Megan SEED/KEGG question

[msteffe1]

Hello.
I'm working with 454 titanium reads for metagenome data.
I've worked with Megan briefly, and it's been great for taxonomic comparison. Since the introduction of version 4, I have had trouble with the functional analysis tools (SEED and KEGG). I've followed the instruction in the manual (blastx, nr or refseq databases) to generate the .rma files. I've tried multiple times, and each time my KEGG and SEED results come back as all hits "not assigned." Is there something I am missing, or are none of my reads really hitting to the databases?

Thanks

[Chuckytah]

Hello.
I'm working with 454 titanium reads for metagenome data.
I've worked with Megan briefly, and it's been great for taxonomic comparison. Since the introduction of version 4, I have had trouble with the functional analysis tools (SEED and KEGG). I've followed the instruction in the manual (blastx, nr or refseq databases) to generate the .rma files. I've tried multiple times, and each time my KEGG and SEED results come back as all hits "not assigned." Is there something I am missing, or are none of my reads really hitting to the databases?

Thanks

Did you have sucess? I'm with almost the same problem

[fznajar]

I am having the exact same problem. I am not sure if SEED option is even highlighted on MEGAN menu. Anybody got success so far?

[DevS]

Why is this happening? I am in the same situation

[DevS]

I am having the exact same problem. I am not sure if SEED option is even highlighted on MEGAN menu. Anybody got success so far?

Hi fznajar,

What database did you blast your sequence against?

[pmb0010]

Hi all!

I am having a similar problem with the SEED/KEGG functional analysis tools. I have uploaded my sequences (blastx using the NCBI nr database) and then am unable to perform any SEED/KEGG analyses (they are just gray). I got it to work once with another file when I first started using MEGAN (a couple weeks ago) but now even that file I am unable to click on the SEED or KEGG Analyzer. Is anyone else having this problem? I know my file is in the correct format (pairwise) and am getting the phylogenetic tree on the first page, just cannot figure out why I am unable to select the SEED/KEGG analyzers.

[pmb0010]

Hi all-

Just found a solution to my above problem that I do not know if it will be helpful to any of you. So when import your blast file (go to file then select import from blast) a box pops with options for import, content, files, LCA Params, and Advance. Under the import tab you can select the file you want to import and the file you want it to save as if you go under the CONTENT tab make sure that the Analyse SEED content and Analyse KEGG content boxes are checked. For some reason mine was not. I do not know if this is the default, but mine were not checked. Once I checked them both the SEED and KEGG options were no longer gray. Hope this might solve some others issues as well.

[Polecat]

I have been having this problem for ages too and am starting to bang my head on the wall.

I can use the SEED and Kegg viewers when I load an example file from the MEGAN site.
I can see everything beautifully for the taxonomic information when I do blastn. I don't understand why though, when I do blast x against the nr database I still can't see my hits.

I was having the grey button problem pmb0010 mentioned and when I follow his instructions I could miraculously see these buttons on my tool bar.

I still get the same problem though. In my test file it might say 49% not assigned, 52% no hits. The no hits is correct, but it is not reading the assigned hits. Any ideas of other things to try?

Here is an example of the blastx structure of the output file:

15 Oct 2012 blastx-2.2.25+ 1000C0VVYACXX_AV1M_CTGTGT_L006_R1_001-trimmed.fasta.1.fasta /blastdb/nr -evalue 0.02 -num_descriptions 1 -num_alignments 1 -num_threads 12
node = --------

BLASTX 2.2.25+


Reference: Stephen F. Altschul, Thomas L. Madden, Alejandro A.
Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.
Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs", Nucleic Acids Res. 25:3389-3402.



Database: All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF
excluding environmental samples from WGS projects
18,538,055 sequences; 6,354,234,804 total letters

Query= HWI-ST1047:86:C0VVYACXX:6:1101:4502:2240 1:N:0:CTGTGT

Length=101
Score E
Sequences producing significant alignments: (Bits) Value

ref|ZP_04532936.1| LOW QUALITY PROTEIN: cell wall-associated hyd... 62.0 4e-08


>ref|ZP_04532936.1| LOW QUALITY PROTEIN: cell wall-associated hydrolase [Escherichia
sp. 3_2_53FAA]
gb|EEH89628.1| LOW QUALITY PROTEIN: cell wall-associated hydrolase [Escherichia
sp. 3_2_53FAA]
Length=156

Score = 62.0 bits (149), Expect = 4e-08
Identities = 29/33 (88%), Positives = 29/33 (88%), Gaps = 0/33 (0%)
Frame = -1

Query 101 PRVHCIFTAISISLSLCWRQRRHHYAIRAGRNL 3
PRVHCIFTA SISLSL WRQ HHYAIRAGRNL
Sbjct 91 PRVHCIFTASSISLSLGWRQPGHHYAIRAGRNL 123



Lambda K H
0.318 0.134 0.401

Gapped
Lambda K H
0.267 0.0410 0.140

Effective search space used: 155148259100

[chevrm]

I am experiencing the same issue as Polecat, also with blastx. Any ideas?

[fznajar]

Sorry for the late reply. I used nr

[NathaJoli]

KEGG in MEGAN5

Hi everyone,

I am a PhD student working on some metagenome data. I've just begin to work with MEGAN, and I have some basics in bioinformatic.

I was wondering if someone could be able to help me on a specific question. I try to use KEGG in MEGAN5. For that you need to give a mapping file from the GI numbers included in the BLAST results to KO numbers. Without this file, the "KEGG button " stays in grey.
I try to give the file which is mentionned on this webpage: http://ab.inf.uni-tuebingen.de/data/...d/welcome.html
But it never works.

I guess I am probably doing something wrong when I am giving this file to the "import from BLAST" window. Or this file is not correct and I need to find an other one.

Is someone can help me?
Thanks a lot

Nathalie

[Polecat]

here we go again..

I know now my issue was not having enough blast hits to let the LCA algorithm work.