Multiple sequence alignment (MSA) remains the most important analytic tool to assess evolutionary relations between proteins and to determine the conserved regions of the sequence that usually harbor structural and functional properties. However, the algorithms that produce MSAs are not perfect and further manual curation carried through by visual inspection is necessary . Inspection is also required to detect or confirm some features of the aligned proteins, such as the presence of a structural domain. Notwithstanding the availability of excellent MSA visualization tools , the growth of the protein database is making difficult these tasks, as well as the calculation of the alignment itself (e.g. Uniref100 , which currently contains over 23 million sequences, has nearly doubled every two years since 2008). Removing highly redundant sequences in a set of homologs helps MSA and its interpretation as well as reducing biases in the protein set when it is taken as a sample for statistical analysis or for machine learning applications. In a previous work, we conceived an algorithm to cluster the whole protein database by aggregating near-full length homologs . Instead of clustering sequences based just on high sequence identity, our approach allows low but significant levels of identity according to BLAST  and is very restrictive in requiring this similarity to be full length without large unmatched portions between the compared sequences. This is to ensure that when two sequences are aggregated they have the same domain structure, which increases the likelihood of both having a similar function . We have implemented this algorithm in a web tool to quickly organize and/or trim a set of proteins in FASTA format.
Given a set of proteins, we use a greedy algorithm to group similar sequences together. First, we pick the longest sequence as the query sequence and we compare it with BLAST to the subset of sequences that either have the same length as the query or are shorter by less than n amino acids (aa). We will retain BLAST matches covering the full length of the query or shorter by less than n / 2 aa from each end. The value of n depends on the length of the query sequence, q, and is by default equal to 32, if q ≥ 200; 20, if 200 > q ≥ 100; 10, if 100 > q ≥ 60; 7, if 60 > q ≥ 40; 5, if 40 > q ≥ 20, and 2 if q < 20. Sequences are considered to match the query sequence if they are sufficiently similar to the query as detected by a BLAST single hit (either continuous or gapped) that covers all (or almost all) of the protein. An identity threshold of 24.8% was used for BLAST hits if q ≥ 80, else a function of q equal to 290.15 ⋅ q − 0.562 for shorter hits, following Sander and Schneider . Query and matched sequences are aggregated and removed from the set and the longest sequence among the remaining ones is chosen as a new query. This procedure is repeated until no sequences remain.
To validate the reduction in number of sequences in FASTA files by our method, we used the OrthoBench, a published benchmark set of manually curated protein families that was designed to assess orthologs’ detection methods  and is available at http://eggnog.embl.de/orthobench/. The OrthoBench consists of 7,981 proteins grouped into 100 protein families classified according to the rate of evolution, domain architecture, low complexity/repeats, lineage-specific loss/duplication, horizontal gene transfer, or alignment quality. We excluded family RefOG065 because among its 19 members 15 of them are also found in RefOG002, and we worked with 7,962 unique sequences in 99 groups (Supplementary Tables 1 and 2). We seeked to evaluate how our algorithm avoids clustering together sequences from different families despite allowing very low sequence identity. For that, we noted the level of reduction of the number of sequences and the number of errors (when sequences belonging to different families are clustered together) as performance indicators. The overall method performance with the default parameters was good, reducing the number of sequences down to 14.41% of the original FASTA size with 60 misclassifications that affect all the same cluster. They are all members of group RefOG086, Membrane GTPase involved in stress response (87 sequences), clustering with a member of the RefOG035 family, ATP-binding cassette (318 sequences). For detailed compression values on individual families, see Supplementary Table 3.
Our default parameters are conservative. We tested how different thresholds of tolerance of length differences between sequences allowed to cluster together would affect performance. Results indicate that moderately increasing tolerance increases the reduction in number of sequences without largely affecting the number of clusters affected and misclassifications (Table 1). Some proteins contain low-complexity regions (LCRs), regions with repetitive sequences and little diversity in their amino acid composition. Hypothesizing that the length of LCRs will be extremely variable within protein families and might cause misclassification of orthologs in different clusters, we tested how detecting and masking these from the original sequences would affect the results. For conservative parameters of LCR detection, our algorithm achieved a similar reduction in the number of sequences without increasing the number of errors (Table 1). As this is heavily dependent on particular protein sets, we decided to provide LCR detection and filtering as a user option. The proportion of misclassifications are only indicative because in the OrthoBench, proteins from different families can be highly divergent, and therefore the number of mistakes could be expected to be smaller than in real-life applications. Indeed the rate of compression for individual families depends obviously of the degree of evolutionary divergence of the sequences, especially when that would involve large changes in protein length or domain shuffling (Table 2). We compared FASTA Herder to two other published methods that use similar greedy clustering strategies, CD-HIT  and kClust . Both methods are very fast and geared toward the compression of large databases. We calculated the compression rates and errors at the lowish identity thresholds that FASTA Herder uses for the same benchmark set. Without restrictions to the coverage, as the minimum of the percentage of the aligned length for the longest sequence, both tools produced very compressed results but usually more errors (Table 3). Limiting the admissible clusters to high levels of coverage produces comparable results for the three tools (Table 3). Another previously published web tool that possess a capability similar to this work is PISCES , a protein database that culls the Protein Data Bank to build the largest possible set of structures that comply with identity cut-offs for sequence and structure. More related to our tool, PISCES accepts as well a FASTA file and culls out sequences by a single identity threshold that can be provided by the user. However PISCES compares all to all sequences, it is much slower and accepts smaller inputs than the other tools.
|Tolerance||Compression (%)||Affected clusters||Misclassifications|
|n||FH||FH with LCR||FH||FH with LCR||FH||FH with LCR|
Tolerance n: Additional number of amino acids permitted over the default values (n = 0 corresponds to the default parameters). Compression (%): number of clusters as percentage of the original FASTA file. FH: without using SEG, FH with LCR: using SEG to detect LCRs with parameters (W = 5 and K1 = 1.0). Affected clusters: number of clusters with misclassified sequences. Misclassifications: total number of misclassified sequences.
|Class||Families||Compression (%)||Similarity range (%)|
|Alignment quality (high)||3||35.88||34.2||99.8|
|Alignment quality (low)||8||37.46||27.6||99.8|
|Horizontal gene transfer||6||6.49||36.1||100|
|Speed of evolution (high)||8||59.11||28.6||88.6|
|Speed of evolution (low)||1||7.69||88.2||100|
Class: class name. Families: number of families in class. Compression (%): average of compression for class families. Similarity range (%): Averages of min and max similarity values for class families.
|Identity (%)||Coverage (%)||Compression (%)||Affected clusters||Misclassifications|
Identity (%): sequence identity thresholds. Coverage (%): Alignment coverage of the leader sequence. Compression (%): percentage of the original size. Affected clusters: number of clusters with misclassified sequences.
Misclassifications: total number of misclassified sequences.
FASTA Herder takes a protein set in FASTA file format and quickly clusters it (e.g. the OrthoBench is clustered in under 2 minutes). Optionally, LCRs, as detected with SEG , can be ignored before clustering. The server also permits to adjust the stringency of the clustering based on the allowed difference in length between sequences to be clustered together, although it may be important to stick to conservative levels when culling sequences from a MSA. We use the BLAST implementation of the BLAST+ suite .
We have implemented an ease-of-use web application to quickly reduce the redundancy of a protein set by clustering homologs of comparable lengths. Although our work is somewhat related to the field of orthology prediction methods (discussed in Ref ), its purpose and scope are different. Orthology detection encompasses the use of whole genomes to identify proteins derived from a single ancestral sequence through speciation events. The aim of our tool is to reduce potentially high redundancy in a FASTA file that may contain proteins belonging to one or more families. This would speed MSA calculation, facilitate MSA inspection, and remove biases that may affect any statistical analysis or computational application involving that set of proteins. In conclusion, we have created a tool to remove redundant sequences from sets of sequences. Simplification of MSAs was our main goal, motivated by the increase in redundant sequences in the databases due to genomic sequencing projects. Our method is simple, works in a matter of seconds with large datasets that are unmanageable by other methods, and includes a number of options (LCR filtering, length thresholds) that make it very flexible.