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HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment


Sequence-based protein function and structure prediction depends crucially on sequence-search sensitivity and accuracy of the resulting sequence alignments. We present an open-source, general-purpose tool that represents both query and database sequences by profile hidden Markov models (HMMs): 'HMM-HMM–based lightning-fast iterative sequence search' (HHblits; Compared to the sequence-search tool PSI-BLAST, HHblits is faster owing to its discretized-profile prefilter, has 50–100% higher sensitivity and generates more accurate alignments.

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Figure 1: Workflow and benchmark comparison.
Figure 2: Structure predictions for Pfam families and the modeling of human Pip49 (also known as FAM69B).

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We acknowledge financial support by the Deutsche Forschungsgemeinschaft (grant SFB646) and by a Gastprofessur grant from Ludwig-Maximilians Universität Munich financed through the Excellence Initiative of the Bundesministerium für Bildung und Forschung.

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Authors and Affiliations



M.R. performed research, J.S. initiated and guided research, A.B. generated the profile-column alphabet, A.H. contributed code for fast file access, and M.R. and J.S. wrote the manuscript.

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Correspondence to Johannes Söding.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 1 and 2 (PDF 3828 kb)

Supplementary Data 1

100 random sequences from the nr database used for run time benchmark. (TXT 55 kb)

Supplementary Data 2

List of query-template pairs for alignment benchmark. (TXT 84 kb)

Supplementary Data 3

3D homology model of PIP49/FAM69B. (TXT 161 kb)

Supplementary Data 4

Training and test set of SCOP domain sequence for sensitivity benchmark. (TXT 174 kb)

Supplementary Data 5

FASTA formatted multiple sequence alignment for human PIP49/FAM69B built by HHblits. (TXT 701 kb)

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Remmert, M., Biegert, A., Hauser, A. et al. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods 9, 173–175 (2012).

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