Hello,
limma's lmFit expects log-transformed expression values as input, and
the returned "log fold-change" is then about the difference between
the
mean expression levels in each group, which given your input data may
very well be 5000 but this is not the proper log fold change at all. I
don't know what kind of data preprocessing you performed but you may
want to make sure that you feed log-transformed data into lmFit.
Regards,
Joern
Ingrid H. G. ?stensen wrote:
> Hi
>
> 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?
>
> Regards,
> Ingrid
>
>
Quoting "Ingrid H. G. ?stensen" <ingrid.h.g.ostensen at="" rr-="" research.no="">:
> Hi
>
> 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?
>
> Regards,
> Ingrid
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.
Jose
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