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Conditional Reciprocal Best BLAST - high confidence ortholog assignment.

What is Conditional Reciprocal Best BLAST?

CRB-BLAST is a novel method for finding orthologs between one set of sequences and another. This is particularly useful in genome and transcriptome annotation.

CRB-BLAST initially performs a standard reciprocal best BLAST. It does this by performing BLAST alignments of query->target and target->query. Reciprocal best BLAST hits are those where the best match for any given query sequence in the query->target alignment is also the best hit of the match in the reverse (target->query) alignment.

Reciprocal best BLAST is a very conservative way to assign orthologs. The main innovation in CRB-BLAST is to learn an appropriate e-value cutoff to apply to each pairwise alignment by taking into account the overall relatedness of the two datasets being compared. This is done by fitting a function to the distribution of alignment e-values over sequence lengths. The function provides the e-value cutoff for a sequence of given length.

CRB-BLAST greatly improves the accuracy of ortholog assignment for de-novo transcriptome assembly (Aubry et al. 2014).

The CRB-BLAST algorithm was designed by Steve Kelly, and this implementation is by Chris Boursnell and Richard Smith-Unna. The original reference implementation from the paper is available for online use at


To install CRB-BLAST, simply use rubygems:

gem install crb-blast


  • NCBI BLAST+ (preferably the latest version) should be installed and in your PATH.
  • Ruby v2.0 or later. If you don't have Ruby, we suggest installing it with RVM.

\curl -sSL | bash -s stable --ruby


CRB-BLAST can be run from the command-line as a standalone program, or used as a library in your own code.

Command-line usage

CRB-BLAST can be run from the command line with:


The options are

    --query, -q <s>:   query fasta file in nucleotide format
   --target, -t <s>:   target fasta file as nucleotide or protein
   --evalue, -e <f>:   e-value cut off for BLAST. Format 1e-5 (default: 1.0e-05)
  --threads, -h <i>:   number of threads to run BLAST with (default: 1)
   --output, -o <s>:   output file as tsv
        --split, -s:   split the fasta files into chunks and run multiple blast
                       jobs and then combine them.
         --help, -l:   Show this message

An example command is:

crb-blast --query assembly.fa --target reference_proteins.fa --threads 8 --output annotation.tsv

Library usage

To include the gem in your code just require 'crb-blast'

A quick example:

blaster ='test/query.fasta', 'test/target.fasta'), 4, true) # to run with an evalue cutoff of 1e-5 and 4 threads

A longer example with each step at a time:

blaster ='test/query.fasta', 'test/target.fasta')
blaster.run_blast(1e-5, 6, true)


The output file for CRB-Blast pulls columns from the blast output.

query - the name of the transcript from the 'query' fasta file
target - the name of the transcript from the 'target' fasta file
id - the percent sequence identity
alnlen - the alignment length
evalue - the blast evalue
bitscore - the blast bitscore
qstart..qend - the coordinates of the alignment in the query from start to end
tstart..tend - the coordinates of the alignment in the target from start to end 
qlen - the length of the query transcript
tlen - the length of the target transcript

Getting help

Please use the issue tracker if you find bugs or have trouble running CRB-BLAST.

Chris Boursnell maintains this software.


This is adademic software - please cite us if you use it in your work.

CRB-BLAST is released under the MIT license.


Conditional Reciprocal Best Blast






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