The Real-time PCR Data Markup Language (RDML) is a structured and universal data standard for exchanging quantitative PCR (qPCR) data and digital PCR (dPCR) data. The data standard should contain sufficient information to understand the experimental setup, re-analyse the data and interpret the results. It was designed to enable transparent exchange of annotated qPCR data between instrument software and third-party data analysis packages, between colleagues and collaborators, and between authors, peer reviewers, journals and readers.
The RDML-Tools center around the RDML data format. The RDML-Tools mainly provide a web page access to the functions of the RDML-Python library, designed to handle RDML files and their dependencies. Users may choose to install the library as a python package calling "python -m pip install rdmlpython". All software is GPL licensed except the RDML-Python library being MIT licensed.
RDML Data Standard
The RDML data standard is a compressed text file in Extensible Markup Language (XML). By design, it collects the information of different experimenters, dyes, samples, targets and thermal cycling conditions separately to experiment/run information, were this information is only referenced by the unique ID. In a final RDML file for example one target might be referenced many times. The RDML-Tools and RDML-Python library were created to handle this dependencies and allow modification of an ID with updating all the references.
RDML evolved over the years and there are several versions available today. The RDML-Tools can convert between the versions. Upload the file to RDML-Edit, activate the edit mode on the main tab and the migration buttons are available on the more tab. Version 1.0 is automatically converted to version 1.1, the lowest supported version by the RDML-Tools. Newer versions offer new and may remove obsolete functionality. Be aware that digital PCR is only supported from version 1.3 on.
RDML was first published in "RDML: structured language and reporting guidelines for real-time quantitative PCR data" in 2009. Version 1.2 and the database RDMLdb were introduced in "RDML-Ninja and RDMLdb for standardized exchange of qPCR data" in 2015.
Import Data into RDML
Importing data is always the second best choice to having a native RDML export in your qPCR machine. If a purchase is planned, consider one of the machines with RDML support.
Without RDML export in your machine, you have to recreate the data first. Run RDML-Edit and create a new file on the main tab and and enable editing with the "enable edit mode" button. For digital PCR, the version needs to be 1.3 or above which can be set on the more... tab.
Most machines offer an export of the raw data. Unfortunately, there is no consent on the format of this export. Follow the instructions for RDML-TableShaper to convert the export into the standard table RDML import format. Select or create a new experiment on the experiments tab. Create a new run and provide the file from TableShaper at "Import Amplification Data".
Currently only Bio-Rad and Stilla export files are supported. Select or create a new experiment on the experiments tab. Create a new run and provide the results or overview file at "Import Digital Data Overview" and the files with the raw well data at "Import Digital Data Wells". Although import without an overview file is possible, naming samples and targets will be very laborious and not recommended.
Last, data for all the samples, targets and thermal cycling conditions have to be added using the edit button on the respective elements.
RDML-Edit is the main tool to view and edit RDML data up to the run level. Selected runs can be views can be visualized with RDML-RunView. With classic qPCR the amplification- or meltcurves are displayed, with digital PCR the positive and negative counts are shown. RDML-TableShaper helps to reformat exported files from machines lacking RDML support. RDML-Validate validates a RDML file against the corresponding schema.
RDML-Edit is a tool to view and edit RDML files. By default RDML-Edit only allows viewing of files. RDML supports many optional parameters to describe a qPCR reaction. RDML-Edit will hide all parameters which were not entered by the user. To modify files enable the edit mode using the button on the main tab. RDML does support different version with different functionality. RDML-Edit can migrate between different versions on the more... tab if the edit mode is activated. Digital PCR is only supported from version 1.3 on. Please read the Data Standard section for an understanding of the basic RDML concepts.
RDML-RunView is a tool to view single runs of a RDML files. Once the RDML file was loaded, the desired experiment and run have to be selected. RDML-RunView will display the plate setup and, with classic qPCR, the amplification- or meltcurves. A well / reaction can be selected which will highlight the corresponding curve. If a curve is selected, the corresponding reaction will be highlighted. Meltcurves can be easily inspected if the color coding is set to target. Setting the color coding to type allows eases the inspection of amplification curves.
RDML-Validate validates a RDML file against the corresponding schema. If the validation fails, RDML-Validate displays a list of errors. Errors point to programming mistakes in the used software to create the RDML file and might be difficult to fix by the user. RDML-Edit offers in edit mode on the main tab two buttons "Recreate Lost Ids" and "Repair RDML File" which might help. If this happens, you should contact the developers of the respective software.
The use of TableShaper
RDML-TableShaper is a tool to shape tables with amplification data for the import with RDML-tools. This is error-prone and second best to original RDML export by the machine. As some machines do not offer RDML export, TableShaper might help. Most qPCR machines export Excel files. Please convert them by opening in Excel (Microsoft) or Calc (Libre Office), selecting the table with the amplification data and save as tab separated text file. The tab separated text file can be modified using TableShaper.
TableShaper follows three consecutive steps:
1. The table separators have to be identified on the first tab. If it does not work out automatically, the separator has to be selected manually. The table columns need to be recognized in the table below.
2. The content need to be recognized in the second tab. If your table format is not found in the predefined settings, you may iteratively select the parameters and load / save them for later use. Adapt the "Number of columns to keep" and "Number of rows to keep" to match the experiments cycle and well numbers and avoid cutoff. "Settings to reshape the table" is the most dramatic shaping option. You can opt to flip the table or to build a table from value list. The result is drawn below and can be used to select the columns for data extraction.
3. Fill the missing content in the third tab. To be able to import the table into RDML, the first six columns have to be filled. Using the fields on the third tab eases this step as squares B2-D7 can be used. Take care in this step as errors in the target id and sample id can not be fixed in RDML later. Save the result as .tsv file.
4. To create an RDML file fill the information on the "Create RDML file" card and hit the button. RDML-Edit will open with the created RDML file and allow to save or view the RDML file.
Alternatively the data can be imported int existing RDML files. Open the RDML file in RDML-Edit (or create new) and enable editing with the "enable edit mode" button. Now change to the experiments tab and open an experiment or create a new experiment. In the experiment, create a new run. Take care to set the ID and PCR-Format fields correctly. Now select the exported .tsv file in "Import Amplification Data". Once saved you can inspect the result using RunView or save it as RDML file on the first tab.