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Input Formats for Differential Scanning Fluorimetry (T_m) Data

The tm_calc.pl script which this site runs can use text files with data for temperature and fluorescence intensity if:

• Columns (fields) are consistently separated by a space, tab, comma, or other punctuation, and rows are separated by an end of line character recognized by Perl
• There is a header line in the first 25 lines of the file containing:
  • a field starting "Tem", e.g. "Temp", "Temp." or "Temperature"
  • fields with sample names, either simple strings e.g. A01 to H12 or a quoted string containing the separator character e.g.
    "50 mM HEPES pH 7.5, 100 mM NaCl, 10% w/v glycerol"
• The header line is immediately followed by data lines containing values for temperature and for fluorescence intensity of each sample, in the same columns as their labels in the header line.

For example, this snippet uses commas as delimiters:

---

...
Cycle,Temp,A01,A02,A03,...
1,20,0.032,0.231,0.003,...
2,21,0.043,0.203,0.005,...
3,22,0.024,0.215,0.013,...
...
71,90,1.34,0.56,0.043,...

...

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The .csv files exported from Opticon Monitor 3 software are also in FIT format; see e.g. the sample input file.

Opticon Monitor binary files

Binary data files (extension .tad or .tad2) can be converted to .csv files either by exporting them via Opticon Monitor 3 software, or by downloading and running the perl script tad2csv.pl

SGC Transform Spreadsheets

Files from six different RT-PCR machines can be converted to a common format by pasting their exported .csv files into the appropriate Excel transform spreadsheet developed by the Structural Genomics Consortium (SGC) . Data in this common format can then be copied and pasted into SGC's analysis spreadsheet to fit a single transition using Graphpad, and using one data point for each degree Celsius.

Or, data in the common SGC format can be exported from the transform spreadsheet to a .csv file and used by this server or by the tm_calc.pl script to fit multiple transitions using all available data. Sample names and Well positions are not preserved in this process, but sample numbers remain consistent.

Transform spreadsheets are available for data in the format produced by the following machines: ABI 7300, ABI 7900HT, ABI StepOnePlus, Agilent Mx3005p, BioRad IQ5 , and BioRad MiniOpticon

SGC invites requests to develop transforms for other formats. Sample files and an instruction manual are also available from SGC.

Basic steps for SGC Transforms

1. Export the data from the machine's software as a .csv file
2. Open that .csv file in Excel or another speadsheet viewer
3. Select the temperature and fluorescence data from the original .csv
4. Copy the data e.g. with "Edit - Copy"
5. Open the appropriate transform spreadsheet
6. Paste the data with "Edit - Paste" onto the first sheet of the transform
7. Go to the second sheet of the transform, "Output",
8. Save the "Output" sheet as .csv:
   • "File - Save As",
   • select comma-separated values (.csv) as file type,
   • use a variant of the original file name e.g. save the transformed sample_name.csv as sample_name.trf.csv.

The new file, e.g. sample_name.trf.csv, can now be used by tm_calc.pl or by this server.

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