I want to know the best protocol for annotating protein from a raw sequence to a functional protein. There are many bioinformatic tools to perform this, but which one is best? If you mention the step-wise protocol, it will be helpful.
If you have the raw NGS sequences. Then before annotation you need to assemble them. Number of tools are available, but I will recommend Trinity(http://trinityrnaseq.sourceforge.net/) as it has the integrated pipeline for the annotation.
A simple way to get started is to use a tool such as ORF finder (Open-Reading-Frame) to identify potential proteins in all six reading frames from raw DNA data.
http://www.ncbi.nlm.nih.gov/gorf/gorf.html
Then you can link directly to potential proteins by clicking on identified ORF's, or you can download the results and blast against a consolidated protein database. I usually start with PFAM, because it contains highly conserved proteins across multiple organisms, so I am more likely to get a reliable hit.
Another option is to use the free-ware version of Blast2Go, but this can be very time consuming. After assembly by Trinity (as suggested above), or another algorithm such as Velvet (https://www.ebi.ac.uk/~zerbino/velvet/) you can break it down into smaller pieces and run Blast2Go. For 2 million reads, it took me about 8 days to use Blast2Go.
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