I'm thinking to use BLASTX to find CDS (protein coding sequence) regions on genomes. How can I set the parameters to let BLASTX put big penalty on a stop codon found? Any experience or any useful link? Thanks.
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Originally posted by rdu View PostI'm thinking to use BLASTX to find CDS (protein coding sequence) regions on genomes. How can I set the parameters to let BLASTX put big penalty on a stop codon found? Any experience or any useful link? Thanks.
Use Scipio or Exonerate - this is exactly what they're designed for.
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Originally posted by tonybolger View PostIn short - Don't
Use Scipio or Exonerate - this is exactly what they're designed for.
Eukaryotic genomes:
Scipio is used for the retrieval of the genome sequence corresponding to a protein query. The tool does not require any annotation data, and is able to correctly identify the gene even if this is spread on several genome contigs and contains mismatches and frameshifts. Because of its post-processing capabilities, Scipio is not only able to correctly identify the gene in the genome corresponding to the protein query but also to correctly identify the homologous genes in the genomes of closely related organisms.
Prokaryotic genomes:
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Originally posted by robs View PostThe problem is that there is not enough information in the question. If he/she is interested in prokaryotic genomes, then Scipio would not be very useful.
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Originally posted by robs View PostThe problem is that there is not enough information in the question. If he/she is interested in prokaryotic genomes, then Scipio would not be very useful.
Eukaryotic genomes:
Scipio is used for the retrieval of the genome sequence corresponding to a protein query. The tool does not require any annotation data, and is able to correctly identify the gene even if this is spread on several genome contigs and contains mismatches and frameshifts. Because of its post-processing capabilities, Scipio is not only able to correctly identify the gene in the genome corresponding to the protein query but also to correctly identify the homologous genes in the genomes of closely related organisms.
Prokaryotic genomes:
http://rast.nmpdr.org/
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Sorry, I didnt get a chance to back here.
Actually, I'm doing this. I downloaded 20 different bacteria genomes from NCBI website. These genome gene annotation files are ready there. Then I cut each genomic DNA sequence to short read length of pieces (~100nt each) to simulate the ngs reads. I was intending to use BLASTX and protein COG database to annotate these short reads to check how the short reads inherit the function annotation. Let me know if it's still not clear.
Thanks for all your suggestions. But I just want to stick to one of NCBI provided tools.
Happy new year to everyone.
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I carried out something similar with 454 reads from a non-model specie, so I had to be aware to point mutations in stretches of homopolymers that could change the frame translation pattern giving rise a false stop codon. My blast parameters were not so restrictive as I think you want. I used a e-value threshold of 0.001 over repetmasked reads using BLASTN (BLASTX was really unsuccesful).
hope this helps and happy new year!
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