Question: Reciprocal blast commandline
gravatar for baurumon
6 months ago by
baurumon10 wrote:

This might be a stupid question but i am new in bioinformatics.

I have few FASTA files which i want to run reciprocal blast. Can anyone please guide me through makeblastdb command line process.

  1. I have downloaded NCBI blast database and extracted nr FASTA file through blastdbcmd

  2. Should i run makeblastdb command with my FASTA file or nr FASTA to create database?

  3. what will be the next step after creating database?
blast tool • 291 views
ADD COMMENTlink modified 6 months ago by ATpoint29k • written 6 months ago by baurumon10

If you follow the reciprocal blast algorithm, e.g. as described at,then:

1.) Extract a set of genes of interest from an existing database. Generate a blast database for this set. 2.) Generate a blast database for your query sequences.

3.) For each gene in the set generated in 1.): a.) Blast it against your query sequences b.) Take the sequence of the best hit and blast it against the original set of genes of interest. If this is the same as the gene of interest, you have a potential ortholog.

This means, you need to create blast databases for both your genes of interest as well as your query set. Since you do two blast calls for each gene of interest, it does not really make sense to use the whole of NCBI nr as genes of interest. It would be more appropriate to take a set from a related organism or a specific set of genes (e.g. plant R-genes etc.)

ADD REPLYlink written 6 months ago by cschu1812.0k

Thank you for the explanation !!!!

ADD REPLYlink written 6 months ago by baurumon10

i used this command to blast in database blastn -query work.fa -db ~/Blast/ssalar/ssalar -outfmt '6 qseqid sseqid sseq' -out newblast_out -word_size 11 -evalue 1e-5 -num_alignments 1 -num_threads 10

and got output as bellow. As you can see i have several hit for one sequence. how can i filter best hit from this file.

enter image description here

ADD REPLYlink written 6 months ago by baurumon10

this issue is solved. thanks

ADD REPLYlink written 6 months ago by baurumon10

Reciprocal blast is generally between a small set of sequences, not NCBI NR.

ADD REPLYlink written 6 months ago by JC9.5k
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