Format of Roche LightCycler output files *.ABT / *.CCC for colour compensation
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7.4 years ago
me • 0

I am automating a workflow for analysis of real-time PCR data sourced from a Roche LightCycler. There are multiple channels for sensing different fluorophores that have a slightly overlapping fluorescence spectrum as detailed here (referred to as "crosstalk"). The detected signals are thus a linear weighting of the true fluorescence, as detailed in Table 1 / Formula 1 here. This is corrected with colour compensation as detailed in the first link.

There is very little (i.e. no) information regarding the format of the older LightCycler file formats ABT (details a particular run) and CCC (details the colour compensation), and I was hoping someone could please provide guidance.

In the ABT file there is a section ^CCC (all section headers appear to start with ^) which includes the location of the colour compensation file and its contents (a 12x4 matrix of numbers that I am trying to make sense of). In my case they appear as (with file paths changed):

1   1   1
-1.33786041651041E-8    3.39600416861281E-6 -0.000392286209994558   0.196738637483626
-6.43980805238762E-9    1.50246180493155E-6 -0.000107523827209977   0.112676603581708
-2.23359269130001E-8    4.9982365413899E-6  -0.000398780255441468   0.0600920934768699
1.55891872931679E-5 -0.00505090913467267    0.162405918150517   34.8790113064609
2.27218464185121E-8 -0.000146878157270101   0.0123033869019081  1.42038161205974
-1.42030623826416E-7    1.40575424253722E-6 0.0012288371257375  0.290852973511342
2.1866259759953E-9  9.8947181762574E-7  -0.00031255171342293    0.199654709634488
-1.40190153175352E-5    0.00289266217140399 -0.188211203807182  5.67986350485893
-4.39402518213953E-6    0.000909318177116326    -0.0584074241424017 1.73152377696672
7.01531463682071E-7 -0.000117467670542772   0.0039676094304943  0.282456463232278
1.37437444939199E-7 -2.4255075537722E-5 0.000917545927808637    0.132638408109113
1.31314492531558E-5 -0.00255990120176339    0.143275086483057   -1.36050870155249

What do these numbers describe?

EDIT: Since originally posting, I accessed a colour compensation file and found that it is identical to the numbers in the ABT.

qPCR crosstalk • 1.9k views

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