Benchmarking the next generation of homology inference tools

Bioinformatics. 2016 Sep 1;32(17):2636-41. doi: 10.1093/bioinformatics/btw305. Epub 2016 Jun 1.


Motivation: Over the last decades, vast numbers of sequences were deposited in public databases. Bioinformatics tools allow homology and consequently functional inference for these sequences. New profile-based homology search tools have been introduced, allowing reliable detection of remote homologs, but have not been systematically benchmarked. To provide such a comparison, which can guide bioinformatics workflows, we extend and apply our previously developed benchmark approach to evaluate the 'next generation' of profile-based approaches, including CS-BLAST, HHSEARCH and PHMMER, in comparison with the non-profile based search tools NCBI-BLAST, USEARCH, UBLAST and FASTA.

Method: We generated challenging benchmark datasets based on protein domain architectures within either the PFAM + Clan, SCOP/Superfamily or CATH/Gene3D domain definition schemes. From each dataset, homologous and non-homologous protein pairs were aligned using each tool, and standard performance metrics calculated. We further measured congruence of domain architecture assignments in the three domain databases.

Results: CSBLAST and PHMMER had overall highest accuracy. FASTA, UBLAST and USEARCH showed large trade-offs of accuracy for speed optimization.

Conclusion: Profile methods are superior at inferring remote homologs but the difference in accuracy between methods is relatively small. PHMMER and CSBLAST stand out with the highest accuracy, yet still at a reasonable computational cost. Additionally, we show that less than 0.1% of Swiss-Prot protein pairs considered homologous by one database are considered non-homologous by another, implying that these classifications represent equivalent underlying biological phenomena, differing mostly in coverage and granularity.

Availability and implementation: Benchmark datasets and all scripts are placed at (


Supplementary information: Supplementary data are available at Bioinformatics online.

MeSH terms

  • Benchmarking*
  • Databases, Protein*
  • High-Throughput Nucleotide Sequencing
  • Proteins
  • Sequence Homology*
  • Sequence Homology, Amino Acid


  • Proteins