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Extracting sequences from tBlastn results(BLAST standalone)

I am using tblastn to search protein queries against custom eukaryotic genome assemblies that lack protein annotations. One suggestion was to parse the hit coordinates and extract the matching genomic regions. Does the blast toolkit make this possible? Does it also make sense to translate the extracted sequences from the genome just so I can do a reciprocal blast to check whether these sequences are homolog candidates?

Would it be more appropriate to first generate a eukaryotic protein annotation for these genomes and then use blastp instead, or is tblastn the preferred approach in this situation?

Thanks in advance for any input. Not sure if this makes sense.

tblastn

Are you trying to find similar sequences in genome assemblies you are testing against? In general doing searches in protein space is always preferable when looking for similar sequences.

One suggestion was to parse the hit coordinates and extract the matching genomic regions. Does the blast toolkit make this possible?

As for extracting tblastn hit sequences, you will need to do that using blasdtdbcmd after parsing the hit coordinates using the blast database of the assemblies.

yeah, I was trying to find similar sequences in the custom database of genomes assemblies (makeblastdb). Only few of them has the faa/gff

As for extracting tblastn hit sequences, you will need to do that using blasdtdbcmd after parsing the hit coordinates using the blast database of the assemblies.

Thank you for this!!

1 answer

On the extraction you can skip the coordinate parsing for most purposes - adding sseq to your -outfmt 6 column list hands you the aligned subject sequence directly. blastdbcmd is still what you want if you need flanking sequence rather than just the aligned block.

The translate-and-reciprocal-blast plan is where I'd be careful. These are eukaryotic genomes, so your tblastn hits are exon fragments: introns break the alignment and one protein query gives you several HSPs strung along the locus. Translating the extracted genomic span translates the introns along with it, so the thing you'd be reciprocal-blasting isn't really a protein.

For that you want a spliced protein-to-genome aligner rather than BLAST. miniprot is the easy modern option, exonerate protein2genome the older one. Either turns your protein query into an actual gene model on the assembly, and that's what's worth translating and checking reciprocally. Also answers your last question - annotating the whole genome first is overkill if you only care about one protein family.

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