[BioC] Limma: Very high logFC values
J.delasHeras at ed.ac.uk
J.delasHeras at ed.ac.uk
Wed Sep 5 17:24:25 CEST 2007
Quoting "Ingrid H. G. Østensen" <Ingrid.H.G.Ostensen at rr-research.no>:
> I am using limma to analyze Illumina expression data (two groups),
> and this time I got some really high logFC values for some genes and
> "low" for others. Example:
> Probe log2 Ratio(logFC) Moderated t-statistic (t) Raw
> p-value Adjusted p-value B
> ILMN_27575 5443.972 27.305 1.81E-06
> 0.009621899 -2.29002
> ILMN_14823 42.5 19.077
> 1.00E-05 0.022251754 -2.32116
> The first gene has intensities in one group (3 samples) around 10
> 000 and in the other group (3 samples) around 5000, and the second
> gene has intensities around 110 in the first group and around 80 in
> the second group.
> I have never seen so high logFC values before, are they realistic?
> Does this values mean that there are big differences combined with
> hight intensities?
I've never seen log2 ratios that high. In fact, I don't thing those
are log ratios at all. They *could* be fold-changes, 'though.
I am not familiar with Illumina, but look at the possible ranges:
If the scanned images are 16-bit, it means you can have raw
intensities measuring anything between 1 to 2^16 (65536). The most
extreme ratio possible would then be 65536/1 (essentially saturated in
one channel and no signal on the other). The log2(65536) is 16.
The only way to get a logFC higher than 16 is if you scan at higher
bit-depths... but the log2FC is still going to be limited by the bit
depth in teh same way: 20-bit images can give you a max logFC of 20.
I don't think you're scanning with >5000-bit resolution.
Maybe the Illumina platform processes the data in some way to expand
the dynamic range, which could then give rise to larger logFCs. But I
am not familiar with Illumina. Is that possible?
Algorithms to combine scans at different PMTs also result in an
expanded dynamic range. Are you using such algorithm? Even in that
case, I can't imagine teh expansion will be large enough to allow a
logFC of >5000.
Fold-changes of 5000 can occur, however, from standard quantitation
from 16-bit images... it would come from a probe/spot with nice signal
on one channel and negligible on the other, after background correction.
So, to cut it short... I'd really make sure I am feeding Limma the
right type of data, as the numbers look almost impossibly large. I'm
pretty sure Illumina scans are 16-bit.
Dr. Jose I. de las Heras Email: J.delasHeras at ed.ac.uk
The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131 6513374
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