Query regarding BLASTKOALA (KEGG) for pathway annotation

Question

[sharmatina189059]

Dear all I have 3652 gene's ids as well as corresponding protein ids of a bacterial genome. I need to annotate these sequences in terms of their pathway. I have used BLASTKOALA , KASS, DAVID and geneSCF for tis purpose, but the problem is only 50% sequences are annotated using KASS and BLASTKOALA. As the 50% annotation is of no use, is there any other tool by which I can annotate my protein sequences for their pathway analysis.

Why BLASTKOALA assigns KO numbers to few genes? is there any parameters we need to set?Did I do any mistake ? Is there any parameters we need to set while implementing BLASTKOALA? I can not go ahead with the 50% pathway annotation. kindly tell me other methods or tool for this purpose.

[Tm]

50% annotation of genes against pathway database is very much common and expected result as many genes and their role in different pathways is still not elucidated.

Answer 1

[natasha.sernova]

You used the following site, am I right?

http://www.kegg.jp/kegg/tool/annotate_sequence.html

This site has its own limits, 2500 genes/sequences.

"Query sequences – Upload a file containing multiple amino acid sequences represented by the FASTA format. The maximum number of sequences is 2500."

You gave it 3652 sequences - it's too many.

You had the following statement at the bottom of the first BlastKOALA page:

"If you have a larger dataset of query sequences, consider splitting the dataset and concatenating the lists of K number assignments that can be downloaded from the result page. The BlastKOALA server accessed from its main page allows you to compare against all KEGG organisms as well as to use a better scoring scheme incorporating Pfam domains. "

[mittu1602]

Yes, @natasha you are correct. I have been working on BlastKOALA for a very long time now. It is tiring but fetched good result. You need to split files and make small chunks @sharmatina189059