Dear List, Martin and Peter,

I'm new to R and Bioconductor, so I was unaware of the fact tileHMM is not a
Bioconductor package... Thanks for pointing it out and sorry :/

Given that Peter answered here, I'll keep on discussing it on the list, this
may interest other people :)

2010/9/23 Peter Humburg <peter.humburg@gmail.com>

> [...]
> So there seems to be a problem with the data.frame containing the probe
> positions. The pos column should contain the start position of the probe
> in the genome.


It does, at least in the .pos file. Here is how it looks like:

 SEQ_ID  CHROMOSOME      PROBE_ID        POSITION        LENGTH  COUNT
NC_000913       NC_000913       ECOLIP1 1       50      1
NC_000913       NC_000913       ECOLIP25        25      50      1
NC_000913       NC_000913       ECOLIP49        49      50      1
NC_000913       NC_000913       ECOLIP73        73      50      1
NC_000913       NC_000913       ECOLIP97        97      50      1


It looks like the number in the probe name may actually
> the position, so that should be fine. But note that there are duplicates
> that you will need to deal with. Looking at the probe sequences these
> look like forward and reverse strand probes.


One single probe is described as FORWARD and REVERSE in the ndf file, but
the pos file counts it once. At the end, the layout object contains both of
the probes.


TileHMM expects a single
> probe per start position. If you are not looking for strand specific
> information you probably should summarize the two probe intensities in
> an appropriate way.
>

When you say "summarize the probe intensities in an appropriate way", you
mean only keep the probe IDs where the probes showing a meaningful signal
after normalization or something like this?

The thing is that I'm searching for binding events wherever they happen, on
whatever strain they happen...

>
>
> > By the way, I have nowhere a correspondence between the
> > probe ID and the gene it matches. So, somehow, blasting all of the probes
> > against the genome seems a bit tedious and not really optimal...
>
> If you have the probe location in the genome there is no need for
> blasting. You can just get annotations for that position (eg with
> biomaRt).
>

Yes, I already did this before. The remark was because of other arrays I was
dealing with before where all this information was in the description file.
But it is out of scope here, sorry for mentionning it.

>
> >
> > Second, when I was looking further in the gff, there are things such as
> the
> > example of the region 90 I pasted above. Here, the region is very big. I
> > checked the probes and so, the start position 101361 corresponds to the
> > probe ID 101361 which lays in the interval 101361-101410 as listed in the
> > ndf file. Moreover, the end position 1013391 corresponds to a probe ID
> > 1013391 which covers the interval 1013391-1013440 according to the ndf
> file.
> >
> > I'm really confused what to think about this stuff and would be very
> > grateful in case someone could explain me how I'm supposed to read this
> gff.
>
> I take it that is much longer than you are expecting for the protein you
> are studying. Could you confirm that the probes are sorted properly?
>

I think so:
R>
head(layout)

       probe.id                                           sequence   x    y
1       ECOLIP1 TTTTAATCCACACAGAGACATATTGCCCGTTGCAGTCAGAATGAAAAGCT 437
1023
2       ECOLIP1 AGCTTTTCATTCTGACTGCAACGGGCAATATGTCTCTGTGTGGATTAAAA 580  820
3 ECOLIP1000016 GTTGATCCGTATGCCAGTAAGTTTGCTGGCTACCACTTAAATAAAACGAA 296  920
4 ECOLIP1000016 TTCGTTTTATTTAAGTGGTAGCCAGCAAACTTACTGGCATACGGATCAAC 711  919
5 ECOLIP1000040 GCAAACTTACTGGCATACGGATCAACAGGATCGGCTATTACAGTTTGGCT 478  918
6 ECOLIP1000040 AGCCAAACTGTAATAGCCGATCCTGTTGATCCGTATGCCAGTAAGTTTGC 106  716
        chr     pos length
1 NC_000913       1     50
2 NC_000913       1     50
3 NC_000913 1000016     50
4 NC_000913 1000016     50
5 NC_000913 1000040     50
6 NC_000913 1000040     50

Similar sorting fashion in the object containing the signals.



> You'll also want to check how many probes there are between the
> beginning and the end of that region. If there are unusually large gaps
> in a region of the genome you want to split the probe sequence to
> exclude these gaps.
>

I used a fragment size of 500 and a max.gap of 300 (the sonication produces
fragments of at about 500 bp). I'll check the number of probes of a given
region showing this extension as I exampled previously.

Thanks for your help :)

>
> Best wishes,
> Peter
>

Best,
Rayna

-- 
"Change l'ordre du monde plutôt que tes désirs."

Mon blog perso/My personal blog : http://hatewasabi.wordpress.com/
Relectrice LinuxFr.org
(http://linuxfr.org/~Malicia/<http://linuxfr.org/%7EMalicia/>
)

PhD Student
"Molecular Evolution and Bioinformatics"
Ludwig-Maximilians University (LMU) of Munich

	[[alternative HTML version deleted]]

