In one paper I read that BlastX was used to remove protein encoding sequences. But didn't really understood how I should proceed.
BLASTX takes your DNA query sequence, translates it into all possible coding frames, and then does a BLAST alignment search to a protein database. You then use the homologous hits from the database search to identify which of your query sequences are likely to come from protein coding regions (and which protein they likely code for).
It is dependent on what data you have. If you are working with NGS data (Contigs / scaffolds) I would recommend using Gene Mark, really fast in comparison to any BLAST analysis you would do. Gene Mark will produce the predicted gene and protein sequences, but it wont tell you what they are (i.e Functionality).
Thank u Michael, Daniel and Joe sir for ur kind suggestions. But, actually m looking for miRNA identification from EST (expressed sequence tags) sequences. So I need to remove the protein encoding sequences. I'll be grateful to u if u can provide me a detail methodology in this regard.
Dear Sunil,
I would like to let you know that there are so many ways you predict miRNa from ESTs . In this regard first you can pre-process your ESTs to get contigs and singleton sequence from ESTs. These sequence will be treated as query for doing BLAST search against known miRNA from MiRBase database. So you need to configure BLAST locally in to a local machine In which CentOS may be installed. After doin BLAST search you can get to know hata re the contigs and singleton sequences are having your pre miRNAs. Then only you need BLASTx of your sequence to remove the proteins coding ESTs(singleton or contigs). After screening the pre-miRNAs check the upstream of 100 from the matched region of your query to look for the matured miRNA based on MEFE and energy (- del G) and the number of miscmatch with the known miRNAs of miRBase.
For better understanding you can can use zhang et al., 2006 paper as you reference.
All d very best and best of luck.
Thank u budheswar Sir for your ideas and suggestions. I have also read some of your article published in bioinformation journal. As u mentioned above I have started following the method described by zhang et al. Since I am a beginner in the field of bioinformatics I am not able to follow the method step by step. Therefore I request you to kindly provide me a step by step methodology in detail so that I can proceed with my sequences. I am not able to follow the methods described in published papers since they are too brief for me to understand.
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