Introduction
============

SSRs, also called simple tandem repeats (STRs) or microsatellites, are DNA segments composed of tandem repetitions of relatively short motifs. They are commonly and easily identified DNA sequences which consist of repeated units one to six base pairs in length.[@b1-aabc-2-023]--[@b4-aabc-2-023] For decades, SSRs were mainly considered to be genetic markers in DNA fingerprinting and diversity studies due to their high rate of polymorphism. Nevertheless, recent studies pointed out that SSR expansions and/or contractions in protein-coding regions bring about a gain or loss of gene function through frameshift mutations.[@b2-aabc-2-023]--[@b4-aabc-2-023] SSR variations in 5'UTRs could affect gene transcription and translation, whereas SSR expansions in 3'UTRs could cause transcription slippage and result in disrupting splicing and possibly disturbing cellular functions. For example, a CGG repeat pattern within the 5'UTR of the *FMR1* gene is expanded in families with fragile X syndrome. When the length of SSR exceeds 200 CGGs, mental retardation occurs due to the absence of the encoded FMR protein.[@b5-aabc-2-023] Another example is a CTG expansion located in the 3'UTR of a *kinase* gene involved in myotonic dystrophy type 1 (*DM1*), a multisystemic dominantly inherited disorder. *DM1* disorder affects skeletal and smooth muscle as well as the eye, heart, endocrine system, and central nervous system.[@b6-aabc-2-023] Furthermore, SSRs in introns affect transcription, mRNA splicing, or export to the cytoplasm, which have been shown that SSRs indeed possess a functional role as cis-regulatory elements. For example, a CA simple sequence repeat within the first 2000 bases in intron 1 enhances *egfr* transcription and involves in breast carcinogenesis.[@b7-aabc-2-023] These functional SSRs, SSRs with biological functions, play an important role in gene regulation.[@b2-aabc-2-023] Consequently, the discovery of potential functional SSRs to decipher gene regulatory networks intrigues biologists.

There are many *in silico* SSR mining tools and databases such as MMDBJ,[@b8-aabc-2-023] Satellog,[@b9-aabc-2-023] MRD,[@b10-aabc-2-023] SSRD,[@b11-aabc-2-023] and EuMicroSatdb. [@b12-aabc-2-023] All of these mining tools are briefly described by Aishwarya and colleagues,[@b12-aabc-2-023] but none of them allows users to retrieve potential functional SSRs using comparative genomics. These available tools either emphasize a collection of SSRs from specific organisms or provide only limited functions for various genome comparisons. Accordingly, it is still tedious for biologists to find functional SSRs from millions of SSR candidates.

Due to functional constraints, DNA regions involved in gene regulation or genome evolution are expected to be conserved among related species. It has been shown that cross-species comparison of DNA sequences can facilitate identification of candidate regulatory elements.[@b13-aabc-2-023] If SSRs possess significant biological functions, they are likely to be located in conserved regions. In this report, the proposed comparative genomics SSR discovery (CG-SSR) web service comprises eleven representative vertebrate species (human, chimpanzee, orangutan, mouse, rat, opossum, rhesus, cow, dog, zebrafish, and medaka) for constructing the fundamental SSR database. It also includes another thirteen species (cat, horse, marmoset, guinea pig, platypus, chicken, lizard, *Xenopus tropicalis*, tetraodon, fugu, stickleback, lamprey, lancelet) for verification of conserved regions of the retrieved SSRs. Users can evaluate the biological significance of SSRs through cross-species conservation because such comparisons are available for the various species selected as the representative model organisms in CG-SSR. CG-SSR also combines other relevant functional genomics information such as GO (Gene Ontology),[@b14-aabc-2-023] InterPro,[@b15-aabc-2-023] and Pfam.[@b16-aabc-2-023] GO provides biological annotation of genes in light of their associated biological processes, cellular components, and molecular functions. The InterPro database contains identifiable features -- protein families, domains, repeats and sites -- found in known proteins that can be applied to novel proteins. Through hidden Markov models and multiple sequence alignments, the Pfam database collects domain information for a large quantity of protein families. Taken together, these functional genomics resources permit biologists to decipher candidate roles for SSRs in gene regulatory networks.

Methods
=======

To construct the database for CG-SSR, vertebrate genome sequences and gene coordinates were obtained from Ensembl Genome Browser.[@b17-aabc-2-023] Whole genomes were scanned based on an efficient correlation method for SSR mining that is composed of two major phases including autocorrelation and overlapping adjacent phases (see Supplementary materials). In the first autocorrelation phase, the CG-SSR performs seed finding, region growing, trimming, recovering, pattern refinement, and noise filtering processes to discover all possible SSR repeats as initial candidates. For the second phase, the system verifies the overlapping records and confirms no redundant patterns by employing merging, cutting and threshold filtering processes. [Figure 1](#f1-aabc-2-023){ref-type="fig"} illustrates the flowchart of CG-SSR searching algorithm and a detailed description of the developed methods and examples can be found in the Supplementary materials.

The core algorithm for discovering SSR patterns from genome sequences employs autocorrelation methodology. The basic concept assumes that the target sequence contains repeat substrings with basic patterns of length N within a range from 1 to 6. If we shift the target sequence with N nucleotides to its right and compare to the original sequence, all repeat patterns can be discovered since they overlapped with their shifted sequence at least within N nucleotides continuously. Based on the observation of such overlapping, we can easily detect the repeating locations by shifting and matching the whole target sequence without knowing the nucleotide contents of repeated patterns. If the continuously matched nucleotides are longer than the shifting length N, at least one repeat with the basic pattern length N is proved to exist in the target sequence. Hence, exact matched strings can be identified and considered as the seeds of perfect SSR candidates. These seeds were then extended by region growing techniques to formulate imperfect SSR sequences. To identify imperfect SSRs with various tolerant conditions, a module applied forward region growing method to perform neighboring comparison. As long as the coordinates and contents of the perfect repeat patterns were obtained, the forward region growing processes were performed by examining the right-hand-side neighboring nucleotides and skipping noise-like nucleotides which did not belong to the perfect repeat patterns. As no extra basic unit pattern could be found by continuously extending the verification on right-hand-side neighboring nucleotides, the searching processes terminated. After the region growing processes, the boundary detection through trimming/recovering operations was applied to delimit the appropriate range of each SSR sequence. In this module, verifying pattern boundaries on both sides was achieved by left-hand-side trimming and right-hand-side recovering processes. The right-hand-side trimming processes were achieved by backward scanning from the rightmost nucleotide of the primitively searched SSR to its last noninterrupted basic pattern. Once the last perfect basic pattern was found, the partial and noninterrupted patterns on its right-hand-side were recalled for its longest representation. For the left-hand-side of SSR string verification, this module examined only on the length of an assigned basic pattern which could not be recovered by insertion, deletion, or substitution examinations employed in the previous autocorrelation phase. In the module of pattern refinement, if a basic unit pattern is a repeated sequence itself, trivially, the pattern will be identified as SSRs within different unit lengths during autocorrelation processes. For example, a tri-nucleotide SSR string of length 60 could be identified as well as a hexa-nucleotide SSR string of length 30 from the first phase computation. Hence, for eliminating the redundant cases of this type, all self-repeating basic patterns were double checked and reduced to its smallest unit size.

After all frameshifts were completed, redundant segments were removed to eliminate possible overlapping patterns. The final noise filtering module was implemented to remove imperfect SSRs which contain impure variations higher than the defined threshold proportion. In the proposed system, two thresholding parameters of minimum length and maximum noise rate were decided in advance for verifying the qualification of a searched SSR record. The first thresholding value confirms the total length of a candidate SSR record which includes the repeats and the tolerant nucleotides, whereas the second thresholding parameter inspects the noise rate of the identified SSR records. The definition of noise rate is indicated as

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where *L~PatternLength~* represents the length of basic unit pattern, *N~RepeatTime~* the number of repeats of complete pattern (exact the same as the basic unit pattern), *N~IncompletePattern~* the number of nucleotides of incomplete patterns (partial subset of the basic unit pattern) in the SSR, and *L~TotalLength~* the total length of the identified SSR. Hence, a perfect SSR possesses "0" noise rate and higher noise rate represents more tolerant nucleotides appeared in the identified SSR strings. The default setting of noise rate for the initial database in CG-SSR is 0.2 which represents 20% of the total nucleotides recognized as the maximum noisy base pairs for each SSR segment.

For the second phase of overlapping adjacent verification, the SSR candidates were re-inspected based on their locations and basic unit patterns. Merging and cutting operations on neighboring SSR candidates enhanced the consecutive relationship and centralized the diverse representations. On the condition of two overlapping SSR candidates, the system firstly verified the overlapped records if they possessed identical basic unit patterns. If two or more SSR records possessed an identical pattern, the recombination of such candidates were concatenated after evaluating the criterion of noise threshold requirements. On the other hand, if these overlapped SSR candidates of the same pattern length did not possess identical basic unit pattern, they usually resulted from the tolerant conditions. Hence, the system combined such two SSR candidates to make sure no entry was redundant. Finally, a threshold filter was applied again to satisfy the requirements of minimum SSR length and maximum noise rate. It is also true that two SSR candidates of different basic unit patterns and lengths might overlap. The ambiguity usually arose on the transistion positions of two consecutive basic patterns. In this system, two SSR strings of different lengths of basic unit patterns would be categorized and stored as two independent SSRs. Only two overlapping adjacent SSRs of the same basic pattern were merged for efficient and effective consideration.

When all of the SSR patterns in each genome were identified respectively, by comparing identical SSR patterns in the conserved regions among various species, the SSR patterns with high occurrence rates were considered as the candidates of important functional SSRs.

Results and discussion
======================

In the retrieval processes, all perfect and imperfect SSRs were verified and annotated with accurate coordinate information of upstream, downstream, 5'UTR, 3'UTR, protein-coding, intron, and intergenic regions. Moreover, for identifying candidates of functional SSRs, the system integrated comparative genomics methods in which information from crossspecies comparison was obtained from the UCSC Genome Browser[@b18-aabc-2-023] to provide coordinates for cross-species conserved regions. Currently, 24 species were included in the CG-SSR system for comparison. Through cross-species conservation, all conserved SSRs were identified and displayed in an explicit table. Furthermore, the biological function of each gene can refer to GO, InterPro, and Pfam resources for further comprehensive analyses. The developed CG-SSR web server is freely available at: <http://cgssr.cs.ntou.edu.tw/>. There are four major functions provided by the system: SSR Discovery, SSR on Transcripts, Comparative Genomics, and SSR Searching Tool.

SSR Discovery
-------------

It provides all loci of perfect and imperfect SSRs on a designated chromosome of a specified genome. Users can allocate precise locations of SSRs by selecting a target species, the number and range of chromosome, parameter of minimum length, and/or specific patterns of interested SSRs.

SSR on Transcripts
------------------

It collects all genes which possess perfect and imperfect SSRs found on their exon regions. Users can allocate precise locations of SSRs by selecting a target species, the number of chromosome, and the transcript IDs. (All transcript IDs were defined in Ensembl release 48, Dec. 2007).

Comparative Genomics
--------------------

It provides a tool for searching all perfect and imperfect SSRs on conserved regions or orthologous genes among various species. From the query keywords or transcript IDs, the system provides precise and complete information of SSRs including coordinates, lengths, basic unit patterns, regions, flanking sequences, and corresponding primers.

SSR Searching Tool
------------------

It provides an online SSR searching tool for discovering all possible SSRs located in the uploaded multiple DNA sequences with respect to required parameter settings.

The detail guidelines of each subsystem can be obtained by clicking on the "tips" icon in each web interface. These guidelines provide helpful information to a user who is looking for a particular point of interest in SSR discovery. In conclusion, abundant resources for functional genomics, cross-species comparison, accurate coordinate annotation, flanking sequences and corresponding primers have been well integrated to provide applicable information for users. In this study, the numbers of retrieved imperfect and perfect SSRs (≥10 base pairs with repeated unit lengths of 1--6 base pairs) by CG-SSR and the number of identifiable gene characteristics and protein families from several well known databases for eleven representative vertebrate species are listed in the [Table 1](#t1-aabc-2-023){ref-type="table"}. All detailed results are available online at the CG-SSR website. [Figure 2](#f2-aabc-2-023){ref-type="fig"} shows the statistical distributions of various repeated unit patterns for eleven model species. As can be seen from the diagram, similarly distributed proportions of different unit lengths of SSR patterns occur for all species and the most amounts of perfect and imperfect SSR repeats is the dinucleotide patterns with the default parameter settings of minimum length of 10 base pairs and noise rate of 20%.

To illustrate the practical applications of CG-SSR for identifying potential functional SSRs through cross-species comparison, several well known functional SSRs were collected and shown in [Table 2](#t2-aabc-2-023){ref-type="table"}. According to comparative genomics rules, functional elements are likely to be located in conserved regions. Notably, several functional SSRs discovered by our system were located in coding regions, UTR regions, and even in intron regions. The identification of functional SSRs in conserved regions of several species is evidence of their common features.

Taking *ATM* gene (ataxia-telengiectasia mutated gene) as an example (Ensembl transcript ID: ENST00000278616), 118 SSRs longer than 20 nucleotides were found. The comparative genomics mechanism provides an efficient way to select potential functional SSRs from among the 118 candidates. For instance, a T repeat exhibited a high degree of conservation among 17 species although it is located in an intron which is generally considered as a nonfunctional region. Interestingly, such a specific intronic mutation of T repeat has been reported to cause aberrant splicing and abnormal transcription in colon tumors.[@b19-aabc-2-023] Similarly, some CAG repeats in coding regions can be found and proved as functional SSRsin the *HD* (Ensembl transcript ID: ENST00000355072), *DRPLA* (ATN1, Ensembl transcript ID: ENST00000356654), *SCA1* (ATXN1, Ensembl transcript ID: ENST00000244769), *SCA2* (ATXN1, Ensembl transcript ID: ENST00000377611), *SCA3* (ATXN3, Ensembl transcript ID: ENST00000340660), and *SCA6* (CACNA1A, Ensembl transcript ID: ENST00000325084). To extract the highly conserved SSRs and verify as the potential functional motifs, users can input the Ensembl transcript ID directly or type the keywords in the query textbox under the "Comparative Genome" website. If an abbreviated keyword cannot be found, users can try the entire gene name to retrieve its corresponding transcript IDs from the specified gene set. Consequently, exactly and partially matched genes were listed in a table. Therefore, one can click on the retrieved transcript IDs and check/uncheck the checkbox of "intron" attribute before sending the query. According to the parameter settings, those retrieved SSRs will be listed in ascending order by chromosome number. To exploit potential functional SSRs of a gene, users are suggested to select SSRs in accordance with the number of conserved species. For example, from the first six genes listed in [Table 2](#t2-aabc-2-023){ref-type="table"}, the CAG repeats appeared in shifted or complementary patterns are considered as potential functional motifs because they were highly conserved in 12 to 22 species from CG-SSR. Indeed, these CAG repeat expansions in coding regions were demonstrated to bring about various neuronal diseases.[@b2-aabc-2-023] These findings suggest that cross-species comparison can be used to identify potential functional SSRs in both coding and noncoding regions.

After retrieving functional SSR candidates, CG-SSR provides functional genomics resources---GO, InterPro, and Pfam---to help users ascertain the potential biological function of each SSR. Using the involvement of *WISP2* in signal transduction as an example (Ensembl transcript ID: ENST00000396767), information provided by GO implicates *WISP2* in cell growth. Interestingly, experimental verification suggests that this A repeat indeed has tumor suppressor function.[@b4-aabc-2-023] Users can efficiently use these functional genomics resources to obtain clues about putative roles of SSRs in gene regulatory networks.

In summary, CG-SSR comprises accurate coordinate, cross-species comparison, and functional genomics resources. It is a comprehensive, efficient and user-friendly online tool for identifying conserved SSRs as potential functional motifs in vertebrates.

Supplementary information: How does CG-SSR searching algorithm work?
====================================================================

Definition of SSR
-----------------

Simple sequence repeats (SSRs), also called microsatellites, are nucleotide segments with basic repeat pattern of 1--6 base pairs in length. The searching algorithm of CG-SSR is designed for discovering all perfect and imperfect (with tolerant) SSRs from various genomic sequences efficiently and effectively.

Algorithm description
---------------------

[Figure 1](#s1-aabc-2-023){ref-type="supplementary-material"} depicts the flowchart of CG-SSR searching algorithms which is composed of two major phases: (I) autocorrelation and (II) overlapping adjacent phases. The first autocorrelation phase including seed finding, region growing, trimming/ recovering, refining, and threshold filtering processes discovers all possible SSR patterns as fundamental candidates. The second overlapping adjacent phase including record merging, record cutting, and threshold filtering processes verifies overlapped segments and confirms no redundant perfect/imperfect SSR patterns. Details of each procedure of these two phases are described in the following sections:

Autocorrelation phase
=====================

Autocorrelation seed finding
----------------------------

The core algorithm for discovering SSR patterns from genome sequences employs autocorrelation methodology. The basic concept assumes that the target sequence contains repeat substrings with basic patterns of length N within a range from 1 to 6. If we shift the target sequence with N nucleotides to its right and compare to its original the original sequence, all repeat patterns can be discovered since they overlapped with their shifted sequence at least within N nucleotides continuously. Based on the observation of such overlapping, we can easily detect the repeating locations by shifting and matching the whole target sequence without knowing the nucleotide contents of repeated patterns. If the continuously matched nucleotides are longer than the shifting length N, at least one repeat with the basic pattern length N is proved to exist in the target sequence. Therefore, the context of the basic pattern can be extracted directly from the target sequence where the repeats appear. The program performs frame shifting processes from length 1 to 6 base pairs to identify mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide SSRs, respectively. [Figure 2](#s2-aabc-2-023){ref-type="supplementary-material"} illustrates an example of autocorrelation processes for identifying seeds of repeat patterns of length N = 3. The number of continuously matched nucleotides reveals the exact length of perfect SSR repeats. However, these discontinuous perfect patterns were interrupted due to various tolerant conditions, therefore categorized as imperfect SSRs. In order to identify both perfect and imperfect SSRs, the proposed algorithms firstly considered the coordinates of perfect repeats and employed the location information as input for the following modules.

Neighboring comparison from region growing
==========================================

To identify imperfect SSRs with various tolerant conditions, the proposed algorithm applied forward region growing method to enable neighboring comparison. As long as the coordinates and contents of the perfect repeat patterns were obtained, the forward region growing processes were performed by examining the right-hand-side neighboring nucleotides and skipping noise-like nucleotides which did not belong to the perfect repeat patterns. The searching processes stopped until no extra basic unit pattern could be found by continuously extending the verification on right-hand-side neighboring nucleotides.

There are three types of sequence tolerance for imperfect SSRs shown in [Figure 3](#s3-aabc-2-023){ref-type="supplementary-material"}: insertion, deletion, and substitution. The insertion case is categorized into two different types: the inserted nucleotides located between two basic unit patterns or appeared inside a basic unit pattern. Deletions and substitutions, apparently recognized by analyzing the length and contents of insertion based on string comparison, can be considered as the insertion in different forms.

Right trimming and left recovering processes
============================================

In this module, verifications on both sides include left-hand-side trimming and right-hand-side recovering processes. Both processes are described as follows:

Right-hand-side trimming
------------------------

Once an SSR sequence was allocated from seed finding and forward region growing, a trimming process was applied to examine its right-hand-side of extended SSR records for a guaranteed representation of noninterrupted pattern. This process was achieved by backward scanning from the right end of the previously searched SSR to its last noninterrupted basic pattern. Once the last perfect basic pattern was found, the following, partial, and noninterrupted patterns on its right side were recalled for its longest representation, such as the "AT" substring on the right end of the designated SSR in [Figure 4a](#s4a-aabc-2-023){ref-type="supplementary-material"}.

Left-hand-side recovering
-------------------------

For the left-hand-side of SSR string verification, the system examined only the length of an assigned basic pattern which could not be recovered by insertion, deletion or substitution examinations employed in the previous autocorrelation phase. An example of left-hand-side recovering process was shown in [Figure 4b](#s4b-aabc-2-023){ref-type="supplementary-material"}. The "ATG" pattern in the leftmost position could not be recognized through the first autocorrelation module, but it could be identified after the verification of left-hand-side recovering process.

Threshold filtering
===================

Two thresholding parameters were applied to verify if an SSR record was qualified for the requirements. One was the parameter of minimum length and the other was the maximum noise rate. The first thresholding filter checked the total length of a candidate SSR record which included the repeat contents and the tolerant nucleotides. The default argument in the system was 10 nucleotides.

The second thresholding parameter inspected the noise rate of an identified SSR record. The definition of noise rate is

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where *L~PatternLength~* represents the length of basic pattern; *N~RepeatTime~* represents the number of repeats of complete pattern; *N~IncompletePattern~* denotes the number of nucleotides of incomplete patterns in the SSR: *L~TotalLength~* is the total length of an identified SSR.

A perfect repeat SSR possesses "0" noise rate, and higher noise rate represents more tolerant nucleotides appeared in an identified SSR strings. The default setting is 0.2 which represents 20% of the total nucleotides recognized as noisy base pairs for the selected SSR string. Examples of noise rate calculation are shown in [Figure 5](#s5-aabc-2-023){ref-type="supplementary-material"}.

Pattern refinement
==================

If a basic unit pattern is a repeat sequence itself, trivially, the pattern will be identified as SSRs with different lengths during autocorrelation processes. For example, a tri-nucleotide SSR string of length 60 could be identified as well as a hexa-nucleotide SSR string of length 30 from the first phase computation. Hence, for eliminating the redundant cases of this type, all self-repeating basic patterns were double checked and reduced to its smallest unit size. In [Figure 6](#s6-aabc-2-023){ref-type="supplementary-material"}, an example of self-repeating SSR was shown and the basic unit pattern "TG" was considered as the fundamental SSR pattern with the smallest unit size for its final representation. Therefore, the SSR string was categorized as a di-nucleotide SSR record instead of a tetra-nucleotide or a hexa-nucleotide.

To achieve robust performance of CG-SSR system, the program skipped the mono-nucleotide SSR seed-finding module. Frameshifting of one nucleotide caused overwhelming false alarm results and wasted too much time on following evaluation processes. However, the mononucleotide SSR could be retrieved by performing from frameshifting of two to six nucleotides and verified by self-repeating analysis. The mononucleotide SSRs could be successfully and efficiently identified after this refinement processes.

It was noticed that a shifting operation applied on an identified SSR sequence formulated another new repeat sequence within a different basic unit pattern. It is shown in [Figure 7](#s7-aabc-2-023){ref-type="supplementary-material"} as an example, "ATG", "TGA", and "GAT" were different basic unit patterns for three SSR sequences. However, it should be defined that these three basic unit patterns were considered as an identical pattern as long as their contexts can be exactly matched after shifting operations.

Overlapping adjacent phase
==========================

In this phase, the SSR candidates were re-inspected based on their locations and basic unit patterns. Merging and cutting operations on neighboring SSR candidates enhance the consecutive relationship and centralize the diverse representations. The proposed filtering operations are described as follows:

Record merging and cutting
--------------------------

On the condition of two overlapping SSR candidates, the system firstly verified the overlapped records if they possessed identical basic unit patterns. If two or more SSR records possessed an identical pattern, the recombination of such candidates were concatenated after evaluating the criterion of noise threshold requirements. On the other hand, if these overlapped SSR candidates of the same pattern length did not possess identical basic unit pattern, they usually resulted from the tolerant conditions. Hence, the system combined such two SSR candidates to make sure no entry was redundant. Finally, a threshold filter was applied again to satisfy the requirements of minimum SSR length and maximum noise rate.

It is also true that two SSR candidates of different basic unit patterns and lengths might overlap. The ambiguity usually arose on the transistion positions of two consecutive basic patterns. In [Figure 8](#s8-aabc-2-023){ref-type="supplementary-material"}, an example of SSR transistion from a tetra-nucleotide SSR to a tri-nucleotide SSR was shown, and the concatenation resulted from the tolerant conditions between two SSR strings. In such system, two SSR strings of different lengths of basic unit patterns would be categorized and stored as two independent SSRs. Only two overlapping adjacent SSRs of the same basic pattern were merged for efficient and effective consideration.

###### 

The flowchart of CG-SSR searching algorithm.

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An example of autocorrelation process.

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All tolerant cases can be considered as special cases of insertions. The substitution and deletion cases were viewed as inserting tolerant segments.

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The right-hand-side trimming process verifies the right-hand-side of SSR to guarantee a complete, noninterrupted representation.

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An example of left-hand-side recovering processes.

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Examples of noise rate calculation.

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Self-repeating basic pattern was verified in the proposed system.

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Different basic unit patterns due to shifting.

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Ambiguity between two basic unit patterns within two different lengths.

This work was supported by the Center for Marine Bioscience and Biotechnology (CMBB) at the National Taiwan Ocean University, Keelung, Taiwan (97529002H1), and the National Science Council in Taiwan, R. O. C. (NSC96-2627- B-019-003 to T.-W. Pai).

**Disclosure**

This SSR is free for use and can be located at the following URL: <http://cgssr.cs.ntou.edu.tw/>. Supplementary information is available at the following URL: <http://cgssr.cs.ntou.edu.tw/si_v2/>. The authors report no conflicts of interest in this work.

![The flowchart of CG-SSR searching algorithm.](aabc-2-023f1){#f1-aabc-2-023}

![Distributions of various repeated unit patterns from mononucleotide to hexanucleotide for 11 vertebrate model species. The number of SSR records was identified based on the parameters of a minimum length of 10 base pairs and a maximum noise rate of 20%.](aabc-2-023f2){#f2-aabc-2-023}

###### 

The total number of verified SSRs in the CG-SSR database, and the number of identifiable gene characteristics and protein families from GO, InterPro, and Pfam databases for 11 representative vertebrate species

  Species                        Human        Chimpanzee   Orangutan    Rhesus       Cow          Dog          Mouse        Rat          Opossum                                        Medaka                                         Zebrafish
  ------------------------------ ------------ ------------ ------------ ------------ ------------ ------------ ------------ ------------ ---------------------------------------------- ---------------------------------------------- ------------
  SSRs                           30,364,358   29,092,001   28,722,387   27,768,093   22,157,544   26,435,804   27,814,234   26,195,617   38,281,261                                     5,963,611                                      15,060,006
  Genes                          55,183       37,006       24,231       40,431       27,194       29,275       43,620       37,591       32,908                                         22,447                                         31,922
  GO records                     226,591      30,208       25,102       26,828       49,510       92,880       232,824      142,334      20,973                                         2,564                                          49,227
  InterPro records               109,750      75,180       54,405       81,741       63,294       61,523       95,177       79,520       89,516                                         50,423                                         81,063
  Pfam records                   56,531       39,782       28,390       42,304       33,534       31,788       49,207       40,762       46,012                                         30,816                                         41,756
  Orthologous genes              182,722      166,630      160,263      173,377      160,224      168,371      198,505      193,363      181,554                                        144,039                                        157,329
  Paralogous genes               87,397       59,186       51,748       123,096      71,592       72,070       161,852      175,790      74,944                                         0[\#](#tfn2-aabc-2-023){ref-type="table-fn"}   216,612
  Comparative genomics species   23           11           7            6            3            5            22           12           0[\$](#tfn3-aabc-2-023){ref-type="table-fn"}   7                                              5
  Conserved region records       18,666,679   11,567,566   5,981,764    7,929,321    3,558,513    8,239,233    17,373,326   12,208,685   0[\$](#tfn3-aabc-2-023){ref-type="table-fn"}   1,612,839                                      1,266,789

**Notes:**

Information of paralogous gene was not available from Ensembl Release 49, Mar. 2008;

Information of conserved region for Opossum was not available from UCSC, 2008.

**Abbreviations:** GO, gene ontology; SSRs, simple sequence repeats; UCSC, University of California, Santa Cruz.

###### 

Illustrations of practical applications of CG-SSR in identifying potential functional SSRs through cross-species comparison. Taking human species as an example, several well known functional SSRs could be retrieved and annotated[@b4-aabc-2-023]

  ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Gene                                                          Ensembltranscript ID   SSR motif        Repeat length (bps)   Region   \# of Conserved species   SSR related biological function                                                                      References
  ------------------------------------------------------------- ---------------------- ---------------- --------------------- -------- ------------------------- ---------------------------------------------------------------------------------------------------- -------------------------------------------------------------------
  *HD*                                                          ENST00000355072        CTG(CAG)         26                    Coding   22                        Expansion causes Huntington's disease (*HD*)                                                         Zoghbi and Orr (2000)[@b20-aabc-2-023]

  *ATN1* (*DRPLA*)                                              ENST00000356654        CAG              53                    Coding   21                        Causes dentatorubropallidoluysian atrophy (*DRPLA*)                                                  Nakamura and colleagues (2001)[@b21-aabc-2-023]

  *ATXN1* (*SCA1*)                                              ENST00000244769        GCA(CAG)         91                    Coding   20                        Causes spinocerebellar ataxias                                                                       Manto (2005)[@b22-aabc-2-023]

  *ATXN2* (*SCA2*)                                              ENST00000377611        CAG              71                    Coding   13                        Causes spinocerebellar ataxias                                                                       Manto (2005)[@b22-aabc-2-023]

  *ATXN3* (*SCA3*)                                              ENST00000340660        CAG              46                    Coding   12                        Causes spinocerebellar ataxias                                                                       Manto (2005)[@b22-aabc-2-023]

                                                                                                                                                                                                                                                                      

  *CACNA1A* (*SCA6*)                                            ENST00000325084        CAG              40                    Coding   15                        Causes spinocerebellar ataxias                                                                       Manto (2005)[@b22-aabc-2-023]

  *AR* (Androgen receptor)                                      ENST00000374690        AGC(CAG)         67                    Coding   11                        Shorter repeat increases hepatitis B virus (*HBV*) -- related hepatocellular carcinoma risk          Yu and colleagues (2001; 2002)[@b23-aabc-2-023],[@b24-aabc-2-023]

  *PABPN1* (Poly(A)-binding protein 2)                          ENST00000397276        GCG              37                    Coding   14                        Oculopharyngeal muscular dystrophy                                                                   Brais and colleagues (1998)[@b25-aabc-2-023]

  *WISP2* (Signal transduction genes)                           ENST00000396767        T(A)             22                    Coding   8                         Tumor-suppressive function                                                                           Markowitz and colleagues (1995)[@b26-aabc-2-023]

  *CALM_HUMAN*                                                  ENST00000356978        AGC(CAG)         21                    5'UTR    15                        Required for *hCALM1* full expression,                                                               Toutenhoofd and colleagues (1998)[@b27-aabc-2-023]

                                                                                                                                                                                                                                                                      

  *FMR1* (Fragile X mental retardation-1)                       ENST00000370475        GCG(CGG)         67                    5'UTR    11                        (CGG)40--200 related in fragile-X-like cognitive/psychosocial impairment                             Franke and colleagues (1998)[@b28-aabc-2-023]

  *AFF3* (*AF4*/*FMR2* family member 3)                         ENST00000317233        GCG(GCC)         26                    5'UTR    8                         Reduced *FMR2* causing abnormal neuronal gene regulation                                             Cummings and Zoghbi (2000)[@b29-aabc-2-023]

  *DMPK* (dystrophia myotonin protein kinase)                   ENST00000291270        CTG              62                    3'UTR    13                        Expansion causes *DM1* disease                                                                       Ranum and Day (2002)[@b6-aabc-2-023]

  *EGFR* (Epidermal growth factor receptor)                     ENST00000275493        CA               51                    Intron   10                        CA repeat enhances *egfr* transcription and involved in breast carcinogenesis                        Tidow and colleagues (2003)[@b7-aabc-2-023]

  *ATM* (Serine-protein kinase *ATM* )                          ENST00000278616        T                22                    Intron   17                        Shortening repeat tract leads to aberrant splicing and abnormal transcription in colon tumor cells   Ejima and colleagues (2000)[@b19-aabc-2-023]

                                                                                                                                                                                                                                                                      

  *ATXN10* (Spinocerebellar ataxia type SCA10)                  ENST00000252934        AGAAT(ATTCT)74                         Intron   8                         Expansion leads to change of function and results in *SCA10* disease                                 Matsuura and colleagues (2000)[@b30-aabc-2-023]

  *FXN* (Frataxin, mitochondrial precursor Friedreich ataxia)   ENST00000377270        CTT(GAA)         18                    Intron   7                         GAA expansion inhibits *FRDA* expression or interferes mRNA formation and lead to *FRDA* disease     Ohshima and colleagues (1998)[@b31-aabc-2-023]\
                                                                                                                                                                                                                                                                      Sakamoto and colleagues (2001)[@b32-aabc-2-023]
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