these types of assay please use the relevant templates to submit the specific assay conditions and corresponding results. To submit data for a different kind of assay not mentioned above, please use the generic genotyping or sequencing template as appropriate.

	Templates	Sample Files
Non-assay Templates
Sample Set	[ Template]	[ Sample File]
Reference Sequence		[ Sample File]
Sequencing Assays
PCR	[ Assay Template] [ Results Template]	[ Sample Assay File] [ Sample Results File]
Generic Sequencing	[ Assay Template] [ Results Template]	[ Sample Assay File] [ Sample Results File]
Genotyping Assays
RFLP	[ Assay Template] [ Results Template]	[ Sample Assay File] [ Sample Results File]
PCR Sizing	[ Assay Template] [ Results Template]	[ Sample Assay File] [ Sample Results File]
Pyrosequencing	[ Assay Template] [ Results Template]
TaqMan	[ Assay Template] [ Results Template]	[ Sample Assay File] [ Sample Results File]
Generic Genotyping	[ Assay Template] [ Results Template]
Miscellaneous Assay
DHPLC	[ Assay Template] [ Results Template]	[ Sample Assay File] [ Sample Results File]

Sample Sets

The sample set template captures basic background information about the individuals (known as "subjects") participating in your study as well as the type of samples which were collected.

Why are there 2 sets of numbers? What is a Subject? What is a Sample?

NOTE: You must request PharmGKB Subject and Samples numbers from PharmGKB prior to submitting data. To do so please email submissions@pharmgkb.org.

Sheet Specific Instructions

Sample Set Sheet

• Enter a unique name for the sample set in the "Sample Set Name" field.
• Enter sample info into the given table - one sample per row. The submitter may enter as many samples as they would like, and are not limited by the amount of rows enclosed.
• In the "Sample PharmGKB ID:" column, enter ID numbers for each sample.
• In the "Subject PharmGKB ID:" column, list the subject that this sample came from - multiple samples may be from the same subject.
• "Sample Timestamp" refers to the time at which the sample was taken.
• "Sample Cell Line" refers to the cell line or cell type that the sample is from. For example blood, buccal cell, HUVEC, Hela.
• "Ontology Term" should be either GO, MeSH or from the PharmGKB phenotype ontology. This cannot be a random term. This can be used to define how a sample was taken (eg. Bronchoalveolar Lavage [E05.510.100]) or more about the type of sample (eg. Neoplasm Metastasis [C23.550.727.650]).
• The "Sample PharmGKB ID" and "Subject PharmGKB ID" fields are required. The others are optional and the submitter should fill out only those columns which are applicable.

Subject Information Sheet

• Enter subject info into the given table - one subject per row.
• Please do NOT include any local IDs for subjects in this template. The submitter may enter as many subjects as they would like, and are not limited by the number of rows listed.
• For the column "Gender", select from the options listed: Male; Female; Hermaphrodite; Unknown.
• For the column "Race (OMB)" select from the options listed: American Indian or Alaskan Native; Asian; Black or African American; Native Hawaiian or Other Pacific Islander; White. This information is required by NIH if you choose to provide any information about race or ethnicity and you are required to use the standardized terminology. For details of this see the definitions from the Office of Management and Budget.
• For the column "Ethnicity (OMB)", select from the options listed: Hispanic or Latino; Not Hispanic or Latino.
• For the column Racial Category you may enter any racial classification. If you choose to enter information in this column you must also provide the relevant OMB mappings in the columns Race (OMB) and Ethnicity (OMB).
• The "Note" column is for any other notes the submitter may like to make about a subject.

Reference Sequence

There is no template for a reference sequence. It is recommended that you prepare your reference sequence as a text file in Word. The sequence must be at between 40bp and 25,000bp in length and contain only IUPAC codes.

When you submit, on the New Reference Sequence page, simply copy and paste the prepared reference sequence into the "sequence" window.

In the Submission Editor, you may also specify the coordinate system you will be using to report positions against the reference sequence. The default coordinate system numbers the first base in the sequence as "1" and has no zero. You may change any of these parameters as needed.

A diagrammatic representation of reference sequence, coordinate system and numbering primers is available here.

DHPLC Assays

DHPLC assays are used to identify samples whose sequence differs from the majority. The reference sequence is used to determine what the majority sequence is, based on the specified analyzed region.

Sheet Specific Instructions

DHPLC Assay Sheet

• Name of assay*: Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, DHPLC analysis for MTHFR C677T.
• Pool Size*: Enter the number of subjects in the run.
• Baseline sample size*: Enter the number of samples tested to determine baseline statistics.
• Analyzed region start position*: Enter the start position on the reference sequence using the coordinate system defined on the webform.
• Analyzed region stop position*: Enter the start and stop positions on the reference sequence using the coordinate system defined on the webform.
• Method information:
  • Name: Enter the general name of this type of method (e.g. DHPLC). If not specified, it defaults to the name of this assay.
  • Template type*: Select from the options listed: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction of DHPLC, including how the results were interpreted.
  • Parameters*: Enter the most important parameters of DHPLC, separated by commas.
  • Protocol Name*: Enter the name of the protocol you followed to perform this particular genotyping assay using DHPLC. For example, MTHFR C677T detection using DHPLC.
  • Protocol Description*: Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

DHPLC Results

Please enter results - one sample per row.

Sheet Specific Instructions

DHPLC Results Sheet

• Sample*: Enter Sample PharmGKB ID. For example, PA131401045.
• DHPLC Result*: Select from the listed: no result, same as majority, variant. If the assay was not successful/yielded unclear results for a sample, enter "no result" in the "Result" column. If a result was obtained, please enter if the genotype was the "same as majority" or a variant.

PCR Assays

This template is for PCR-based sequencing assays, not for amplification prior to other types of assays. (Information on amplification prior to RFLP or other types of assays can be included in the method and protocol description.)

The start and stop positions of the amplicon must lie on the reference sequence (link to description). If primers are defined, they must not have regions that overlap, the forward primer must be upstream of the reverse primer, and the amplicon must lie within the outermost extent of the primers. For example, if the primers start and stop at positions (5, 10) and (20, 25) respectively, the largest amplicon region can be (5, 25).

The start position is the 5' most base position of the region and the stop position is the 3' most base.

Sheet Specific Instructions

PCR Assay Sheet

• Name of assay*: Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, PCR-based sequencing analysis for MTHFR C677T.
• Amplicon start position on reference sequence*: Define the start position of the amplicon on the reference sequence. Please use the coordinate system as defined on the webform
• Amplicon stop position on reference sequence*: Define the stop positions of the amplicon on the reference sequence. Please use the coordinate system as defined on the webform.
• Forward primer sequence: Enter the sequence for the forward primer. The definitions of the primers are not required but are strongly encouraged. If primers aren't defined, warnings will be generated when this file is validated, and the submitter will be required to accept the warnings before the file can be uploaded to the PharmGKB website.
• Forward primer start position: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. For the majority of primers, the number entered here should be 1. Where the primers contain restriction sites or a short extension this may be different, for example if the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Amplicon stop position on reference sequence:
• Reference sequence start position: Positions are based on the coordinate system defined on the webform.
• Reference sequence stop position: Positions are based on the coordinate system defined on the webform.
• Reverse primer sequence: Enter the sequence for the reverse primer.
• Reverse primer start position: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. If the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Reverse primer stop position:
• Reference sequence start position: Positions are based on the coordinate system defined on the webform.
• Reference sequence stop position: Positions are based on the coordinate system defined on the webform.
• Method information:
  • Name: Enter the general name of this type of method (e.g. PCR-based sequencing). If not specified, it defaults to the name of this assay.
  • Template type*: Select one from the list: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction of the type of PCR used, including how the results were interpreted.
  • Parameters*: Enter the most important parameters of the type of PCR used, separated by commas. For example, annealing temperature, number of cycles.
  • Protocol Name*: Enter the name of the protocol you followed to perform this particular genotyping assay using PCR. For example, MTHFR C677T PCR-based Sequencing.
  • Protocol Description*: Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

PCR Results

There are 2 sheets in this form. Please report information regarding variants in the PCR Results sheet, and the interrogated range in the Interrogated Ranges sheet.

Sheet Specific Instructions

PCR Results Sheet

The submitter is required to enter at least one row per sample defined in the Sample Set. Please enter one sample/variant pair per row.

• Sample*: Enter Sample PharmGKB ID. For example, PA131401045.
• Sequence both strands*: Select Yes or No.
• Result*: Select from the list: no result, no variant, variant. If the assay was not successful or yielded unclear results for a sample, enter "no result" in the "Result" column. If a variant was found, please enter "variant" in the "Result" column and give values in the "Variant..." columns. If no variant was found for a sample, enter "no variant" in the "Result" column. A "no variant" result implies that the subject is homozygous per the reference sequence over the interrogated ranges for the sample (or the entire analyzed region if no interrogated ranges are specified).
• Variant Position: Enter the position according to the reference sequence.
• Variant Allele 1: Enter the actual base of the variant (i.e. A,T,C,G or -, where "-" represents deletion). Entering "wild type" or "mutant" will result in an error message on the webform
• Variant Allele 2: Enter the actual base of the variant (i.e. A,T,C,G or -).

Interrogated Ranges Sheet

Please enter one sample/interrogated range pair per row.

• Sample ID: Enter Sample PharmGKB ID. For example, PA131401045.
• For the "Interrogated range..." columns: please enter the start/stop positions according to the reference sequence and the coordinate system defined on the webform
• Interrogated ranges are the regions of the sequence that were confidently assayed. If no interrogated range is entered, it is assumed that the entire amplicon was read accurately. If the entire amplicon could not be sequenced, the interrogated range should define the regions that were successfully sequenced. All variants reported must lie within the reported interrogated range(s) for the sample.

Genotyping Assay

This form should only be used if the assay is not one of the predefined genotyping assays (i.e. DHPLC, RFLP, pyrosequencing or TaqMan). A genotyping assay is defined here as any assay that can only detect the change of one base pair each time when the assay is run. To report assays that a few base pairs can be detected in one assay, please contact PharmGKB at submissions@pharmgkb.org.

Sheet Specific Instructions

Genotyping Assay Sheet

The submitter is required to enter at least one row per sample defined in the Sample Set. Please enter one sample/variant pair per row.

• Name of assay*: Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, Heteroduplex generator analysis for MTHFR C677T.
• Interrogated position*: Enter the position being assayed according to the reference sequence and coordinate system defined on the webform.
• Method information:
  • Name: Enter the general name of this type of method (e.g. Heteroduplex Generator). If not specified, it defaults to the name of this assay.
  • Type: Select one from the list: computation, hybridization, other, SSCP, unknown.
  • Template type*: Select one from the list: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction of the method used, including how the results were interpreted.
  • Parameters*:Enter the most important parameters of Heteroduplex generator method used, separated by commas.
  • Protocol Name*:Enter the name of the protocol you followed to perform this particular genotyping assay using Heteroduplex generator. For example, MTHFR C677T detection using HG.
  • Protocol Description*: Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

Genotyping Results

Please enter results one sample per row.

Sheet Specific Instructions

Genotyping Results Sheet

The submitter is required to enter at least one row per sample defined in the Sample Set. Please enter one sample/variant pair per row.

• Sample*: Enter sample PharmGKB ID. For example, PA131401045.
• Result*: Enter whether the assay was successful: yes or no. If you enter "yes" in this column you need to supply the Variant Alleles observed.
• Variant Position: Enter the variant position according to the reference sequence and coordinate system defined on the webform.
• For the Variant Allele columns: Enter the actual base of the variant (i.e. A,T,C,G or -, where "-" represents deletion). Entering "wild type" or "mutant" will result in an error message on the webform. If the assay was not successful/yielded unclear results for a sample, leave the "allele" columns blank.

Sequencing Assay

This is a generic form for sequencing assays, and should only be used if the assay is not one of the predefined sequencing assay types (PCR assays or pyrosequencing) or if limited information is known about the protocol, for example sequencing performed by a sequencing facility or historical data for which method information has been lost. Note that for this assay, the reference sequence is considered the "wild type" sequence, meaning that if no variant is reported for a subject, the genotype is the same as the reference sequence within the interrogated range.

Sheet Specific Instructions

Sequencing Assay Sheet

• Name of assay*:Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, Sequencing analysis for MTHFR C677T.
• For analyzed region start/stop position*: Enter the start and stop positions of the region being assayed according to the reference sequence and coordinate system defined on the webform.
• Method information:
  • Name: Enter the general name of this type of method (e.g. Sequencing). If not specified, it defaults to the name of this assay.
  • Template type*: Select one from the list: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction to the sequencing method used, including how the results were interpreted.
  • Parameters*: Enter the most important parameters of Heteroduplex generator method used, separated by commas.
  • Protocol Name*:Enter the name of the protocol you followed to perform this particular genotyping assay. For example, Sequencing assay for MTHFR C677T.
  • Protocol Description*: Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

Sequencing Results

There are 2 sheets in this form. Please report information regarding variants in the Sequencing Results sheet and the interrogated range in the Interrogated Ranges sheet.

Sheet Specific Instructions

Sequencing Results Sheet

Please enter results one sample/variant pair per row. The submitter is required to enter at least one row per sample defined in the Sample Set.

• Sample*: Enter sample PharmGKB ID. For example, PA131401045.
• Sequence both strands*: Select Yes or No.
• Result*: Select from the list: no result, no variant, variant. If the assay was not successful or yielded unclear results for a sample, enter "no result" in the "Result" column. If a variant was found, please enter "variant" in the "Result" column and give values in the "Variant..." columns. If no variant was found for a sample, enter "no variant" in the "Result" column. A "no variant" result implies that the subject is homozygous per the reference sequence over the interrogated ranges for the sample (or the entire analyzed region if no interrogated ranges are specified).
• Variant Position: Enter the position according to the reference sequence.
• Variant Allele 1: Enter the actual base of the variant (i.e. A,T,C,G or -, where "-" represents deletion).Entering "wild type" or "mutant" will result in an error message on the webform.
• Variant Allele 2: Enter the actual base of the variant (i.e. A,T,C,G or -). Entering "wild type" or "mutant" will result in an error message on the webform

Interrogated Ranges Sheet

Please enter one sample/interrogated range pair per row.

• Sample ID: Enter Sample PharmGKB ID. For example, PA131401045.
• For the "Interrogated range..." columns: please enter the start/stop positions according to the reference sequence and the coordinate system defined on the webform
• Interrogated ranges are the regions of the sequence that were confidently assayed. If no interrogated range is entered, it is assumed that the entire amplicon was read accurately. If the entire amplicon could not be sequenced, the interrogated range should define the regions that were successfully sequenced. All variants reported must lie within the reported interrogated range(s) for the sample.

RFLP Assay

Information on amplification prior to RFLP assays can be included in the method and protocol description

Sheet Specific Instructions

RFLP Assay Sheet

• Name of assay*: Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, RFLP analysis for MTHFR C677T. For example, Heteroduplex generator analysis for MTHFR C677T.
• Interrogated position*: Enter the position being assayed according to the reference sequence and coordinate system defined on the webform.
• Target genotype*: Enter the genotype this assay is designed to detect (i.e. A, C, G, T, or -, where "-" represents deletion).
• Method information:
  • Name: Enter the general name of this type of method (e.g. RFLP). If not specified, it defaults to the name of this assay.
  • Template type*: Select one from the list: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction of the RFLP method, including how the results were interpreted.
  • Parameters*: Enter the most important parameters of RFLP, separated by commas.
  • Protocol Name*:Enter the name of the protocol you followed to perform this particular genotyping assay using RFLP. For example, MTHFR C677T detection using RFLP.
  • Protocol Description*:Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

RFLP Results

Please enter results one sample per row.

Sheet Specific Instructions

RFLP Results Sheet

The submitter is required to enter at least one row per sample defined in the Sample Set. Please enter one sample/variant pair per row.

• Sample*: Enter sample PharmGKB ID. For example, PA131401045.
• Result*: Enter whether the assay was successful: yes or no. If you enter "yes" in this column you need to supply the Variant Alleles observed.
• Variant Position: Enter the variant position according to the reference sequence and coordinate system defined on the webform.
• For the Variant Allele columns: Enter the actual base of the variant (i.e. A,T,C,G or -, where "-" represents deletion). Entering "wild type" or "mutant" will result in an error message on the webform. If the assay was not successful/yielded unclear results for a sample, leave the "allele" columns blank.

PCR Sizing Assay

This template is for PCR-based sizing assay in which primers are designed around a region already known to contain repeats. That region is amplified by PCR and run out on a gel. The gel bands are used to estimate the number of repeats in the region.

Sheet Specific Instructions

PCR Sizing Assay Sheet

• Name of assay*: Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, PCR-based sizing analysis for GC repeats in ABCB1. For example, Heteroduplex generator analysis for MTHFR C677T.
• Repeated DNA sequence: Enter the repeated sequence this assay is designed to detect.
• Analyzed region start position on reference sequence*: Define the start position of the amplicon according to the reference sequence and coordinate system defined on the webform.
• Analyzed region stop position on reference sequence*: Define the stop
  positions of the analyzed sequence on the reference sequence. Please use the coordinate system.
• Forward primer sequence: Enter the sequence for the forward primer. The definitions of the primers are not required but are strongly encouraged. If primers aren't defined, warnings will be generated when this file is validated, and the submitter will be required to accept the warnings before the file can be uploaded to the PharmGKB website.
• Forward primer start position*: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. For the majority of primers, the number enter here should be 1. When the primers contain restriction sites or a short extension this may be different, for example, if the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Forward primer stop position:
• Reference sequence start position: Positions are based on the coordinate system defined on the webform.
• Reference sequence stop position: Positions are based on the coordinate system defined on the webform.
• Reverse primer sequence: Enter the sequence for the reverse primer.
• Reverse primer start position: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. For the majority of primers, the number enter here should be 1. When the primers contain restriction sites or a short extension this may be different, for example, if the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Reverse primer stop position:
• Reference sequence start position: Positions are based on the coordinate system defined on the webform.
• Reference sequence stop position: Positions are based on the coordinate system defined on the webform.
• Method information:
  • Name: Enter the general name of this type of method (e.g. PCR-based sequencing). If not specified, it defaults to the name of this assay.
  • Template type*: Select one from the list: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction of PCR-based sizing used, including how the results were interpreted. For example, "This is a PCR-based sizing assay in which primers are designed around a region already known to contain GC repeats. That region is amplified by PCR and run out on a gel. The sizes of the gel bands are used to estimate the number of repeats in the region".
  • Parameters*: Enter the most important parameters of the PCR-based sizing method, separated by commas. For example, annealing temperature, number of cycles.
  • Protocol Name*:Enter the name of the protocol you followed to perform this particular genotyping assay using PCR. For example, PCR-based sizing for GC repeats in ABCB1.
  • Protocol Description*:*: Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

PCR Sizing Results

Please enter results one sample per row.

Sheet Specific Instructions

PCR Sizing Results Sheet

• Sample*: Enter sample PharmGKB ID. For example, PA131401045.
• Result*: Enter whether the assay was successful: yes or no. If you enter "yes" in this column you need to supply the Variant Alleles observed.
• Variant Position: Enter the variant position according to the reference sequence and coordinate system defined on the webform.
• For the Variant Allele columns: Enter the actual bases of the repeats (i.e. GCGC). Entering "wild type" or "mutant" will result in an error message on the webform. If the assay was not successful/yielded unclear results for a sample, leave the "allele" columns blank.

Pyrosequencing Assay

Sheet Specific Instructions

Pyrosequencing Assay Sheet

• Name of assay*: Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, pyrosequencing analysis for MTHFR C677T.
• Interrogated position*: Enter the position being assayed according to the reference sequence and coordinate systema defined on the webform.
• Analyzed region start position*: Define the start position of the amplicon on the reference sequence. Please use the coordinate system as defined on the webform.
• Analyzed region stop position*: Define the stop positions of the analyzed sequence on the reference sequence (links to description). Please use the coordinate system defined on the webform.
• Forward primer sequence: Enter the sequence for the forward primer. The definitions of the primers are not required but are strongly encouraged. If primers aren't defined, warnings will be generated when this file is validated, and the submitter will be required to accept the warnings before the file can be uploaded to the PharmGKB website.
• Forward primer start position: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. For the majority of primers, the number enter here should be 1. When the primers contain restriction sites or a short extension this may be different, for example, if the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Forward primer stop position:
• Reference sequence start position: Positions are based on the coordinate system defined on the webform.
• Reference sequence stop position: Positions are based on the coordinate system defined on the webform.
• Reverse primer sequence: Enter the sequence for the reverse primer.
• Reverse primer start position: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. For the majority of primers, the number enter here should be 1. When the primers contain restriction sites or a short extension this may be different, for example, if the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Reverse primer stop position:
• Pyrosequencing primer sequenceReference sequence start position*: Enter sequence of the primer that is used on the biotinylated strand.
• Pyrosequencing primer orientation*: Enter either forward or reverse.
• Method information:
  • Name: Enter the general name of this type of method (e.g. Pyrosequencing). If not specified, it defaults to the name of this assay.
  • Template type*: Select one from the list: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction of the Pyrosequencing method used, including how the results were interpreted.
  • Parameters*: Enter the most important parameters of this Pyrosequencing method, separated by commas. For example, annealing temperature, number of cycles.
  • Protocol Name*: Enter the name of the protocol you followed to perform this particular genotyping assay using pyrosequencing. For example, pyrosequencing for MTHFR C677T.
  • Protocol Description*:Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

Pyrosequencing Results

Please enter results one sample per row.

Sheet Specific Instructions

Pyrosequencing Results Sheet

• Sample*: Enter sample PharmGKB ID. For example, PA131401045.
• Result*: Enter whether the assay was successful: yes or no. If you enter "yes" in this column you need to supply the Variant Alleles observed.
• Variant Position: Enter the variant position according to the reference sequence and coordinate system defined on the webform.
• For the Variant Allele columns: Enter the actual base of the variant (i.e. A,T,C,G or -, where "-" represents deletion). Entering "wild type" or "mutant" will result in an error message on the webform. If the assay was not successful/yielded unclear results for a sample, leave the "allele" columns blank.

TaqMan Assay

Note: It is assumed a MGBNFG 3' quencher and minor groove binder molecule is added to the 3' end of the probe. If this assumption is not correct, please specify in the probe dye fields. If a commercial primer was used, and the sequence is not know, please give the catalogue number and company for the primer in the Protocol Description.

Sheet Specific Instructions

TaqMan Assay Sheet

• Name of assay*: Enter a unique name for this assay, a name reflects particularly the reported measurement. For example, TaqMan analysis for MTHFR C677T.
• Interrogated position*: Enter the position being assayed according to the reference sequence and coordinate system defined on the webform.
• Reference probe dye: For example 6FAM^TM dye.
• Reference probe sequence: Enter the sequence of the probe annealing to the wild type sequence.
• Reference probe target genotype: Enter the genotype the probe is designed to detect (i.e. A, T, C, G).
• Variant probe dye: For example VIC dye.
• Variant probe sequence: Enter the sequence of probe annealing to the sequence with variant.
• Variant probe target genotype: Enter the genotype the probe is designed to detect (i.e. A, T, C, G).
• Forward primer sequence: Enter the sequence for the forward primer. The definitions of the primers are not required but are strongly encouraged. If primers aren't defined, warnings will be generated when this file is validated, and the submitter will be required to accept the warnings before the file can be uploaded to the PharmGKB website.
• Forward primer start position: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. For the majority of primers, the number enter here should be 1. When the primers contain restriction sites or a short extension this may be different, for example, if the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Forward primer stop position:
• Reference sequence start position: Positions are based on the coordinate system defined on the webform.
• Reference sequence stop position: Positions are based on the coordinate system defined on the webform.
• Reverse primer sequence: Enter the sequence for the reverse primer.
• Reverse primer start position: The region in the primer that anneals to the reference sequence. The first base in the primer's sequence is position 1. For the majority of primers, the number enter here should be 1. When the primers contain restriction sites or a short extension this may be different, for example, if the primer anneal to the reference sequence from the 4th base, the number entered here should be 4.
• Reverse primer stop position:
• Reference sequence start position: Positions are based on the coordinate system defined on the webform.
• Reference sequence stop position: Positions are based on the coordinate system defined on the webform
• Method information:
  • Name: Enter the general name of this type of method (e.g. TaqMan). If not specified, it defaults to the name of this assay.
  • Template type*: Select one from the list: diploid, clone, other and unknown.
  • Multiple PCR amplification tested: Select from the options listed: yes, no, unknown, NA.
  • Multiple clones tested: Select from the options listed: yes, no, unknown, NA.
  • Description*: Give a brief introduction of the TaqMan method used, including how the results were interpreted.
  • Parameters*: Enter the most important parameters of this TaqMan method, separated by commas. For example, annealing temperature, number of cycles.
  • Protocol Name*: Enter the name of the protocol you followed to perform this particular genotyping assay usingTaqMan. For example, TaqMan for MTHFR C677T.
  • Protocol Description*:Enter the whole protocol so that others can repeat your analysis using the protocol you provide here.
  • Buffer Name: Enter the commercial name of the main buffer you used in your protocol.
  • Buffer Description: Give a brief description of the above main buffer, including vendor's names, state and country.

TaqMan Results

Please enter results one sample per row.

Sheet Specific Instructions

TaqMan Results Sheet

• Sample*: Enter sample PharmGKB ID. For example, PA131401045.
• Result*: Enter whether the assay was successful: yes or no. If you enter "yes" in this column you need to supply the Variant Alleles observed.
• Variant Position: Enter the variant position according to the reference sequence and coordinate system defined on the webform.
• For the Variant Allele columns: Enter the actual base of the variant (i.e. A,T,C,G or -, where "-" represents deletion). Entering "wild type" or "mutant" will result in an error message on the webform. If the assay was not successful/yielded unclear results for a sample, leave the "allele" columns blank.