RFDD help pages
Raw data file format
At the end of the experimental part of RFDD technique, you should have done all the PCRs. At this point starts the bioinformatic part by the creation of the files that RFDD website will need to analyse your experimentation. Though it will be the only painstaking part for you, you have to care a special intention.
Manual filtering
First, you will have to filter by yourself the PCRs that seem to be good against ones that might have run worth. It means in most cases that you will have to compare for each trinucleotides and enzymes, the electrophoregrams between the two conditions and expect them to be globally and intuitively similar.
Files format
RFDD website works on PCR files. Each PCR (i.e. each condition / trinucleotide / enzyme) must have only one corresponding file.
To create a file, you need to export the row data of each electrophoregram you filtered to a text file.
To do so, it depends on the capillary electrophoresis software you use and your computer system.
For Genscan, copy the two collumns corresponding to the amplicon size and the fluorescence level and paste them in that order in a spreadsheet file (with Excel on Windows or Mac). Then save it as a plain text file, separated by a tabulation (using "save as" and choosing text for "format" and tabulation for "separation" in Excel). No header (i.e. column names), just values.
It will gives a result similar to that screenshot :
File names
When saving as plain text your PCR row data, you need to rigorously name the files or they will not be taken in account.
The naming rules are as following :
Note that discriminant trinucleotide must be in small caps, each field is separated by an underscore (and nothing else), and the name is terminated by ".txt" . Furthermore, enzyme name must comply to the internationnal standard (see this link ).
For exemple, a PCR done under ACT discriminant trinucleotide, HhaI restriction enzyme and a tumoral condition, will lead to file named :
It may appear laborious because you will need to avoid any mistakes over at most 256 files (i.e. 2 conditions * 2 enzymes * 64 trinucleotides).
But after that, RFDD web site does everything else !
Upload archive
When you have finished the previous steps, let all the file in a folder as on the following screenshot to compress them in a ZIP archive.
To compress you should have to right click on the folder a choose "compress in ...".
Do not include anything else than PCR files in that archive. You can let at most a folder.
Give the ZIP file the name you want.
Go to the "analysis" -> "alignement menu and select the archive on your computer to be uploaded. Then click on "Submit Validation" boutton to next choose your alignment parameters.
Note
We engage in a strict confidenciality on the data you uploaded on the web site. There's a temp folder management system that remove any files that are one day old.
