BACKGROUND
biomedical research has been greatly accelerated by the advances in sequencing technologies, especially genomic research.
recently, next-generation sequencing  technology, including roche  <dig>  illumina ga and abi solid platforms, has emerged as a powerful tool for generating high-throughput sequencing data.
systematic evaluation revealed that these three platforms could possess high sequencing sensitivity because of the large number of reads obtained  <cit> .
therefore, ngs technology has been applied in many studies, including transcriptome profiling  <cit> , snp identification  <cit> , genome sequencing and re-sequencing  <cit> , biomarker detection  <cit> , and metagenomics  <cit> .
ngs technology was also applied in mirna identification and profiling studies.
morin and colleagues identified  <dig> novel human mirna genes and made a list of mirnas differentially expressed between embryo cell libraries  <cit> .
glazov discovered  <dig> new chicken mirnas and  <dig> mirtrons  <cit> .
in addition, wheeler not only sequenced mirnas from several metazoan genomes but also studied mirna’s evolution status  <cit> .
in a typical analysis pipeline, the generated ngs sequence reads are first subject to adaptor trimming and then mapping back to reference sequences, including genomes, scaffolds or transcripts.
several tools, including blast  <cit> , razers  <cit> , seqmap  <cit> , soap <dig>  <cit> , bwa  <cit> , maq  <cit>  and bowtie  <cit> , have been used for such mapping.
following the mapping step, the ngs reads are further processed to meet specific experimental interrogations.
while it is essential to process the mappable reads in subsequent studies, a fraction of sequence reads cannot be mapped back to reference sequences.
in many cases, these un-mappable reads are imputed to sequencing errors and discarded without further consideration.
with the rapid increase of ngs reads, we intend to examine the possible biological relevance and possible sources of un-mappable reads.
therefore, we have developed the un-mappable reads solution  pipeline in this study.
although un-mappable reads could originate from platform-specific technique errors, there have been reports demonstrating the possibilities of viral genomic sequences or cryptic splicing isoforms in ngs data  <cit> .
eukaryotic organisms are often infected by different viruses, leading to stable symbiosis or parasitism.
as parasites, the infecting viruses rule the infected cells to produce their own genetic materials.
therefore, the collected rna samples could be contaminated by viral transcripts when tissue or cells are lysed, which produces un-mappable reads when only the host cell genome is used for mapping.
kreuze et al detected virus infection by deep sequencing of viral small rnas  <cit> .
they concluded that ngs technology can be a method for diagnosis and discovery of virus infections.
wu et al also reached a similar conclusion  <cit> .
in kreuze’s study, in addition to the expected infecting viruses, unexpected novel virus reads and unidentified sequence reads also accounted for a large fraction of all reads.
the results from these studies demonstrate that the genomic sequences from infecting viruses may contribute to un-mappable reads, and ngs technology is useful for systematic examination of putative viral genomes.
another possible source of un-mappable reads is cryptic splicing isoforms.
during gene expression, eukaryotic genes usually undergo mrna splicing by removing introns and merging exons.
the sequence reads located at the exon-exon junctions of novel alternative splicing isoforms can be mapped back neither to the genome nor to reference mrnas.
for example, trapnell et al.
could identify novel wobble splicing junctions from ngs reads  <cit> .
however, there are no specific tools for discovering cryptic alternative splicing exon-exon junctions from large numbers of ngs reads.
at present, there is no biological user-friendly bioinformatic tool or service available focusing on the scanning of viral genomic regions or novel alternative splicing exon-exon junctions from un-mappable reads.
we believe that such a tool would be beneficial for biological science researchers.
methods and materials
collection of genomes and sequences reads
for mapping sequence reads back to viral genomes, we first downloaded  <dig> viral genomic sequences from ncbi refseq  <dig>  <cit> .
according to the categories of their hosts, these viral genomes were classified into five classes, including animals, plants, fungi, protozoan plus algae and bacteria plus archaea.
we also downloaded the genomic sequences of several animal species from the ucsc genome browser database  <cit>  for extracting exon-exon junctions.
the genomic versions of these species are listed in additional file  <dig>  in this study, sequence reads from ngs technology of several libraries were used.
the sequencing platform, rna source species, and rna source tissue of these libraries are listed in additional file  <dig>
extraction of exon-exon junction sequences
during maturation, eukaryotic genes usually undergo messenger rna splicing, producing many alternative splicing isoforms from one gene.
ucsc mapped these splicing isoforms back to genomes, and determined the boundaries and genomic coordinates of exons, recording the information in refflat files.
as shown in fig.
<dig>  from such coordinate information, we may exactly define the boundaries of exons and introns.
further, we may also define the exon-end fragments at exon’s both termini, start  or end .
by extracting and assembling the exon-end fragments, from continuous or discrete exons, we collected 60-nt exon-exon junctions .
therefore, they are either continuous or discrete eejs, where the former denote known splicing patterns and the latter denote novel ones.
as a result, the number of 60-nt eejs is  for each transcript, where n is the number of exon in each transcript.
by doing so, we collected 60-nt eejs for  <dig> species.
the refflat versions, number of eejs and scientific names of these  <dig> species are available in additional file  <dig>
sequence reads processing and mapping
ngs technologies have produced millions of reads, some of which may occur with high frequency.
such high-occurrence reads cause redundancy problems, and should be solved first.
therefore, we developed an in-house tool, called non-redundant reads producer , to solve this redundancy problem.
nrp identifies unique sequence reads from input data, assigned a new id and tabulates the occurrence frequency  of each unique read.
after nrp processing, non-redundant un-mappable reads may be mapped back to viral genomes or eejs by umars.
in the studies involved in mapping sequence reads back to genomes, 100% identity is usually demanded  <cit> .
because viral genomes usually have higher mutation rates than eukaryotic ones, we allowed one nucleotide variation, including mismatch and gap, when mapping back to viral genomes or to eej sequences.
the mapping procedures in this study were done with blast  <cit> .
prediction of viral mirnas
after the mapping procedure, the viral genomic loci mapped by sequence reads are considered as candidate mirnas.
these genomic loci and their flanking sequences were extracted, followed by alignment using mirna identification pipeline  <cit> .
for each candidate mirna, the pipeline first calculated the values of ten features, which serve as discrimination indices in a support vector machine  algorithm.
then, the svm was used as a classifier to classify candidate mirnas into positive or negative sets.
cdna preparation
in this study, we used sequence reads from l <dig> library  to scan the eejs of novel alternative splicing isoforms, followed by experimental validation of the detected eejs in  <dig> human tissues.
bellow we described how to prepare cdnas from these tissues.
human tissue poly rnas  or total rnas  purchased from clontech  were reverse-transcribed by transcriptor reverse transcriptase , primed by oligo  <dig> according to the supplier's instructions.
after the reverse transcription reaction, the mixtures were phenol-extracted once, followed by chloroform extraction.
excess primers were removed by applying the mixtures to chroma spin- <dig>  gel filtration column.
the purified cdnas were properly diluted and subjected to polymerase chain reaction  as the amplification templates.
in this study, cdnas from  <dig> tissues were investigated and they were labeled as follow: m: dna marker, 1: blood, 2: bone marrow, 3: brain, 4: colon, 5: heart, 6: kidney, 7: liver, 8: lung, 9: ovary, 10: pancreas, 11: placenta, 12: skeletal muscle, 13: small intestine, 14: stomach, 15: testis, 16: whole fetus, 17: breast tumor, 18: cervix tumor, 19: colon tumor, 20: kidney tumor, 21: lung tumor, 22: ovary tumor, 23: gastric tumor, 24: pcr no-template control.
experimental validation of discrete eej
the dtetcted eejs were verified by pcr amplification, followed by capillary sequencing confirmation.
primer pair sequences were picked from each couple of the “discrete” eej-spanning exons and were listed in additional file  <dig>  pcr components include mainly 1mm dntp, 1μm primer separately,  <dig> u takara taq dna polymerase  per 10μl reaction volume, and the diluted cdna.
the thermal reaction was set at 94°c for  <dig> minutes,  <dig> cycles  of denaturing at 94°c for  <dig> seconds, annealing at 58°c for  <dig> seconds, and extension at 72°c for  <dig> seconds, finally at 72°c for  <dig> minutes.
pcr products were separated by 3% nusieve  conventional tae-agarose gel, and visualized through the ultraviolet light source.
the detected and the estimated target size regions of the gel were cut-out and the nucleic-acid contents were purified by viogene gel purification reagents.
minor bands eluted were further subjected to additional  <dig> pcr cycles with the same pair of primer.
the amplified nucleic acid fragments were directly sequenced by abi 3730xl dna analyzer .
RESULTS
umars pipeline and interface
the main concept of umars is to provide a user-friendly solution for biologists to retrieve valuable information from discarded ngs reads that could not be mapped back to a reference genome.
we have initially produced two major datasets for interrogation, a virus genome sequence collection and an animal exon-exon junction sequence collection.
since many ngs studies intend to study mirnas, we also provided an additional portal for a mirna discovery pipeline.
the overall umars schema is illustrated in fig.
<dig>  the first step of umars deals with the redundancy problem of ngs reads.
for convenience and efficiency in data processing and network traffic, we developed an in-house tool, called non-redundant reads producer , to solve the problem.
the reads uploaded to umars must be processed by nrp in advance or be presented in the format of nrp output.
nrp accepts the files containing unmapped read sequences in the fasta format.
an nrp standalone program can be downloaded from the umars website.
next, the uploaded non-redundant ngs reads could be further processed by either umars:eej or umars:vir.
the purpose of umars:eej is to identify novel alternative splicing exon-exon junctions  from un-mappable reads.
the sequences of all possible eejs of  <dig> species were collected in advance.
in umars:eej, uploaded reads are mapped to eejs.
to avoid random sequence matches, besides our mapping criteria , a mapping match must overlap both exons for at least five nucleotides, not skewing too much to either exon.
following the mapping procedure, umars tabulats detected eejs and their expression levels.
the detected eejs are reported as either continuous or discrete eejs.
continuous eejs represent known mrna transcripts.
however, discrete eejs could represent novel splicing isoforms.
the purpose of umars:vir is to identify possible virus genomic regions from un-mappable ngs reads.
in umars:vir, the uploaded ngs reads are mapped to all  <dig>  known virus genomes.
following the mapping procedure, umars tabulates detected virus species and their expression levels.
the detected viral genomic regions may locate at intergenic, protein-coding gene, pre-mirna regions and so on according to the annotations of refseq  <dig> and mirbase  <dig>  such information of genomic annotation is also provided by the umars service.
several viruses are reported to encode viral mirnas, regulating expression of host genes and playing important roles in host immune misfunctions  <cit> .
therefore, umars:vir may further have the option to detect viral mirnas by an additional mirna identification pipeline from viral intergenic genomic regions.
the umars service is freely available to the academic community.
users may access umars via http://musk.ibms.sinica.edu.tw/umars/.
the non-redundant reads uploaded into umars must not exceed  <dig> mb in size.
such size limitation has nothing to do with pipeline performance but reduces the network load.
as shown in fig.
3a, in umars:vir, users can select the host category  of their expected virus.
in umars:eej, users can select the species corresponding to the eejs .
the umars results will be sent back to the users via e-mail.
cn denotes copy number of total reads mappable to the virus; rn denotes the number of distinct genomic regions mapped by reads.
the values in parentheses denote the corresponding values when no mismatch was allowed in the mapping procedure.
because their high similarity, most of the reads of type  <dig> ebv were also mapped back to type  <dig> ebv.
case study and demonstration of umars:vir
to demonstrate the utility of umars, we have analyzed ngs reads using umars:vir.
in the first case, we investigated the un-mappable reads from the human npc cells  infected with epstein-barr virus .
we examined whether the expected ebv genome could be detected by umars:vir.
as a result, eight viruses were detected under our mapping criteria.
as shown in table  <dig>  the expected ebv matches dominated over other un-expected viruses in terms of expression level, which shows that umars:vir can be used to detect infections by a specific virus from un-mappable reads.
besides ebv, there were seven un-expected viruses detected, most of which infect primates, and all of them belong to the herpes virus family.
as demonstrated in table  <dig>   <dig>  reads were mapped back to ebv under our mapping criteria.
these ebv reads spanned the ebv genome at  <dig> genomic regions.
the genomic contents, intergenic or intragenic, and detailed information about these regions are listed in additional file  <dig>  as shown in table  <dig> and additional file  <dig>   <dig> % of these regions are located at the inter-genic regions;  <dig> % at the ebv pre-mirna regions; and only  <dig> % at the protein-coding regions.
as well,  <dig> out of  <dig> protein-coding genes and  <dig>  out of  <dig> pre-mirnas were detected, respectively.
this mirna dominance phenomenon was observed because the l <dig> sample rna was extracted with a small rna kit rather than with a transcriptome kit.
the regions mapped by reads were classified into three categories according to the annotation of refseq  <dig> and mirbase  <dig>  pre-mirna regions dominated over other regions in terms of copy number and region number.
because of the strong sequencing intensity, additional mature mirnas from the same precursors, including isomirs at the same arm and minor forms of mature mirna at the opposite arm, are usually detected from ngs reads  <cit> .
after arranging mirna reads in order within their corresponding pre-mirnas, we observed many isomirs at all of the  <dig> detected pre-mirnas .
compared with ebv pre-mirnas in mirbase  <dig>  the reference mature mirnas do not always represent the most abundant reads.
in addition, according to mirbase  <dig> annotation, mir-bart <dig>  mir-bart <dig> and mir-bart <dig> encode mature mirnas only at their 3’, 5’ and 3’ arm respectively.
however, we detected additional mature mirnas at the 5’ arm of mir-bart <dig>  the 3’ arm of mir-bart <dig> and the 5’ arm of mir-bart <dig>  moreover, the 5’ arm of mir-bart <dig> and the 3’ arm of mir-bart <dig> encode more reads than the original arms.
this result is similar to that in wheeler’s report  <cit>  and should be noted in future data updates of the mirbase.
case study and demonstration of umars:eej
in addition to virus infections, splicing exon-exon junctions of gene transcripts may also contribute to un-mappable reads.
we, therefore, had the un-mappable reads from human h <dig> cells  processed by umars:eej.
as a result,  <dig>  un-mappable reads were mapped back to  <dig>  eejs, extracted from  <dig>  transcripts .
on further examination,  <dig> % of the detected eejs were continuous ones, which indicated they were the major mrna transcription forms.
the digits in parenthesis denote the values when no mismatch was allowed in the mapping procedure.
continuous eej dominated over discrete ones in terms of read copy number and est abundance.
however, there were  <dig>  discrete eejs detected by umars:eej from the l <dig> library.
we randomly selected five detected eejs for pcr validation and three of them were successfully validated.
referring to the ucsc genome browser, some discrete eejs had est evidence, although these splicing isoforms were not deposited in refseq.
as shown in fig.
4a, four ests, including bu <dig>  d <dig>  d <dig> and d <dig>  matched the detected eej from nm_ <dig> , which was assembled from the exon-end fragments of exon  <dig> and exon  <dig>  we further designed a pcr experiment in which the forward and reverse primers were located at exon  <dig> and exon  <dig>  respectively .
as a result, we succeeded in detecting the expected fragment  of the alternative transcript in most of the  <dig> tissues , which demonstrated that the eej, nm_ <dig> , occurred almost universally.
the sequencing result also proved the authenticity of this discrete transcript .
another two detected eejs, nm_ <dig> and nm_ <dig>  were also successfully validated .
nm_ <dig> and nm_ <dig> are splicing isoforms of clstn <dig>  the eej  from nm_ <dig> is identical to the eej  from nm_ <dig>
myl <dig>  encodes a myosin alkali light chain and is associated with cell migration  <cit> .
it was also reported that fibroblasts promote the growth of breast tumor cells by enhancing the expression of several genes, including myl <dig>  <cit> .
in this study, c alternative transcript and d transcript of myl <dig> have similar expression levels in most normal tissues .
however, the d transcript dominates over c alternative transcript in most tumor tissues, including breast tumor .
it is possible that these alternative splicing isoforms function differently with each other and are associated with tumor genesis.
CONCLUSIONS
with the rapid increase of sequencing data, umars can detect more and more un-expected splicing isoforms which may provide us insights deeper into gene functions and relations to disease.
although ngs technology has been considered a powerful sequencing tool in biological research, large-scale studies, such as those using microarrays, seem to produce un-expected data unavoidably.
such un-expected data could be background noise, and should be eliminated for data accuracy.
in this study, we have shown that some un-mappable reads are not caused by sequencing errors.
they can originate from viral infection or transcript splicing.
our umars pipeline provides another way to examine and recycle the un-mappable reads that are commonly discarded as garbage.
although we have proposed two possible sources for generating un-mappable redas, a fraction of un-mappable reads still failed to be detected by umars.
more effort should be expended in investigating the biological relevance and possible sources of un-mappable reads.
authors’ contributions
scl and wcc executed this study and wrote the draft of this manuscript.
wcc and cnh were responsible for construction of umars interface.
chl did the jobs of cdna preparation and pcr experiments.
ysj and hcc provided the solid read data.
kwt and chc participated in discussion on research design.
wcl supervised the study and edited the manuscript.
competing interests
the authors declare that they have no competing interests.
supplementary material
additional file 1
the refflat version, genome version, number of transcript, number of exon-exon junction and scientific names of the  <dig> species.
click here for file
additional file 2
the sequencing platform, rna source species, rna source tissue of these libraries.
click here for file
additional file 3
primer sequences involved in this study.
click here for file
additional file 4
the mapped reads and their corresponding genomic loci.
in the region info column, “-” denoted intergenic regions without known gene annotation.
sn.
denoted serial numbers of each mapping match.
click here for file
additional file 5
the detected isomirs of ebv pre-mirnas.
click here for file
additional file 6
pcr experimental validation of the detected eej, nm_ <dig> .
the description of the marker lane and the abbreviations are the same with the ones of fig.
4c.
the forward and reverse primers were located at exon  <dig> and exon  <dig>  respectively.
the pcr result showed that the expected eej  can be experimentally detected.
besides, the major c and the minor d transcripts, the c alternative transcript  was also detected.
both of the detected c alternative and d transcript have est evidences.
the sequencing result provided the authenticity of the detected eejs.
click here for file
additional file 7
pcr experimental validation of the detected eej, nm_ <dig> .
the description of the marker lane and the abbreviations are the same with the ones of fig.
4c.
the forward and reverse primers were located at exon  <dig> and exon  <dig>  respectively.
the pcr result showed that the expected eej  can be experimentally detected.
a c alternative transcript  was also detected.
in addition the d <dig> transcript , we also detected a d <dig> transcript .
the sequencing result provided the authenticity of the detected eejs.
click here for file
additional file 8
the information of all detected discrete eejs.
values in this table are tab separated and can be opened with excel.
click here for file
acknowledgements
this work was supported by grants from academia sinica and national science council of taiwan.
this article has been published as part of bmc bioinformatics volume  <dig> supplement  <dig>  2011: selected articles from the ninth asia pacific bioinformatics conference .
the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/12?issue=s <dig>

 biomedical research has been greatly accelerated by the advances in sequencing technologies, especially genomic research. recently, next-generation sequencing technology, including roche illumina ga and abi solid platforms, has emerged as a powerful tool for generating high-throughput sequencing data. systematic evaluation revealed that these three platforms could possess high sequencing sensitivity because of the large number of reads obtained. therefore, ngs technology has been applied in many studies, including transcriptome profiling, snp identification, genome sequencing and re-sequencing, biomarker detection, and metagenomics. ngs technology was also applied in mirna identification and profiling studies. morin and colleagues identified novel human mirna genes and made a list of mirnas differentially expressed between embryo cell libraries. glazov discovered new chicken mirnas and mirtrons. in addition, wheeler not only sequenced mirnas from several metazoan genomes but also studied mirna’s evolution status. in a typical analysis pipeline, the generated ngs sequence reads are first subject to adaptor trimming and then mapping back to reference sequences, including genomes, scaffolds or transcripts. several tools, including blast, razers, seqmap, soap, bwa, maq and bowtie, have been used for such mapping. following the mapping step, the ngs reads are further processed to meet specific experimental interrogations. while it is essential to process the mappable reads in subsequent studies, a fraction of sequence reads cannot be mapped back to reference sequences. in many cases, these un-mappable reads are imputed to sequencing errors and discarded without further consideration. with the rapid increase of ngs reads, we intend to examine the possible biological relevance and possible sources of un-mappable reads. therefore, we have developed the un-mappable reads solution pipeline in this study. although un-mappable reads could originate from platform-specific technique errors, there have been reports demonstrating the possibilities of viral genomic sequences or cryptic splicing isoforms in ngs data. eukaryotic organisms are often infected by different viruses, leading to stable symbiosis or parasitism. as parasites, the infecting viruses rule the infected cells to produce their own genetic materials. therefore, the collected rna samples could be contaminated by viral transcripts when tissue or cells are lysed, which produces un-mappable reads when only the host cell genome is used for mapping. kreuze et al detected virus infection by deep sequencing of viral small rnas. they concluded that ngs technology can be a method for diagnosis and discovery of virus infections. wu et al also reached a similar conclusion. in kreuze’s study, in addition to the expected infecting viruses, unexpected novel virus reads and unidentified sequence reads also accounted for a large fraction of all reads. the results from these studies demonstrate that the genomic sequences from infecting viruses may contribute to un-mappable reads, and ngs technology is useful for systematic examination of putative viral genomes. another possible source of un-mappable reads is cryptic splicing isoforms. during gene expression, eukaryotic genes usually undergo mrna splicing by removing introns and merging exons. the sequence reads located at the exon-exon junctions of novel alternative splicing isoforms can be mapped back neither to the genome nor to reference mrnas. for example, trapnell et al. could identify novel wobble splicing junctions from ngs reads. however, there are no specific tools for discovering cryptic alternative splicing exon-exon junctions from large numbers of ngs reads. at present, there is no biological user-friendly bioinformatic tool or service available focusing on the scanning of viral genomic regions or novel alternative splicing exon-exon junctions from un-mappable reads. we believe that such a tool would be beneficial for biological science researchers. methods and materials collection of genomes and sequences reads for mapping sequence reads back to viral genomes, we first downloaded viral genomic sequences from ncbi refseq. according to the categories of their hosts, these viral genomes were classified into five classes, including animals, plants, fungi, protozoan plus algae and bacteria plus archaea. we also downloaded the genomic sequences of several animal species from the ucsc genome browser database for extracting exon-exon junctions. the genomic versions of these species are listed in additional file in this study, sequence reads from ngs technology of several libraries were used. the sequencing platform, rna source species, and rna source tissue of these libraries are listed in additional file extraction of exon-exon junction sequences during maturation, eukaryotic genes usually undergo messenger rna splicing, producing many alternative splicing isoforms from one gene. ucsc mapped these splicing isoforms back to genomes, and determined the boundaries and genomic coordinates of exons, recording the information in refflat files. as shown in fig. from such coordinate information, we may exactly define the boundaries of exons and introns. further, we may also define the exon-end fragments at exon’s both termini, start or end. by extracting and assembling the exon-end fragments, from continuous or discrete exons, we collected 60-nt exon-exon junctions. therefore, they are either continuous or discrete eejs, where the former denote known splicing patterns and the latter denote novel ones. as a result, the number of 60-nt eejs is for each transcript, where n is the number of exon in each transcript. by doing so, we collected 60-nt eejs for species. the refflat versions, number of eejs and scientific names of these species are available in additional file sequence reads processing and mapping ngs technologies have produced millions of reads, some of which may occur with high frequency. such high-occurrence reads cause redundancy problems, and should be solved first. therefore, we developed an in-house tool, called non-redundant reads producer, to solve this redundancy problem. nrp identifies unique sequence reads from input data, assigned a new id and tabulates the occurrence frequency of each unique read. after nrp processing, non-redundant un-mappable reads may be mapped back to viral genomes or eejs by umars. in the studies involved in mapping sequence reads back to genomes, 100% identity is usually demanded. because viral genomes usually have higher mutation rates than eukaryotic ones, we allowed one nucleotide variation, including mismatch and gap, when mapping back to viral genomes or to eej sequences. the mapping procedures in this study were done with blast. prediction of viral mirnas after the mapping procedure, the viral genomic loci mapped by sequence reads are considered as candidate mirnas. these genomic loci and their flanking sequences were extracted, followed by alignment using mirna identification pipeline. for each candidate mirna, the pipeline first calculated the values of ten features, which serve as discrimination indices in a support vector machine algorithm. then, the svm was used as a classifier to classify candidate mirnas into positive or negative sets. cdna preparation in this study, we used sequence reads from l library to scan the eejs of novel alternative splicing isoforms, followed by experimental validation of the detected eejs in human tissues. bellow we described how to prepare cdnas from these tissues. human tissue poly rnas or total rnas purchased from clontech were reverse-transcribed by transcriptor reverse transcriptase, primed by oligo according to the supplier's instructions. after the reverse transcription reaction, the mixtures were phenol-extracted once, followed by chloroform extraction. excess primers were removed by applying the mixtures to chroma spin- gel filtration column. the purified cdnas were properly diluted and subjected to polymerase chain reaction as the amplification templates. in this study, cdnas from tissues were investigated and they were labeled as follow: m: dna marker, 1: blood, 2: bone marrow, 3: brain, 4: colon, 5: heart, 6: kidney, 7: liver, 8: lung, 9: ovary, 10: pancreas, 11: placenta, 12: skeletal muscle, 13: small intestine, 14: stomach, 15: testis, 16: whole fetus, 17: breast tumor, 18: cervix tumor, 19: colon tumor, 20: kidney tumor, 21: lung tumor, 22: ovary tumor, 23: gastric tumor, 24: pcr no-template control. experimental validation of discrete eej the dtetcted eejs were verified by pcr amplification, followed by capillary sequencing confirmation. primer pair sequences were picked from each couple of the “discrete” eej-spanning exons and were listed in additional file pcr components include mainly 1mm dntp, 1μm primer separately, u takara taq dna polymerase per 10μl reaction volume, and the diluted cdna. the thermal reaction was set at 94°c for minutes, cycles of denaturing at 94°c for seconds, annealing at 58°c for seconds, and extension at 72°c for seconds, finally at 72°c for minutes. pcr products were separated by 3% nusieve conventional tae-agarose gel, and visualized through the ultraviolet light source. the detected and the estimated target size regions of the gel were cut-out and the nucleic-acid contents were purified by viogene gel purification reagents. minor bands eluted were further subjected to additional pcr cycles with the same pair of primer. the amplified nucleic acid fragments were directly sequenced by abi 3730xl dna analyzer.  umars pipeline and interface the main concept of umars is to provide a user-friendly solution for biologists to retrieve valuable information from discarded ngs reads that could not be mapped back to a reference genome. we have initially produced two major datasets for interrogation, a virus genome sequence collection and an animal exon-exon junction sequence collection. since many ngs studies intend to study mirnas, we also provided an additional portal for a mirna discovery pipeline. the overall umars schema is illustrated in fig. the first step of umars deals with the redundancy problem of ngs reads. for convenience and efficiency in data processing and network traffic, we developed an in-house tool, called non-redundant reads producer, to solve the problem. the reads uploaded to umars must be processed by nrp in advance or be presented in the format of nrp output. nrp accepts the files containing unmapped read sequences in the fasta format. an nrp standalone program can be downloaded from the umars website. next, the uploaded non-redundant ngs reads could be further processed by either umars:eej or umars:vir. the purpose of umars:eej is to identify novel alternative splicing exon-exon junctions from un-mappable reads. the sequences of all possible eejs of species were collected in advance. in umars:eej, uploaded reads are mapped to eejs. to avoid random sequence matches, besides our mapping criteria, a mapping match must overlap both exons for at least five nucleotides, not skewing too much to either exon. following the mapping procedure, umars tabulats detected eejs and their expression levels. the detected eejs are reported as either continuous or discrete eejs. continuous eejs represent known mrna transcripts. however, discrete eejs could represent novel splicing isoforms. the purpose of umars:vir is to identify possible virus genomic regions from un-mappable ngs reads. in umars:vir, the uploaded ngs reads are mapped to all known virus genomes. following the mapping procedure, umars tabulates detected virus species and their expression levels. the detected viral genomic regions may locate at intergenic, protein-coding gene, pre-mirna regions and so on according to the annotations of refseq and mirbase such information of genomic annotation is also provided by the umars service. several viruses are reported to encode viral mirnas, regulating expression of host genes and playing important roles in host immune misfunctions. therefore, umars:vir may further have the option to detect viral mirnas by an additional mirna identification pipeline from viral intergenic genomic regions. the umars service is freely available to the academic community. users may access umars via http://musk.ibms.sinica.edu.tw/umars/. the non-redundant reads uploaded into umars must not exceed mb in size. such size limitation has nothing to do with pipeline performance but reduces the network load. as shown in fig. 3a, in umars:vir, users can select the host category of their expected virus. in umars:eej, users can select the species corresponding to the eejs. the umars results will be sent back to the users via e-mail. cn denotes copy number of total reads mappable to the virus; rn denotes the number of distinct genomic regions mapped by reads. the values in parentheses denote the corresponding values when no mismatch was allowed in the mapping procedure. because their high similarity, most of the reads of type ebv were also mapped back to type ebv. case study and demonstration of umars:vir to demonstrate the utility of umars, we have analyzed ngs reads using umars:vir. in the first case, we investigated the un-mappable reads from the human npc cells infected with epstein-barr virus. we examined whether the expected ebv genome could be detected by umars:vir. as a result, eight viruses were detected under our mapping criteria. as shown in table the expected ebv matches dominated over other un-expected viruses in terms of expression level, which shows that umars:vir can be used to detect infections by a specific virus from un-mappable reads. besides ebv, there were seven un-expected viruses detected, most of which infect primates, and all of them belong to the herpes virus family. as demonstrated in table reads were mapped back to ebv under our mapping criteria. these ebv reads spanned the ebv genome at genomic regions. the genomic contents, intergenic or intragenic, and detailed information about these regions are listed in additional file as shown in table and additional file % of these regions are located at the inter-genic regions; % at the ebv pre-mirna regions; and only % at the protein-coding regions. as well, out of protein-coding genes and out of pre-mirnas were detected, respectively. this mirna dominance phenomenon was observed because the l sample rna was extracted with a small rna kit rather than with a transcriptome kit. the regions mapped by reads were classified into three categories according to the annotation of refseq and mirbase pre-mirna regions dominated over other regions in terms of copy number and region number. because of the strong sequencing intensity, additional mature mirnas from the same precursors, including isomirs at the same arm and minor forms of mature mirna at the opposite arm, are usually detected from ngs reads. after arranging mirna reads in order within their corresponding pre-mirnas, we observed many isomirs at all of the detected pre-mirnas. compared with ebv pre-mirnas in mirbase the reference mature mirnas do not always represent the most abundant reads. in addition, according to mirbase annotation, mir-bart mir-bart and mir-bart encode mature mirnas only at their 3’, 5’ and 3’ arm respectively. however, we detected additional mature mirnas at the 5’ arm of mir-bart the 3’ arm of mir-bart and the 5’ arm of mir-bart moreover, the 5’ arm of mir-bart and the 3’ arm of mir-bart encode more reads than the original arms. this result is similar to that in wheeler’s report and should be noted in future data updates of the mirbase. case study and demonstration of umars:eej in addition to virus infections, splicing exon-exon junctions of gene transcripts may also contribute to un-mappable reads. we, therefore, had the un-mappable reads from human h cells processed by umars:eej. as a result, un-mappable reads were mapped back to eejs, extracted from transcripts. on further examination, % of the detected eejs were continuous ones, which indicated they were the major mrna transcription forms. the digits in parenthesis denote the values when no mismatch was allowed in the mapping procedure. continuous eej dominated over discrete ones in terms of read copy number and est abundance. however, there were discrete eejs detected by umars:eej from the l library. we randomly selected five detected eejs for pcr validation and three of them were successfully validated. referring to the ucsc genome browser, some discrete eejs had est evidence, although these splicing isoforms were not deposited in refseq. as shown in fig. 4a, four ests, including bu d d and d matched the detected eej from nm_, which was assembled from the exon-end fragments of exon and exon we further designed a pcr experiment in which the forward and reverse primers were located at exon and exon respectively. as a result, we succeeded in detecting the expected fragment of the alternative transcript in most of the tissues, which demonstrated that the eej, nm_, occurred almost universally. the sequencing result also proved the authenticity of this discrete transcript. another two detected eejs, nm_ and nm_ were also successfully validated. nm_ and nm_ are splicing isoforms of clstn the eej from nm_ is identical to the eej from nm_ myl encodes a myosin alkali light chain and is associated with cell migration. it was also reported that fibroblasts promote the growth of breast tumor cells by enhancing the expression of several genes, including myl. in this study, c alternative transcript and d transcript of myl have similar expression levels in most normal tissues. however, the d transcript dominates over c alternative transcript in most tumor tissues, including breast tumor. it is possible that these alternative splicing isoforms function differently with each other and are associated with tumor genesis.  with the rapid increase of sequencing data, umars can detect more and more un-expected splicing isoforms which may provide us insights deeper into gene functions and relations to disease. although ngs technology has been considered a powerful sequencing tool in biological research, large-scale studies, such as those using microarrays, seem to produce un-expected data unavoidably. such un-expected data could be background noise, and should be eliminated for data accuracy. in this study, we have shown that some un-mappable reads are not caused by sequencing errors. they can originate from viral infection or transcript splicing. our umars pipeline provides another way to examine and recycle the un-mappable reads that are commonly discarded as garbage. although we have proposed two possible sources for generating un-mappable redas, a fraction of un-mappable reads still failed to be detected by umars. more effort should be expended in investigating the biological relevance and possible sources of un-mappable reads. authors’ contributions scl and wcc executed this study and wrote the draft of this manuscript. wcc and cnh were responsible for construction of umars interface. chl did the jobs of cdna preparation and pcr experiments. ysj and hcc provided the solid read data. kwt and chc participated in discussion on research design. wcl supervised the study and edited the manuscript. competing interests the authors declare that they have no competing interests. supplementary material additional file 1 the refflat version, genome version, number of transcript, number of exon-exon junction and scientific names of the species. click here for file additional file 2 the sequencing platform, rna source species, rna source tissue of these libraries. click here for file additional file 3 primer sequences involved in this study. click here for file additional file 4 the mapped reads and their corresponding genomic loci. in the region info column, “-” denoted intergenic regions without known gene annotation. sn. denoted serial numbers of each mapping match. click here for file additional file 5 the detected isomirs of ebv pre-mirnas. click here for file additional file 6 pcr experimental validation of the detected eej, nm_. the description of the marker lane and the abbreviations are the same with the ones of fig. 4c. the forward and reverse primers were located at exon and exon respectively. the pcr result showed that the expected eej can be experimentally detected. besides, the major c and the minor d transcripts, the c alternative transcript was also detected. both of the detected c alternative and d transcript have est evidences. the sequencing result provided the authenticity of the detected eejs. click here for file additional file 7 pcr experimental validation of the detected eej, nm_. the description of the marker lane and the abbreviations are the same with the ones of fig. 4c. the forward and reverse primers were located at exon and exon respectively. the pcr result showed that the expected eej can be experimentally detected. a c alternative transcript was also detected. in addition the d transcript, we also detected a d transcript. the sequencing result provided the authenticity of the detected eejs. click here for file additional file 8 the information of all detected discrete eejs. values in this table are tab separated and can be opened with excel. click here for file acknowledgements this work was supported by grants from academia sinica and national science council of taiwan. this article has been published as part of bmc bioinformatics volume supplement 2011: selected articles from the ninth asia pacific bioinformatics conference. the full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/12?issue=s 
we are investigating possible biological relevance and possible sources of un-mappable reads. the umars service is freely available to the academic community and can be accessed via http://musk.ibms.sinica.edu.tw/umars/. for mapping un-mappable reads, we first collected viral genomes and sequences of exon-exon junctions. they can originate from viral infection or transcript splicing. un-mappable reads solution is a user-friendly web service focusing on retrieving valuable information from sequence reads that cannot be mapped back to reference genomes. therefore, we developed umars to scan for virus genomic fragments or exon-exon junctions of novel alternative splicing isoforms from un-mappable reads. second, we also detected exon-exon junctions from un-mappable reads. further experimental validation also ensured the authenticity of the umars pipeline. by demonstrating the results of two umars alignment cases, we show the applicability of umars. in a typical analysis, adaptor-trimmed ngs reads were first mapped back to reference sequences, including genomes or transcripts. however, a fraction of ngs reads failed to be mapped back to the reference sequences. recently, next-generation sequencing technology has emerged as a powerful tool for generating high-throughput sequencing data and has been applied to many kinds of biological research. then, we constructed umars pipeline as an automatic alignment interface. our umars pipeline provides another way to examine and recycle the un-mappable reads that are commonly discarded as garbage.
 un-mappable reads solution is a user-friendly web service focusing on retrieving valuable information from sequence reads that cannot be mapped back to reference genomes. recently, next-generation sequencing technology has emerged as a powerful tool for generating high-throughput sequencing data and has been applied to many kinds of biological research. in a typical analysis, adaptor-trimmed ngs reads were first mapped back to reference sequences, including genomes or transcripts. however, a fraction of ngs reads failed to be mapped back to the reference sequences. such un-mappable reads are usually imputed to sequencing errors and discarded without further consideration. methods we are investigating possible biological relevance and possible sources of un-mappable reads. therefore, we developed umars to scan for virus genomic fragments or exon-exon junctions of novel alternative splicing isoforms from un-mappable reads. for mapping un-mappable reads, we first collected viral genomes and sequences of exon-exon junctions. then, we constructed umars pipeline as an automatic alignment interface.  by demonstrating the results of two umars alignment cases, we show the applicability of umars. we first showed that the expected ebv genomic fragments can be detected by umars. second, we also detected exon-exon junctions from un-mappable reads. further experimental validation also ensured the authenticity of the umars pipeline. the umars service is freely available to the academic community and can be accessed via http://musk.ibms.sinica.edu.tw/umars/.  in this study, we have shown that some un-mappable reads are not caused by sequencing errors. they can originate from viral infection or transcript splicing. our umars pipeline provides another way to examine and recycle the un-mappable reads that are commonly discarded as garbage. 11- january the ninth asia pacific bioinformatics conference inchon, korea 