BACKGROUND
short tandem repeats , also known as short sequence repeats or microsatellites, are genome segments composed of short repeating sequences.
the length of the fundamental repeat unit varies from one to six nucleotides  <cit> .
strs are highly abundant in many different organisms, and are distributed in both genic and intergenic regions <cit> .
repeat structures expand or are deleted mainly due to replication slippage, which leads to highly polymorphic str patterns among individuals  <cit> ; these polymorphic features make str motifs suitable genetic markers  <cit> .
several str markers have been applied to individual/paternity identification and species/subspecies differentiation  <cit> , while some strs are involved in gene regulatory pathways.
abnormal expansion of such functional strs located within coding regions frequently cause various types of disease  <cit> .
even when located within non-coding regions, strs might also act as important functional regulatory elements  <cit> .
therefore, discoveries of polymorphic strs among different sequenced samples might be helpful for detecting useful genetic markers, while findings of well-conserved strs might lead to their identification as functional elements for gene regulation networks.
in traditional approaches, genomic str markers have typically been discovered by analyzing dna sequences through in silico methods and verified by pcr  <cit> .
various in silico tools are available for detecting both perfect and imperfect strs within a single species  <cit> .
in recent years, a revolutionary development in sequencing technology called next generation sequencing  has greatly impacted the growth and speed of genetic research.
with relatively low costs and increased throughput, research at the genomic and transcriptomic levels has now become affordable and practical  <cit> .
traditional est libraries applied to est-str discovery have been gradually replaced by ngs approaches, known as rna-seq techniques, which provide extensive coverage at the whole-transcriptome level  <cit> .
recent publications have shown that ngs plays a low-cost and time-efficient role in polymorphic str marker discovery, even without providing reference sequences  <cit> .
the latest tools have also focused on str marker discovery through ngs read analysis.
for example, qdd is an open-source str search tool package that provides a pipeline from raw ngs reads to str identification and corresponding primer design  <cit> .
hoffman and nichols also proposed a manual method for in silico str marker screening  <cit> .
their experiments with antarctic seals demonstrated the effectiveness of in silico str marker discovery across individual ngs samples.
the lobstr program was developed by gymrek et.al., who constructed a comprehensive survey of str variations from ngs-derived personal genomes  <cit> .
an automated method for detecting str polymorphisms from ngs data reads could utilize the high throughput advantages of ngs without the influence of manually examined factors.
in addition, we also developed a prototype system for detecting polymorphic strs within human genomes based on the conception of an str template profile  <cit> .
however, due to our limited knowledge, there are no online web applications that allow users to compare personal genomes or specify genes for a comprehensive str analysis.
therefore, we sought to develop an efficient identification system that is capable of detecting conserved and polymorphic strs across different individual sequence reads.
the proposed method could detect str polymorphisms without curated procedures, and could be directly applied for the efficient identification of conserved and polymorphic str markers and accelerate functional analysis of regulatory str motifs.
RESULTS
we have performed a statistical analysis of the str distributions in several datasets including chromosomal genes, combined dna index system  genes, disease-related genes, cross-species homologous genes, and genes that are unique to humans.
the two major reasons for performing statistical analyses on different gene sets were:  to determine the most frequently appearing lengths of polymorphic/conserved str patterns, and  the most frequently occurring regions of polymorphic str motifs.
to understand the extent of variation in the identified strs, the distribution scale ranged from  <dig> to  <dig> bp.
in addition, we selected the interval from  <dig> to  <dig> bp to analyze conserved degrees of identified strs.
retrieved strs from the different datasets are shown and discussed in the following sub-sections.
codis marker analysis
codis is a collection of investigated and verified dna markers provided by the u.s. federal bureau of investigation  to criminal justice services.
thirteen str markers  were examined in this program.
from the verification results, our proposed system could successfully detect and list all  <dig> str markers from  <dig> genomes, including six individual genomes from the  <dig> genomes trio project and one from the ensembl reference genome.
all retrieved str markers are listed in table  <dig>  and it should be noted that both str markers within the gene vwa  defined by codis contain multiple short repeat patterns, and the adopted cgssr program could successfully identify the three str markers "acag", "agat", and "tcca" for vwa.
ensg00000182578
ensg00000011376
ensg00000249816
ensg00000180176
ensg00000171560
ensg00000171791
ensg00000115705
ensg00000110799
ensg00000075213
ensg00000168367
d5s818
d13s317
d21s11
these genomes include the reference genome from ensembl and  <dig> genomes  from the  <dig> genomes trio project.
the first ceu family: na <dig>  na <dig>  and na12878; the second yri family: na <dig>  na <dig>  and na <dig>  enbl represents the genome from the ensembl database; na <dig> represents the ceu daughter, na <dig> the ceu father, and na <dig> the ceu mother; na <dig> represents the yri mother, na <dig> the yri father, and na <dig> the yri daughter.
it should be noted that both str markers of vwa were identified as  <dig> separate strs in this table.
it was also observed that most polymorphic strs within a family group agreed with inherited characteristics, i.e., the daughter's alleles were inherited from either one of her parents.
based on codis str markers for comparing these two families from the trio project, the results show that  <dig> of  <dig> str loci displaying identical repeat patterns/numbers among all selected individuals, and only one or two str patterns possessing minor differences in length could be found between parents and daughter in both families.
these results strongly suggest that distinguishability at the individual level in the post-ngs era would likely improve if more distinct str markers were added to support codis.
disease gene analysis
to verify the accuracy and efficiency of the proposed system in detecting str markers, we selected  <dig> well-known genes containing disease-related strs.
all identified strs occurred in different genetic locations including protein coding regions, 5′ utrs, 3′ utrs and introns; large variations in repeat number might be causally related to serious genetic diseases according to previous medical reports.
table  <dig> lists all details including gene names, str patterns and their genetic locations, expansion/deletion mechanisms, disease names, and references  <cit> .
range
related
thirteen disease-related str motifs located within coding, utr, and intron regions were selected as test markers.
the normal ranges of repeat number and variation types  of strs causing disease phenotypes are listed according to published references.
the polymorphisms of disease-related strs within all individuals were detected and compared, as shown in table  <dig>  the results show that  <dig> of  <dig> polymorphic strs among all  <dig> individuals could be identified, and most repeat numbers fall within the normal range.
however, three str markers could not be retrieved from two individual samples .
the unsuccessful str detection was mainly due to missing nucleotides in the consensus sequences.
figure  <dig> displays the undetected str patterns by showing alignment results of the target strs and corresponding flanking sequences between the reference profile and individual sequences.
we observed that the individual consensus sequences were filled with the character "n" at the expected repeat locations; this might be due to ngs sequencing flaws or errors caused by the sequence alignment map  tool consensus output data.
these examples of unsuccessful detection also indicate that the performance of the proposed system depends on the accuracy of ngs sequencing and reconstruction processes.
in table  <dig>  none of the remaining successfully retrieved strs showed abnormal patterns consistent with lethal diseases.
most of these regulatory strs were identified in all individuals and were matched with family inheritance characteristics.
nevertheless, from the resulting tables we observed that two str patterns located within the coding regions of the dmpk and ar genes were not consistent with heredity principles.
this phenomenon might be a result of mixed sequencing data from heterozygous alleles.
more recently developed assembly and reference mapping methods might be capable of distinguishing heterozygous alleles and overcoming such problems  <cit> .
0*
0*
0*
0*
enbl represents ensembl; na <dig> represents the ceu child, na <dig> the ceu father, and na <dig> the ceu mother; na <dig> represents the yri mother, na <dig> the yri father, and na <dig> the yri child.
the * indicates that the specific str could not be found due to sequencing errors or inconsistent reference mapping processes.
this table shows that the str pattern for the "fmr1" gene displayed obvious variations among different individuals.
polymorphic str distributions for homologous and human-unique genes
to discover and distinguish important features of polymorphic strs extracted from orthologous genes and human-unique genes within a human genome, we performed a statistical analysis of str distributions from previously collected gene sets.
in table  <dig>  the average occurrence rate of polymorphic strs in all  <dig> homologous genes is  <dig>  , which is less than the percentage of polymorphic strs in  <dig> human-unique genes with a rate of  <dig>  .
this observation suggests that characteristics of strs in homologous genes are highly conserved among various species.
in other words, if homologous genes possessing highly variable strs were conserved across species, this might lead to effects on important genetic functions.
in addition, we compared the variation rates of codis str markers, which were higher than the percentage of homologous genes but lower than the rates of human-unique genes.
we speculate that the polymorphic str patterns of these  <dig> human-unique genes should provide more identifiable str markers than codis-selected genes, and might not be related to genetic functions for human beings or provide distinguishable features for different individuals.
human-unique genes exhibited the highest variation rate compared to codis and homologous genes.
.
to observe the levels of str marker variation within homologous genes, we calculated maximum deviation  and average deviation  in base pairs.
the definitions of max.
dev.
and avg.
dev.
are denoted in eq  and eq , respectively.
max.
dev.
represents the largest number of repeat differences  of a specified str within the identical genes from any two individuals, and avg.
dev.
is obtained by taking an average-of-length difference  between all corresponding strs within the identical genes from all possible pair combinations among  <dig> individuals.
max.dev.a=max|aisk|-min|ajsk|,i≠j,sk∈ai,ajforallk
avg.dev.=∑i,i≠j7∑j7|aisk|-|ajsk| <dig>
where |aisk| is denoted as the repeat length of the str sk within the selected "a" gene from the ith individual, while |ajsk| represents for the jth individual.
we found that a total of  <dig> polymorphic str patterns were detected in  <dig> human-unique genes, in which most of the patterns were located within "intron" regions.
these results were similar to those for the codis str markers.
additional file  <dig> lists the sorted strs according to the avg.
dev.
and max.
dev.
to illustrate the differences in repeat length for each person, we selected two str patterns with large differences among  <dig> individuals.
in addition, we selected examples of polymorphic str patterns with family inheritance relationships from all detected strs.
two aligned results are shown in figure  <dig>  it is interesting to observe in table  <dig> that the polymorphic strs from codis gene sets were well-conserved in different families and individuals: a total of  <dig> defined str biomarkers within  <dig> genes displayed exactly the same repeat pattern and length, and only one or two polymorphic strs could be identified between any two individuals.
hence, how to increase distinguishability at different levels becomes an interesting challenge.
here we illustrate two str examples in figure  <dig> that showed variations in polymorphic strs at different levels; such str motifs could be further experimentally evaluated and applied to identify different individuals or groups.
according to the str variations among  <dig> human genomes, we tried to define  <dig> distinct types for comparing polymorphic strs.
the first type of polymorphic str represents a set of suitable strs for distinguishing each individual, including the query sequences coming from members of the same family.
the second type of polymorphic str demonstrates a set of identified str biomarkers obeying inheritance and could be applied to different groups.
the last type of specific str provides a set of suggested strs that reveal characteristics that are identical for the trio families but different from the other groups.
a total of  <dig> specific str biomarkers were defined from the identified  <dig> polymorphic strs within human-unique genes.
additional file  <dig> lists all  <dig> relevant str biomarkers, of which  <dig> markers appeared as a type of single nucleotide polymorphism .
all of these  <dig> strs demonstrate relatively high potential as distinguishable str biomarkers at different levels.
polymorphic str distributions in chromosomes
polymorphic strs identified from each chromosome were analyzed and compared for  <dig> individuals.
the total number of genes is  <dig>  in this study, of which  <dig> were not successfully detected due to serious sequence variations and/or query genes located at defined boundaries.
moreover, a successful rate of  <dig> % was achieved for polymorphic str analyses in this study.
in addition, we did not consider str motifs in the y chromosome since it belongs exclusively to males.
hence, we only performed a statistical analysis of the str distribution of both polymorphic and conserved strs among all acquired genomes.
figures 3a and 3b show the distributions of polymorphic strs and conserved strs within each defined chromosome, respectively.
in both figures, the x-axis represents the chromosome number, the y-axis represents the number of differences between varied strs, and the z-axis denotes the accumulated percentages of polymorphic/conserved strs in each selected chromosome.
the highest bars in the last row  in figure 3a represent all accumulated percentages of polymorphic strs for each chromosome, while the highest bars in the last row  in figure 3b represent all accumulated percentages of conserved strs.
the highest percentages of the corresponding bars from two figures should total 100% for each chromosome.
for example, the percentage of polymorphic strs in chromosome  <dig> is calculated by taking the total number of polymorphic strs  within chromosome  <dig> divided by the total number of identified strs  within chromosome  <dig>  and the average percentage of polymorphic strs in the first chromosome for  <dig> human genomes is nearly  <dig> %.
similarly, the average percentage of conserved strs for chromosome  <dig> is obtained by taking the total number of conserved strs  divided by the total number of identified strs .
after taking the average from  <dig> human genomes, the percentage of conserved strs is approximately  <dig> % for chromosome  <dig>  in other words, in chromosome  <dig>  the total number of conserved strs is more than 7-fold greater than the number of polymorphic strs.
it should be noted that the ratio of conserved strs to polymorphic strs is quite consistent for each chromosome, and the average fold change for all the different chromosomes is about  <dig> .
furthermore, we also evaluated the total length of strs , total length of selected genes , total number of genes , total number of strs , total number of polymorphic strs , density of polymorphic strs, and occurrence ratio of polymorphic strs in each chromosome.
these data are summarized in table  <dig>  which shows that the highest density of polymorphic strs was found on chromosomes  <dig> and  <dig> , and the lowest density was observed on chromosome  <dig> .
it should be noted that the occurrence ratio of polymorphic strs in each chromosome is distributed evenly within the range from  <dig> % to  <dig> %.
however, these data show non-random associations between strs and genes that could be observed from the distributions of the number of strs, the gene number and gene length on each chromosome.
for example, the total numbers of strs retrieved from chromosomes  <dig> and  <dig> are  <dig> and  <dig>  respectively, but the total numbers of genes are  <dig> and  <dig>  respectively.
as another example, the total numbers of strs retrieved from chromosomes  <dig> and  <dig> are  <dig> and  <dig>  but the total gene lengths are  <dig>  mbp and  <dig>  mbp, respectively.
greater gene lengths or higher numbers of genes do not imply the existence of repeat segments.
chr = chromosome; tlstr represents the total length of strs in a query chromosome; tlgene is the total length of selected genes; tngene is the total number of genes; tnstr is the total number of strs; tnpstr is the total number of polymorphic strs.
alternatively, highly variable str patterns among  <dig> human genomes can be determined by assessing the extent of str variations using a manhattan-like scatter plot for all human chromosomes.
the quality setting for all identified str patterns is defined as  <dig>  for this plot.
through the manhattan plot , several polymorphic str motifs exhibiting very high variation were readily apparent, and these extremely varied cases could be considered as the first choice for str biomarker candidates.
if a higher normalization threshold value for variation were assigned, fewer polymorphic str biomarker candidates would be retrieved from the plot.
for example, when the threshold value of variation was set to "6", the system replied with  <dig> important polymorphic str candidates.
these selected str candidates are located within ensg <dig>  ensg <dig>  ensg <dig>  ensg <dig>  and ensg <dig> on chromosomes  <dig>   <dig>   <dig>   <dig>  and x, respectively.
the isp online web system
we designed a comprehensive web-based system called isp for efficiently identifying polymorphic strs among different individuals.
several useful functions were designed for users to retrieve and verify all potentially important str biomarkers and compare personal strs to  <dig> published human genomes.
users can enter an ensembl gene id, gene descriptions, gene names, or any related keywords, and the system immediately responds with query results for the appropriate gene selection.
users can then select an interesting gene and a pop-up dialog for str quality and str variation settings is displayed on the web page.
for real-time analysis, only two quality values of  <dig>  and  <dig>  are currently available, and variation degrees are automatically decided and unlocked for selection depending on the selected genes.
a quality of  <dig>  indicates that all identified strs are perfect strs, while a quality of  <dig>  indicates that an identified str contains less than 10% noise including mutations, insertions, and deletions.
variation degree is calculated as the true difference in base pairs between any two polymorphic strs.
in the proposed system, users can provide customized sequences for str polymorphism analysis.
once the query sequences are uploaded, the system will apply blast+ to align the query sequence against the reference human genome.
once the query sequence is successfully aligned to one of the collected human genes, the newly identified strs within the query sequence are compared to all  <dig> human genomes for polymorphic str analyses.
the query results are exactly the same as described above.
here, the threshold for identity in blast+ was set at 99%.
such a relatively high threshold value avoids ambiguous situations caused by non-human sequences.
finally, the compared results are displayed via a tabulated interface and sent via email.
for security reasons the url was designed with embedded encryption.
the system also includes four test gene sets including disease-related genes, codis genes, homologous genes, and genes related to a go term of go: <dig>  corresponding statistical reports stored in microsoft excel files are provided in the developed system.
for online queries for interesting genes, users can click on the folder "isp datasets", and four different gene sets and their corresponding identified polymorphic strs are available for each individual gene.
to comprehensively analyze polymorphic strs for all human genes, the folder "chromosome statistics" provides  <dig> excel files, each of which contains the total number of strs, total number of polymorphic strs, total length of selected genes, total length of strs, percentages of exact genetic locations of all detected strs, percentages of different variation degrees for all polymorphic strs, and two different degrees of str quality .
all these statistics can be downloaded directly from the interface.
one example of the polymorphic str distributions on chromosome  <dig> with perfect str quality settings is shown in figure  <dig>  when comparing the yellow bars in the last row, the group percentage of polymorphisms of mono-nucleotide str motifs appears with the highest gene number, while the tri-nucleotide str motifs comprise the lowest percentage of genes.
polymorphic strs located within the coding regions  exhibit the lowest rates since the variations appearing within translated proteins might lead to different protein structures and induce deleterious effects on protein function.
the longest variation type of str among the  <dig> human genomes is the di-nucleotide str motif, which occurs within the intron regions of chromosome  <dig>  statistics for all chromosomes with different quality settings may be downloaded directly from the developed web site.
to comprehensively display the identified polymorphic strs and provide detailed information on selected genes, the system has a look-up table.
in this table, users can easily find detailed descriptions of the selected gene and identified strs.
this web page includes ensembl gene id, gene name, pattern of polymorphic strs, transcript id, and str locations within the corresponding chromosome.
in addition, the system also provides sequence files for two assembled families and reference sequences from ensembl.
because of alternative splicing mechanisms in genomes, genetic regions of identified str patterns might be affected and result in different conclusions for different transcripts.
to observe all possible scenarios, the system presents all polymorphic strs according to transcript id.
users can click on any transcript id and the identified results are immediately shown on the web page.
to rapidly identify polymorphic str patterns, users can click on a detected polymorphic pattern within the gene information table to display a corresponding message that is annotated with the locations framed in red.
to display global sequence alignments of the identified strs among the  <dig> individuals, clicking on the identified str pattern or "alignment result" automatically displays the alignment results.
through these alignment procedures, users can verify and understand the polymorphic distribution of strs among sample genomes.
the multiple sequence alignments are generated in the system by clustalw  <cit> .
CONCLUSIONS
in this study, an automated workflow for discovering str polymorphisms from individual ngs sequencing data was proposed and the developed system is freely available at http://isp.cs.ntou.edu.tw/.
the proposed algorithms started with performing reference mapping or de novo assembly of the imported ngs sequences, and the coordinate calibration was defined by mapping onto the ensembl reference human genome.
an integrated str template profile was initially created to overcome the insertions and deletions that occurred in the reference genome or other target genomes.
all possible polymorphic str patterns could be detected automatically and precisely according to the aligned coordinate system.
in this paper, polymorphic strs from several different gene sets were applied to demonstrate the proof-of-concept, including the gene set selected by codis, the disease-related gene set caused by str variations, the cross-species homologous gene set, and a human unique gene set as our evaluation datasets.
in addition, all str polymorphisms that were found within the  <dig> genomes trio project  were comprehensively identified and downloadable from the designed website.
we also performed statistical analyses on both polymorphic and conserved strs in each chromosome , and occurrence frequencies for polymorphic str variations between cross-species homologous genes and human-unique genes were compared for investigating the relationships between functional features or identifiable features of str biomarkers.
therefore, str variation frequencies for human-unique genes were clearly higher than those for cross-species conserved homologous genes, despite both gene sets exhibiting similar str distributions and densities.
this result provides an important implication in that mutations of str elements tend not to appear within highly conserved genes among different organisms during evolutionary processes, and these cross-species conserved strs could be considered more functionally related strs.
in other words, the polymorphic strs that appeared within human-unique genes could be regarded as good candidates for identifiable biometric features.
focusing on the selected  <dig> polymorphic strs from human-unique genes, three different categories were logically analyzed and suggested according to the  <dig> human genomes .
interestingly, we found some str variation characteristics from human-unique genes possessing distinguishable features that could support codis str verification.
furthermore, from genome-wide analysis and selection, we found a set of  <dig> polymorphic strs retrieved from human-unique genes that displayed relatively higher distinguishability compared to other identified strs.
in order to understand the distributions of polymorphic strs within each chromosome , we compared densities of polymorphic strs within each chromosome, and the results show that chromosome  <dig> had the highest density of polymorphic strs, while chromosome  <dig> had the lowest density.
the developed system has shown that our proposed methods could detect any polymorphic str markers efficiently, and the proposed method could take advantage of ngs high-throughput sequencing technology and detect polymorphic strs without manually curated and compared works.
in order to efficiently provide a clear view of query results for polymorphic strs for each gene, we have pre-processed all genes within all chromosomes .
users will be able to perform customized sequence comparisons online for identifying all polymorphic strs within a specified gene.
in addition, users can upload their own query sequences to compare str variations with  <dig> human genomes.
we believe that the developed system can facilitate research involving the detection of novel str biomarkers and the discovery of regulatory str elements.

 short tandem repeats, also known as short sequence repeats or microsatellites, are genome segments composed of short repeating sequences. the length of the fundamental repeat unit varies from one to six nucleotides. strs are highly abundant in many different organisms, and are distributed in both genic and intergenic regions. repeat structures expand or are deleted mainly due to replication slippage, which leads to highly polymorphic str patterns among individuals; these polymorphic features make str motifs suitable genetic markers. several str markers have been applied to individual/paternity identification and species/subspecies differentiation, while some strs are involved in gene regulatory pathways. abnormal expansion of such functional strs located within coding regions frequently cause various types of disease. even when located within non-coding regions, strs might also act as important functional regulatory elements. therefore, discoveries of polymorphic strs among different sequenced samples might be helpful for detecting useful genetic markers, while findings of well-conserved strs might lead to their identification as functional elements for gene regulation networks. in traditional approaches, genomic str markers have typically been discovered by analyzing dna sequences through in silico methods and verified by pcr. various in silico tools are available for detecting both perfect and imperfect strs within a single species. in recent years, a revolutionary development in sequencing technology called next generation sequencing has greatly impacted the growth and speed of genetic research. with relatively low costs and increased throughput, research at the genomic and transcriptomic levels has now become affordable and practical. traditional est libraries applied to est-str discovery have been gradually replaced by ngs approaches, known as rna-seq techniques, which provide extensive coverage at the whole-transcriptome level. recent publications have shown that ngs plays a low-cost and time-efficient role in polymorphic str marker discovery, even without providing reference sequences. the latest tools have also focused on str marker discovery through ngs read analysis. for example, qdd is an open-source str search tool package that provides a pipeline from raw ngs reads to str identification and corresponding primer design. hoffman and nichols also proposed a manual method for in silico str marker screening. their experiments with antarctic seals demonstrated the effectiveness of in silico str marker discovery across individual ngs samples. the lobstr program was developed by gymrek et.al., who constructed a comprehensive survey of str variations from ngs-derived personal genomes. an automated method for detecting str polymorphisms from ngs data reads could utilize the high throughput advantages of ngs without the influence of manually examined factors. in addition, we also developed a prototype system for detecting polymorphic strs within human genomes based on the conception of an str template profile. however, due to our limited knowledge, there are no online web applications that allow users to compare personal genomes or specify genes for a comprehensive str analysis. therefore, we sought to develop an efficient identification system that is capable of detecting conserved and polymorphic strs across different individual sequence reads. the proposed method could detect str polymorphisms without curated procedures, and could be directly applied for the efficient identification of conserved and polymorphic str markers and accelerate functional analysis of regulatory str motifs.  we have performed a statistical analysis of the str distributions in several datasets including chromosomal genes, combined dna index system genes, disease-related genes, cross-species homologous genes, and genes that are unique to humans. the two major reasons for performing statistical analyses on different gene sets were: to determine the most frequently appearing lengths of polymorphic/conserved str patterns, and the most frequently occurring regions of polymorphic str motifs. to understand the extent of variation in the identified strs, the distribution scale ranged from to bp. in addition, we selected the interval from to bp to analyze conserved degrees of identified strs. retrieved strs from the different datasets are shown and discussed in the following sub-sections. codis marker analysis codis is a collection of investigated and verified dna markers provided by the u.s. federal bureau of investigation to criminal justice services. thirteen str markers were examined in this program. from the verification results, our proposed system could successfully detect and list all str markers from genomes, including six individual genomes from the genomes trio project and one from the ensembl reference genome. all retrieved str markers are listed in table and it should be noted that both str markers within the gene vwa defined by codis contain multiple short repeat patterns, and the adopted cgssr program could successfully identify the three str markers "acag", "agat", and "tcca" for vwa. ensg00000182578 ensg00000011376 ensg00000249816 ensg00000180176 ensg00000171560 ensg00000171791 ensg00000115705 ensg00000110799 ensg00000075213 ensg00000168367 d5s818 d13s317 d21s11 these genomes include the reference genome from ensembl and genomes from the genomes trio project. the first ceu family: na na and na12878; the second yri family: na na and na enbl represents the genome from the ensembl database; na represents the ceu daughter, na the ceu father, and na the ceu mother; na represents the yri mother, na the yri father, and na the yri daughter. it should be noted that both str markers of vwa were identified as separate strs in this table. it was also observed that most polymorphic strs within a family group agreed with inherited characteristics, i.e., the daughter's alleles were inherited from either one of her parents. based on codis str markers for comparing these two families from the trio project, the results show that of str loci displaying identical repeat patterns/numbers among all selected individuals, and only one or two str patterns possessing minor differences in length could be found between parents and daughter in both families. these results strongly suggest that distinguishability at the individual level in the post-ngs era would likely improve if more distinct str markers were added to support codis. disease gene analysis to verify the accuracy and efficiency of the proposed system in detecting str markers, we selected well-known genes containing disease-related strs. all identified strs occurred in different genetic locations including protein coding regions, 5′ utrs, 3′ utrs and introns; large variations in repeat number might be causally related to serious genetic diseases according to previous medical reports. table lists all details including gene names, str patterns and their genetic locations, expansion/deletion mechanisms, disease names, and references. range related thirteen disease-related str motifs located within coding, utr, and intron regions were selected as test markers. the normal ranges of repeat number and variation types of strs causing disease phenotypes are listed according to published references. the polymorphisms of disease-related strs within all individuals were detected and compared, as shown in table the results show that of polymorphic strs among all individuals could be identified, and most repeat numbers fall within the normal range. however, three str markers could not be retrieved from two individual samples. the unsuccessful str detection was mainly due to missing nucleotides in the consensus sequences. figure displays the undetected str patterns by showing alignment results of the target strs and corresponding flanking sequences between the reference profile and individual sequences. we observed that the individual consensus sequences were filled with the character "n" at the expected repeat locations; this might be due to ngs sequencing flaws or errors caused by the sequence alignment map tool consensus output data. these examples of unsuccessful detection also indicate that the performance of the proposed system depends on the accuracy of ngs sequencing and reconstruction processes. in table none of the remaining successfully retrieved strs showed abnormal patterns consistent with lethal diseases. most of these regulatory strs were identified in all individuals and were matched with family inheritance characteristics. nevertheless, from the resulting tables we observed that two str patterns located within the coding regions of the dmpk and ar genes were not consistent with heredity principles. this phenomenon might be a result of mixed sequencing data from heterozygous alleles. more recently developed assembly and reference mapping methods might be capable of distinguishing heterozygous alleles and overcoming such problems. 0* 0* 0* 0* enbl represents ensembl; na represents the ceu child, na the ceu father, and na the ceu mother; na represents the yri mother, na the yri father, and na the yri child. the * indicates that the specific str could not be found due to sequencing errors or inconsistent reference mapping processes. this table shows that the str pattern for the "fmr1" gene displayed obvious variations among different individuals. polymorphic str distributions for homologous and human-unique genes to discover and distinguish important features of polymorphic strs extracted from orthologous genes and human-unique genes within a human genome, we performed a statistical analysis of str distributions from previously collected gene sets. in table the average occurrence rate of polymorphic strs in all homologous genes is, which is less than the percentage of polymorphic strs in human-unique genes with a rate of. this observation suggests that characteristics of strs in homologous genes are highly conserved among various species. in other words, if homologous genes possessing highly variable strs were conserved across species, this might lead to effects on important genetic functions. in addition, we compared the variation rates of codis str markers, which were higher than the percentage of homologous genes but lower than the rates of human-unique genes. we speculate that the polymorphic str patterns of these human-unique genes should provide more identifiable str markers than codis-selected genes, and might not be related to genetic functions for human beings or provide distinguishable features for different individuals. human-unique genes exhibited the highest variation rate compared to codis and homologous genes.. to observe the levels of str marker variation within homologous genes, we calculated maximum deviation and average deviation in base pairs. the definitions of max. dev. and avg. dev. are denoted in eq and eq, respectively. max. dev. represents the largest number of repeat differences of a specified str within the identical genes from any two individuals, and avg. dev. is obtained by taking an average-of-length difference between all corresponding strs within the identical genes from all possible pair combinations among individuals. max.dev.a=max|aisk|-min|ajsk|,i≠j,sk∈ai,ajforallk avg.dev.=∑i,i≠j7∑j7|aisk|-|ajsk| where |aisk| is denoted as the repeat length of the str sk within the selected "a" gene from the ith individual, while |ajsk| represents for the jth individual. we found that a total of polymorphic str patterns were detected in human-unique genes, in which most of the patterns were located within "intron" regions. these results were similar to those for the codis str markers. additional file lists the sorted strs according to the avg. dev. and max. dev. to illustrate the differences in repeat length for each person, we selected two str patterns with large differences among individuals. in addition, we selected examples of polymorphic str patterns with family inheritance relationships from all detected strs. two aligned results are shown in figure it is interesting to observe in table that the polymorphic strs from codis gene sets were well-conserved in different families and individuals: a total of defined str biomarkers within genes displayed exactly the same repeat pattern and length, and only one or two polymorphic strs could be identified between any two individuals. hence, how to increase distinguishability at different levels becomes an interesting challenge. here we illustrate two str examples in figure that showed variations in polymorphic strs at different levels; such str motifs could be further experimentally evaluated and applied to identify different individuals or groups. according to the str variations among human genomes, we tried to define distinct types for comparing polymorphic strs. the first type of polymorphic str represents a set of suitable strs for distinguishing each individual, including the query sequences coming from members of the same family. the second type of polymorphic str demonstrates a set of identified str biomarkers obeying inheritance and could be applied to different groups. the last type of specific str provides a set of suggested strs that reveal characteristics that are identical for the trio families but different from the other groups. a total of specific str biomarkers were defined from the identified polymorphic strs within human-unique genes. additional file lists all relevant str biomarkers, of which markers appeared as a type of single nucleotide polymorphism. all of these strs demonstrate relatively high potential as distinguishable str biomarkers at different levels. polymorphic str distributions in chromosomes polymorphic strs identified from each chromosome were analyzed and compared for individuals. the total number of genes is in this study, of which were not successfully detected due to serious sequence variations and/or query genes located at defined boundaries. moreover, a successful rate of % was achieved for polymorphic str analyses in this study. in addition, we did not consider str motifs in the y chromosome since it belongs exclusively to males. hence, we only performed a statistical analysis of the str distribution of both polymorphic and conserved strs among all acquired genomes. figures 3a and 3b show the distributions of polymorphic strs and conserved strs within each defined chromosome, respectively. in both figures, the x-axis represents the chromosome number, the y-axis represents the number of differences between varied strs, and the z-axis denotes the accumulated percentages of polymorphic/conserved strs in each selected chromosome. the highest bars in the last row in figure 3a represent all accumulated percentages of polymorphic strs for each chromosome, while the highest bars in the last row in figure 3b represent all accumulated percentages of conserved strs. the highest percentages of the corresponding bars from two figures should total 100% for each chromosome. for example, the percentage of polymorphic strs in chromosome is calculated by taking the total number of polymorphic strs within chromosome divided by the total number of identified strs within chromosome and the average percentage of polymorphic strs in the first chromosome for human genomes is nearly %. similarly, the average percentage of conserved strs for chromosome is obtained by taking the total number of conserved strs divided by the total number of identified strs. after taking the average from human genomes, the percentage of conserved strs is approximately % for chromosome in other words, in chromosome the total number of conserved strs is more than 7-fold greater than the number of polymorphic strs. it should be noted that the ratio of conserved strs to polymorphic strs is quite consistent for each chromosome, and the average fold change for all the different chromosomes is about. furthermore, we also evaluated the total length of strs, total length of selected genes, total number of genes, total number of strs, total number of polymorphic strs, density of polymorphic strs, and occurrence ratio of polymorphic strs in each chromosome. these data are summarized in table which shows that the highest density of polymorphic strs was found on chromosomes and, and the lowest density was observed on chromosome. it should be noted that the occurrence ratio of polymorphic strs in each chromosome is distributed evenly within the range from % to %. however, these data show non-random associations between strs and genes that could be observed from the distributions of the number of strs, the gene number and gene length on each chromosome. for example, the total numbers of strs retrieved from chromosomes and are and respectively, but the total numbers of genes are and respectively. as another example, the total numbers of strs retrieved from chromosomes and are and but the total gene lengths are mbp and mbp, respectively. greater gene lengths or higher numbers of genes do not imply the existence of repeat segments. chr = chromosome; tlstr represents the total length of strs in a query chromosome; tlgene is the total length of selected genes; tngene is the total number of genes; tnstr is the total number of strs; tnpstr is the total number of polymorphic strs. alternatively, highly variable str patterns among human genomes can be determined by assessing the extent of str variations using a manhattan-like scatter plot for all human chromosomes. the quality setting for all identified str patterns is defined as for this plot. through the manhattan plot, several polymorphic str motifs exhibiting very high variation were readily apparent, and these extremely varied cases could be considered as the first choice for str biomarker candidates. if a higher normalization threshold value for variation were assigned, fewer polymorphic str biomarker candidates would be retrieved from the plot. for example, when the threshold value of variation was set to "6", the system replied with important polymorphic str candidates. these selected str candidates are located within ensg ensg ensg ensg and ensg on chromosomes and x, respectively. the isp online web system we designed a comprehensive web-based system called isp for efficiently identifying polymorphic strs among different individuals. several useful functions were designed for users to retrieve and verify all potentially important str biomarkers and compare personal strs to published human genomes. users can enter an ensembl gene id, gene descriptions, gene names, or any related keywords, and the system immediately responds with query results for the appropriate gene selection. users can then select an interesting gene and a pop-up dialog for str quality and str variation settings is displayed on the web page. for real-time analysis, only two quality values of and are currently available, and variation degrees are automatically decided and unlocked for selection depending on the selected genes. a quality of indicates that all identified strs are perfect strs, while a quality of indicates that an identified str contains less than 10% noise including mutations, insertions, and deletions. variation degree is calculated as the true difference in base pairs between any two polymorphic strs. in the proposed system, users can provide customized sequences for str polymorphism analysis. once the query sequences are uploaded, the system will apply blast+ to align the query sequence against the reference human genome. once the query sequence is successfully aligned to one of the collected human genes, the newly identified strs within the query sequence are compared to all human genomes for polymorphic str analyses. the query results are exactly the same as described above. here, the threshold for identity in blast+ was set at 99%. such a relatively high threshold value avoids ambiguous situations caused by non-human sequences. finally, the compared results are displayed via a tabulated interface and sent via email. for security reasons the url was designed with embedded encryption. the system also includes four test gene sets including disease-related genes, codis genes, homologous genes, and genes related to a go term of go: corresponding statistical reports stored in microsoft excel files are provided in the developed system. for online queries for interesting genes, users can click on the folder "isp datasets", and four different gene sets and their corresponding identified polymorphic strs are available for each individual gene. to comprehensively analyze polymorphic strs for all human genes, the folder "chromosome statistics" provides excel files, each of which contains the total number of strs, total number of polymorphic strs, total length of selected genes, total length of strs, percentages of exact genetic locations of all detected strs, percentages of different variation degrees for all polymorphic strs, and two different degrees of str quality. all these statistics can be downloaded directly from the interface. one example of the polymorphic str distributions on chromosome with perfect str quality settings is shown in figure when comparing the yellow bars in the last row, the group percentage of polymorphisms of mono-nucleotide str motifs appears with the highest gene number, while the tri-nucleotide str motifs comprise the lowest percentage of genes. polymorphic strs located within the coding regions exhibit the lowest rates since the variations appearing within translated proteins might lead to different protein structures and induce deleterious effects on protein function. the longest variation type of str among the human genomes is the di-nucleotide str motif, which occurs within the intron regions of chromosome statistics for all chromosomes with different quality settings may be downloaded directly from the developed web site. to comprehensively display the identified polymorphic strs and provide detailed information on selected genes, the system has a look-up table. in this table, users can easily find detailed descriptions of the selected gene and identified strs. this web page includes ensembl gene id, gene name, pattern of polymorphic strs, transcript id, and str locations within the corresponding chromosome. in addition, the system also provides sequence files for two assembled families and reference sequences from ensembl. because of alternative splicing mechanisms in genomes, genetic regions of identified str patterns might be affected and result in different conclusions for different transcripts. to observe all possible scenarios, the system presents all polymorphic strs according to transcript id. users can click on any transcript id and the identified results are immediately shown on the web page. to rapidly identify polymorphic str patterns, users can click on a detected polymorphic pattern within the gene information table to display a corresponding message that is annotated with the locations framed in red. to display global sequence alignments of the identified strs among the individuals, clicking on the identified str pattern or "alignment result" automatically displays the alignment results. through these alignment procedures, users can verify and understand the polymorphic distribution of strs among sample genomes. the multiple sequence alignments are generated in the system by clustalw.  in this study, an automated workflow for discovering str polymorphisms from individual ngs sequencing data was proposed and the developed system is freely available at http://isp.cs.ntou.edu.tw/. the proposed algorithms started with performing reference mapping or de novo assembly of the imported ngs sequences, and the coordinate calibration was defined by mapping onto the ensembl reference human genome. an integrated str template profile was initially created to overcome the insertions and deletions that occurred in the reference genome or other target genomes. all possible polymorphic str patterns could be detected automatically and precisely according to the aligned coordinate system. in this paper, polymorphic strs from several different gene sets were applied to demonstrate the proof-of-concept, including the gene set selected by codis, the disease-related gene set caused by str variations, the cross-species homologous gene set, and a human unique gene set as our evaluation datasets. in addition, all str polymorphisms that were found within the genomes trio project were comprehensively identified and downloadable from the designed website. we also performed statistical analyses on both polymorphic and conserved strs in each chromosome, and occurrence frequencies for polymorphic str variations between cross-species homologous genes and human-unique genes were compared for investigating the relationships between functional features or identifiable features of str biomarkers. therefore, str variation frequencies for human-unique genes were clearly higher than those for cross-species conserved homologous genes, despite both gene sets exhibiting similar str distributions and densities. this result provides an important implication in that mutations of str elements tend not to appear within highly conserved genes among different organisms during evolutionary processes, and these cross-species conserved strs could be considered more functionally related strs. in other words, the polymorphic strs that appeared within human-unique genes could be regarded as good candidates for identifiable biometric features. focusing on the selected polymorphic strs from human-unique genes, three different categories were logically analyzed and suggested according to the human genomes. interestingly, we found some str variation characteristics from human-unique genes possessing distinguishable features that could support codis str verification. furthermore, from genome-wide analysis and selection, we found a set of polymorphic strs retrieved from human-unique genes that displayed relatively higher distinguishability compared to other identified strs. in order to understand the distributions of polymorphic strs within each chromosome, we compared densities of polymorphic strs within each chromosome, and the results show that chromosome had the highest density of polymorphic strs, while chromosome had the lowest density. the developed system has shown that our proposed methods could detect any polymorphic str markers efficiently, and the proposed method could take advantage of ngs high-throughput sequencing technology and detect polymorphic strs without manually curated and compared works. in order to efficiently provide a clear view of query results for polymorphic strs for each gene, we have pre-processed all genes within all chromosomes. users will be able to perform customized sequence comparisons online for identifying all polymorphic strs within a specified gene. in addition, users can upload their own query sequences to compare str variations with human genomes. we believe that the developed system can facilitate research involving the detection of novel str biomarkers and the discovery of regulatory str elements. 
we have developed an online system that efficiently identifies polymorphic strs and provides novel distinguishable str biomarkers for different levels of specificity. in addition, we analyzed str variations for highly conserved homologous genes and human-unique genes. in total polymorphic strs were identified from human-unique genes, among which strs were retrieved and clustered into three different groups for efficient comparison. high-throughput next generation sequencers have fostered new cutting-edge computing techniques for genome-scale analyses, and cross-genome comparisons have facilitated the efficient identification of polymorphic str markers for various applications. assembled contigs from next generation sequencing data were aligned and calibrated according to selected reference sequences. candidate polymorphic strs within a personal genome could be easily retrieved and compared to the constructed str profile through query keywords, gene names, or assembled contigs. short tandem repeats are abundant in human genomes. to verify identified polymorphic strs, human genomes from the genomes project were employed for comprehensive analyses, and str markers from the combined dna index system and disease-related str motifs were also applied as cases for evaluation. an automated and efficient system for detecting human polymorphic strs at the genome scale is proposed in this study.
 short tandem repeats are abundant in human genomes. numerous strs have been shown to be associated with genetic diseases and gene regulatory functions, and have been selected as genetic markers for evolutionary and forensic analyses. high-throughput next generation sequencers have fostered new cutting-edge computing techniques for genome-scale analyses, and cross-genome comparisons have facilitated the efficient identification of polymorphic str markers for various applications.  an automated and efficient system for detecting human polymorphic strs at the genome scale is proposed in this study. assembled contigs from next generation sequencing data were aligned and calibrated according to selected reference sequences. to verify identified polymorphic strs, human genomes from the genomes project were employed for comprehensive analyses, and str markers from the combined dna index system and disease-related str motifs were also applied as cases for evaluation. in addition, we analyzed str variations for highly conserved homologous genes and human-unique genes. in total polymorphic strs were identified from human-unique genes, among which strs were retrieved and clustered into three different groups for efficient comparison.  we have developed an online system that efficiently identifies polymorphic strs and provides novel distinguishable str biomarkers for different levels of specificity. candidate polymorphic strs within a personal genome could be easily retrieved and compared to the constructed str profile through query keywords, gene names, or assembled contigs. short tandem repeat genomes projectcodisnext generation sequencinggenetic diseaseorthologous genehuman-unique gene15- december the 25th international conference on genome informatics tokyo, japan 