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with OPTICS

P. Usha Madhuri and Dr.S.P.Rajagopalan

Abstract— Protein sequence clustering has been widely used as a part of the analysis of protein structure and function. In most cases

single link or graph-based clustering algorithms have been applied. Clustering of proteins using SEQOPTICS (sequence clustering with

OPTICS), which is based on OPTICS (Ordering Points To Identify the Clustering Structure),is an attractive approach due to its emphasis

on visualization of results and support for interactive work such as choosing parameters. Ordering Points To Identify the Clustering

Structure can be implemented for Visualization of the sequence clustering structure. The system was evaluated by comparison with other

existing methods.

—————————— ——————————

1. INTRODUCTION

contain most protein sequences and are very popular in

XTRACTING useful information from biological biological research.

biological sequences databases. Among

biological sequences, protein sequences are an

especially interesting category since protein is

As more and more protein sequences become

available, we can better study protein structure and

function. One of the most important computational

methods is sequence clustering [5, 6]. The goal of

functionally essential in life and its alphabet is large clustering protein sequences is to get a biologically

(20 amino acids). There are several well-known protein meaningful partitioning. Clustering a large set of

databases: Pfam[1] is a collection of protein families protein sequences offers several advantages: Proteins

and domains which contains multiple protein are usually grouped into families based on the sequence

alignments of these families; National Center for similarity clustering, which provides some clues about

Biotechnology Information(NCBI)[2] protein sequence the general features of that family and evolutionary

database is an integrated, text-based search and evidence of proteins; Clustering also helps to infer the

retrieval system that is very often used in biological biological function of a new sequence by its similarity

research; Swiss-Prot[3] is a protein sequence database to some function-known sequences; Moreover, protein

which strives to provide a high level of annotation, a clustering can be used to facilitate protein 3-

minimal level of redundancy and high level of dimensional structure discovery, which is very

integration with other databases; The Protein important for understanding protein’s function.

Information Resource(PIR)[4] serves as an integrated Recently developed clustering methods have been

public resource of functional annotation of protein successful in clustering a large number of sequences

data to support genomic/proteomic research and simultaneously. ProClust[7] used a graph based

scientific discovery. These databases are often used as approach and consider multi-domain sequences.

data source for protein sequence clustering study. In SYSTER[8] overcomes the problem of an asymmetric

this paper we use two data sets from Pfam since it is a distance matrix by computing a local pairwise

semi-automatic protein family database, which aims to alignment after performing a BLAST[9] search.

be comprehensive as well as accurate. Swiss- Prot and GeneRage[10] is a fast method for clustering large

NCBI protein databases are also used because they protein data sets. ProtoMap[11] applies some more

elaborate considerations.

———————————————— Among protein sequence clustering methods,

P. Usha Madhuri, Research Scholar, Dr.M.G.R. University, Chennai the simplest and most widely used category are based

on hierarchical clustering algorithm (single linkage)[12].

Dr.S.P.Rajagopalan, Emeritus Professor, Department of Computer It aggregates all the sequences linked by a level of

Applications, Dr.M.G.R University, Chennai.

similarity above a given threshold, so that within a

cluster any sequence is linked to at least one other

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sequence. This approach may yield fairly good results, alignments and Hidden Markov Models(HMMs). Pfam

but often a majority of sequences are grouped into one multiple alignments come in two forms. In the first,

single huge cluster resulting from a massive chain “seed” alignments are representative, non-redundant

effect due to multi-domain proteins. sets of sequences that are checked reasonably carefully

Blastclust program, one part of BLAST in a manual alignment editor.

package from NCBI, is an example of single linkage

protein sequence clustering. Another category, graph-

Pairwise

based clustering algorithms, are also commonly

Distance

employed due to the clustering quality. BAG[13] is a

Computing

sequence clustering algorithms based on graph theory.

OPTICS (Ordering Points To Identify the

Clustering Structure)[14] is a density-based clustering

method and is popular because it orders the data into a

density-based clustering structure corresponding to a Data Sets SEQOPTIC

broad range of parameter settings. For density-based Extraction S Clustering

methods, it is difficult to decide the input parameters

that the algorithm is sensitive to. OPTICS is a good

solution to density-based cluster ordering. Although it

does not produce clusters explicitly, OPTICS generates Result

an augmented ordering of data sets representing its Analysis

density-based clustering structure, and this structure

can be visualized. Since OPTICS does not limit cluster Figure. 1 SEQOPTICS Overview

extraction to global parameters, it is possible to extract

cluster information interactively as well as

SEQOPTICS expands the use of OPTICS, a

automatically.

method that has not been used in protein sequence

For any protein sequences clustering method a

analysis. Figure 1 shows the overview of our method.

suitable distance measure needs to be chosen. Some

First, data sets are extracted from data sources (mostly

functionally related sequences share little or no

protein databases), then mixed and randomized. Three

discernible sequence similarity and detection of these

data sources are Pfam, Swiss-Prot and NCBI. Secondly,

relationships is difficult. The general practice to carry

the pairwise distances between any two proteins are

out protein sequence clustering is based on pair-wise

computed. Here we used a normalized Smith-

sequence similarity/dissimilarity computed by

Waterman score. Several other options may be chosen,

algorithms such as Smith-Waterman[15]. Some other

such as BLAST or FASTA, for distance measure. Then

protein distance measurement such as BLAST[9],

the OPTICS algorithm is adopted to execute the

FASTA[16] are also very commonly taken in existing

clustering and the clustering structure is graphically

methods.

presented. Finally the clustering results are analyzed

How to evaluate clustering results quality is

and compared to some other methods by Jaccard

an important issue in clustering analysis. For two-

coefficient.

dimensional data, it is clear that we can plot the data

and read the distribution to tell how good the

clustering results are. But in high dimension data or 2.1 DATA SETS

sequence clustering, direct visualization is normally The alignments are HMM-generated automatic

not feasible. In protein sequence clustering, a popular alignments of every homologous domain [1]. Two other

measure of clustering quality is based on how well the data sets are from NCBI and Swiss-Prot separately.

clusters identified by the clustering algorithm match Each protein sequence is labelled by its original

the protein families defined in some database by notation. This labelling defines the assumed “true”

biological experts[8]. Another method is to compare clusters.

SEQOPTICS with some existing methods by using

certain validation techniques [17].

For example, if a sequence is extracted with

“IGA1” from NCBI, then it is labelled as “IGA1” and

2 METHODS assumed to be in “IGA1” cluster. The size of each data

Four data sets are extracted from different protein set ranges from 197 to 319 sequences just for testing our

repositories as shown in table 1. Two of them are from method.For each data set, protein sequences from

Pfam since it is a protein families database of

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different families are mixed and randomized to understanding how OPTICS works: An object p is in the

exclude manual clustering. ε- neighborhood of q if the distance from p to q is less

than ε; A core object has at least MinPts neighbors in its

TABLE 1 ε-neighborhood. The reachability distance of p is the

PROTEIN SEQUENCE DATA SETS smallest distance such that p is density-reachable from

a core object o. A cluster is a set of density-connected

objects which is maximal with respect to density-

reachability.

A reachability plot is a bar chart that shows

each object’s reachability distance in the order the object

was processed which demonstrates the cluster structure

of data. The final clusters can be extracted by either ε-

cutoff or steepness of the plot. For more detailed

information about OPTICS algorithm, please refer the

original paper[14].

SEQOPTICS is implemented with a distance

measure of sequences based on Smith-Waterman

Note: The number in parenthesis is the number of algorithm. The core OPTICS part was tested with the

sequences in each family

data sets from the author. Two parameters need to be

chosen, ε and MinPts. In this paper, since the distance

2.2 COMPUTING DISTANCE between any two protein sequences is between 0 and 1,

Our approach, consonant with others, starts with a we can use a single ε for all data set, for example, set ε

distance measure. When data sets are from different as 0.99, which is slightly smaller than 1. The MinPts

protein families, it is a common practice to use a used here is 10 just for experiment purposes. For the

normalized pairwise local alignment score by Smith- whole protein database, ε can still use any value

Waterman dynamic programming algorithm. There between 0.95 to 0.99, MinPts should be set as the

are several parameters in Smith-Waterman, for average number of sequences in a family.

example, scoring matrix, open gap penalty and There are two main advantages to apply

extending gap penalty. Different scoring matrices OPTICS in protein sequences clustering analysis: 1)

including BLOSUM50 and PAM250 have been tried. OPTICS can find the local density region; 2) OPTICS

BLOSUM50, which is also used in FASTA[16], is used produces an augmented ordering of the database

in this paper. The open gap penalty taken is 12 and the representing its density based clustering structure and

extending gap penalty is 2. The final similarity score this ordering can be visualized, for example, in

between two protein sequences is then normalized by reachability plot. The cluster ordering actually contains

the following: information about every cluster, i.e., OPTICS enables

S ( a , b) the extraction of not only “traditional” cluster

SN (a, b) information, but also intrinsic clustering structure.

Min( S (a, a ), S (b, b))

where S(a, b) is the Smith-Waterman local alignment

score between two sequences a and b; S(a, a) is the

3 RESULTS

score of sequence a to itself; S(b, b) is the score of SEQOPTICS is used to cluster the data sets. These

sequence b to itself; and SN(a, b) is the normalized provide some clues about clustering structure. The final

score. The distance between two protein sequences is density-based cluster sets are defined from the ordering

defined as: Distance (a, b) = 1 − SN (a, b); reachability distance. To judge the resulting clustering

set’s biological accuracy, we need to compare it to a

With this normalization, every distance score is “true” cluster set. However, there is no generally

between 0 and 1. If other scoring methods are used accepted “true” cluster set.

instead of Smith-Waterman, the distance measure All automatical protein clustering methods are

needs to be adjusted appropriately. based on “all against all” sequence comparison. Real

clusters need to be verified by biological expertise.

Since it is impossible to have “real” clustering, we have

2.3 OPTICS CLUSTERING to assume the original database clusters are the “real”

Some preliminary remarks on OPTICS have been given clusters. That is the way of most automatic protein

in the introduction. Some definitions might help us clustering does.

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3.1 VISUALIZATION OF THE CLUSTER STRUCTURE a is “true positive”, i.e., the number of

sequence pairs clustered together in both sets,

A reachability distance plot was made for each data

which can be define as :

set. In the figure, the horizontal axis represents the

ordering of each sequence, the vertical axis represents a ( i , j ) M ij 1 Tij 1, i j

the reachability distance, and each valley stands for a b is “false negative”, i.e., the number of

cluster set. For data set 1, there are five valleys in sequence pairs clustered together in the true

Figure 2: The first two valleys are composed of cluster set, but not in the current

sequences from cytochrom B562; The third valley clustersolution defined as:

consists of sequences from glucokinase; The fourth b ( i , j ) M ij 0 Tij 1, i j

valley contains sequences from GABAR family; The

fifth valley are sequences from bac globin family. c is “false positive”, i.e., the number of

sequence pairs clustered in the current

solution, but not in the true cluster set, defined

3.2 EXTRACTION OF THE CLUSTERS as:

The final density-based clusters were extracted by c ( i , j ) M ij 1 Tij 0 , i j

using a cutoff value. For example, in Figure 2, the d is “true negative”, i.e., the number of

cutoff value is set as 0.860 (the line reachability sequence pairs not clustered in either current

distance = 0.860). Under this cutoff condition, each solution or the true cluster set, defined as :

valley in Figure 2 between two sequences with

d ( i , j ) M ij 0 Tij 0 , i j

reachability distance higher than the cutoff is a cluster.

The sequence starting a valley with reachability There are many validation techniques in references[18].

distance higher than the cutoff is also in the same Three parameter based on the above are as follows:

cluster as rest sequences in the valley. Precision is defined as:

a

P ------ (1)

(a c)

Recall is defined as:

a

R ------ (2)

(a b)

Jaccard Coefficient is defined as:

a

S ------ (3)

(a b c)

All three values range between 0 and 1.The

better the clustering, the bigger the values. In a perfect

clustering which is identical to the true cluster, P = 1, R

= 1, and S = 1. Most existing sequence clustering

Figure 2. Cluster Structure of Date Set 1: Pfam (Protein

Familiy) methods perform well in terms of Precision but not in

Recall. This is shown in later validation result in Table 2

Any sequence with reachability distance along with additional comparative values.

higher than the cutoff is noise if it does not start a

valley. Therefore, in Figure 2, there are four clusters The clusters were formed from the same data

give the cutoff value 0.860. Similarly, there are four sets with two other clustering methods, blastclust [9]

clusters in Figure 3 given cutoff 0.745, six clusters in and BAG [13], using default parameters of these

Figure 4 given cutoff 0.860. methods. BAG is a graph based clustering method and

most existing methods are based on graph clustering.

Blastclust is chosen because it is from NCBI blast

3.3 VALIDATION OF THE CLUSTER SET package. This hierarchical sequence clustering method

A cluster set of n data points from experiment can be is very popular in biological research.

n * ( n 1)

represented by m values in a triangular

2 Using the Jaccard Coefficient, Precision and

matrix M, where for i < j, Mi j = 1, if and only if i and j Recall, we developed comparison values shown in the

are in the same cluster and Mi j = 0 otherwise. If T is Table 2. From the Table 2, we see that SEQOPTICS

the matrix of the “true” clusters, two cluster sets produces good results relative to each original cluster

(“true” and “experimental”) can be compared based on set in terms of Jaccard Coefficient. Every SEQOPTICS

the following numbers: Jaccard Coefficient is higher than 0.65 and the highest

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being 0.85. It is also seen in the table that SEQOPTICS adequacy of our approach for small-scale data and the

outperforms BAG and blastclust on all the data sets usefulness of the cluster structure visualization.

chosen on Jaccard Coefficient. According to Ankerst[14], one good feature of OPTICS

is that it is unnecessary to limit oneself to a single set of

TABLE 2 global parameters. An augmented cluster ordering

COMPARISON OF CLUSTERING RESULTS OF BAG, contains information equivalent to density based

BLASTCLUST AND SEQOPTICS METHODS clusterings corresponding to a broad range of

parameter settings; as such, the cluster ordering is a

versatile base for both automatic and interactive cluster

analysis. A second good feature lies in the visualization

of the data set distribution. Depending on data set size,

one can either represent the cluster-ordering

graphically for a small data set, or, employ an alternate

technique (appropriate) for large data sets. In this

paper, we demonstrated in SEQOPTICS the

visualization of cluster structure is meaningful.

SEQOPTICS has proved its value for small data sets

(<1000 sequences)in this paper. If we are applying this

method to a large data set, such as whole database,

The performance with BAG exceeds blastclust future improvements are necessary to make it more

for the same reason. However, BAG and blastclust useful. We may list specifically: 1) use some other

tends to give more clusters than the “true” clusters, distance measure for protein sequence distance, e.g.,

explaining why the Precision of those two methods on BLAST or FASTA; 2) apply parallel computing tools, for

all data sets are 1. But neither of these two performs example, Message Passing Interface(MPI) for large data

well in terms of Recall. Overall, SEQOPTICS performs sets; 3) implement visualization techniques for large

better than BAG and blastclust and seems a promising data sets; 4) consider incremental cluster ordering

method in terms of both clustering quality coupled algorithms since protein databases are very frequently

with its graphical representation of clustering being updated.

structure.

REFERENCES

4 EVALUATION OF THE EXECUTION TIME

[1] Alex Bateman, Ewan Birney, Lorenzo Cerruti, Richard Durbin, The

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where n is the number of sequences and l is the 2002.

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5 CONCLUSION

[5] Evgenia V Kriventseva, Margaret Biswas, and Rolf Apweiler.

In this paper a programmed system, SEQOPTICS, for Clustering and analysis of protein families. Current Opinion in

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described. A core portion(phase) of the system is based [6] S. Mohseni-Zadeh, P. Brezellec, and J. L. Risler. Cluster-c, an

on OPTICS clustering and visualization method, algorithm for the large-scale clustering of protein sequences based on

which we believe is being used here for the first time in the extraction of maximal cliques. Computational Biology and

protein sequence clustering. Prior to this phase, it is Chemistry, 28:211–218, 2004.

necessary to compute the distance between (protein) [7] P. Pipenbacher, A. Schliep, S. Schneckener, A. Schonhuth, D.

sequences. A normalized Smith-Waterman score is Schomburg, and R. Schrader. ProClust: improved clustering of

used in this paper to compute the required distance. protein sequences with an extended graph-based approach.

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thesis, der Universitat Bielefeld, 2002.

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[16] W R Pearson and D J Lipman. Improved tools for biological

sequence analysis. Proc. Natl. Acad. Sci. U.S.A., 85:2444–2448, 1988.

[17] Maria Halkidi, Yannis Batistakis, and Michalis Vazirgiannis. On

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[18] M. Halkidi, Y. Batistakis, and M Vazirgiannis. On clustering

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[19] B. Amit and B. Baldwin. Algorithms for scoring coreference chains,

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In MUC6 ’95: pages 45–52, Morristown, NJ, USA, 1995.

University Degrees achieved Master of Computer Applications

in the year of 2003 , from University of Madras, Chennai.

Pursuing Ph.D under the supervisor Dr.S.P.Rajagopalan,

Emeritus Professor, Dr.M.G.R University, Chennai. Currently

working as Assistant professor in Velammal Engineering College,

Chennai. A paper title An overview of Basic Clustering Algorithms

was published in Internal Journal of Computer Science and

System Analysis. Associated with ISTE professional association.

of Computer Applications, Dr.M.G.R University, Chennai.

Syndicate member of University of Madras. Published more than

150 Papers in National and Internaltional Journals. Also

published more than 50 books in maths and computer science

disciplines. Achieved Best Teacher award.

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