micrornas ( mirnas ) , a small rna species of 22 nt in length , control gene expression and are involved in the regulation of a variety of biological processes including development , differentiation , cell proliferation , metabolism and inflammation , as well as in human diseases ( 1 ) . it is estimated that more than 60% of human protein - coding genes contain mirna target sites with their 3 utr regions ( 2 ) . mirnas can function as oncogenes or tumor suppressors , as do mir-21 and let-7 , respectively , and improved knowledge of them has been one of the defining developments in cancer research over the past decade . the abnormal expression of mirnas and dysregulation of factors that regulate mirnas contribute to the progression of tumor ( 3 ) . owing to the stability of mirnas within clinical samples , mirnas have been regarded as potential prognostic indicators in cancers ( 4 ) and as biomarkers for cancer classification ( 58 ) . many mirna signatures have been proposed for patient prognosis and clinical response and are being investigated in clinical trials ( 9 ) . the function of mirnas can be efficiently and specifically inhibited by artificial mirna mimics or antagomirs , supporting their potentials as novel therapy tactics for diseases ( 913 ) . mrx34 , a synthetic mir-34a mimic loaded in liposome , is the first mirna - based therapy and has entered the clinics for cancer therapy in 2013 ( 14 ) . in the past few years , because of the increasing use of next generation sequencing ( ngs ) , enormous amounts of small rna sequencing data have been generated from large - scale cancer projects such as the cancer genome atlas ( tcga ) and the international cancer genome consortium ( icgc ) . these data help researchers to investigate the roles of mirnas in cancer progression . however , translating such massive amounts of data into information that can be easily interpreted and accessed remains a challenge . previously , we developed ym500 ( 15 ) , a database that includes integrated pipelines for mirna quantification , isomir identification , arm switching discovery and novel mirna prediction from smrna - seq . ym500 provides researchers with integrated mirna - related information with various graphical visualization pages from 468 human and 141 mouse smrna - seq datasets via a user - friendly web interface . no particular biological question or disease in addition to including more smrna - seq results ( > 8000 tcga cancer - related datasets ) , we focus on human cancer research in this version . other new features also include novel mirna identification and validation : novel mirnas , i.e. those not included in mirbase r21 , were identified from thousands of samples , and a new strategy was developed for filtering out false - positive discoveries . especially , dozens of clip - seq datasets are provided as experimental evidence for in silico predicted novel mirnas . regarding isomirs , meta - analysis , which allows researchers to identify differentially expressed mirnas and arm - switching events in two customer - defined groups of samples . there are 8105 smrna - seq datasets from tcga , including those of primary tumors , paired normal tissues , peripheral blood mononuclear cell ( pbmc ) , recurrent tumors and metastatic tumors , incorporated into ym500v2 ( supplemental table s1 ) . raw data were downloaded from cghub ( https://cghub.ucsc.edu/ ) and were pre - processed by in - house scripts . to avoid the disturbance of reads with poor quality , we filtered out reads that do not meet the following criterion : phred score > 30 in > 90% sequence . about 3055% of reads were excluded in this step , but millions of reads still remained for each dataset . as shown in supplemental figure s1 , this filtration step dramatically improves data quality , which is important for novel mirna and isomir identification . the other pre - processing steps were the same as those followed in our previous study ( 15 ) . clinical information of each sample was manually curated by proficient clinicians based on clinical data obtained in tcga . in this version , 22 clip - seq datasets are incorporated as experimental evidence for the putative novel mirnas , and a new strategy was developed to reduce the false - positive rate . it has been noted that there are numerous advantages in conducting novel mirna prediction by performing a single analysis of the pooled data ( 1618 ) . after pre - processing , we pooled 8105 and 468 smrna - seq datasets from tcga and the gene expression omnibus ( human datasets in ym500 ) , respectively , comprising approximately 11.1 billion reads from 193 million unique sequences . when analyzing the sequences , we found that there are 76.7% of sequences with only one read in all the samples and 0.41% of sequences with more than 100 reads in 8600 samples ( supplemental figure s2 ) . we assume that if a sequence does exist in humans , multiple reads from multiple independent experiments will appear to support its existence . thus , to reduce the false - positive rate , only sequences that met the criterion of being supported by > 100 reads in > 10 independent datasets were used for novel mirna prediction according to the pipeline described in our previous study ( 15 ) . in brief , the pooled dataset was used for novel mirna prediction by mirdeep2 ( 19 ) , mireap ( 20 ) and miranalyzer ( 21 ) . after unifying the prediction results of the three algorithms and filtering out novel mirnas similar to known transcripts , 3467 putative novel mirnas were predicted by at least one algorithm and 1408 of those were supported by clip - seq data ( supplemental figure s3 ) . the alignment results of both smrna - seq and clip - seq are provided in the web interface for each putative novel mirna ( figure 1 ) . a representation of example of  novel mirna , indicating the alignment results of the reads from smrna - seq ( a ) and clip - seq ( b ) . ym500v2 adds a new function , meta - analysis , which allows researchers to identify differentially expressed mirs and arm - switching events from two user - defined , specific sets of samples . we utilize an r / bioconductor package , deseq ( 22,23 ) and the algorithm described in our previous study ( 15 ) to identify differentially expressed mirs and arm - switching events , respectively . as shown in supplemental figure s4 , users can select one or multiple datasets and define what sample type they would like to investigate , such as  primary solid tumor or solid tissue normal. in addition , we also provide a list of clinical criteria , such as icd - o-3 histology , tumor stage , distant metastasis and lymph node status , to help researchers to select a subgroup of well - defined cancer samples according to one or multiple clinical parameters . after selecting two groups of samples , users could overview the detailed clinical information of the two groups of selected samples before submitting this job to the server for real - time calculation . the user would then receive a notification email with a result i d , and could then see a visualization of the results ( figure 2 ) in the  result and download section when the job is completed . isomir section to help researchers realize the expression profile of an isomir across distinct tissues . mir-21 - 5p+ca , an isomir of mir-21 - 5p adenylated by papd5 reported to lead the degradation of mir-21 - 5p ( 24 ) , is used as an example ( figure 3 ) . figure 3a shows the percentage of samples that expressed this isomir in various tissues , and figure 3b indicates the mean expression of a specific isomir . two new charts for isomirs . panel ( a ) shows the percentage of samples that expressed the isomir in various tissues , and panel ( b ) indicates the mean expression of a specific isomir . the decreasing cost of sequencing technology has led to large amounts of small rna sequencing data from cancer - related studies . to make the most of these valuable yet massive amounts of data , we have updated our ym500 database to make it focus on the human cancer mirnome . there are more than 8000 cancer - related samples incorporated into ym500v2 , and a new function meta - analysis , which allows researchers to fully utilize these thousands of cancer samples , is also added . with the updated database , researchers will have the opportunity , for example , to identify a specific set of mirnas for a distinct question if they could define samples according to a clarified biological or clinical goal . to achieve this , our meta - analysis function allows a user to select two groups of samples using dozens of clinical criteria and then identify differentially expressed mirnas and arm - switching events for the two groups ( figure 2 ) . putative novel mirnas in ym500v2 were identified from thousands of samples , and more importantly , a new strategy was developed for filtering out false - positive discoveries . compared with the previous version of ym500 , ym500v2 has a lower number of novel mirnas ( from 11 000 to 3500 ) , but has more novel mirnas identified by three prediction algorithms ( from 90 to 189 ) . moreover , 40.61% ( 1408/3467 ) of the putative novel mirnas could be supported by clip - seq data , and a web interface displays the alignment results of both smrna - seq and clip - seq . accumulating evidence suggests that a mirna locus could produce a series of isomirs during the mirna biogenesis process ( 2427 ) . the phenomenon of isomirs contributes largely to the dynamic mirnome and coinstantaneously presents a challenge for mirna study . a few isomirs have been proven to be functional regulatory molecules ( 24,2731 ) , and more studies about isomirs should be performed and taken into consideration ( 32 ) . several in silico tools have been developed for identifying isomirs from smrna - seq data ( 3336 ) , and such tools will greatly promote the discovery of isomirs and their relevant functional roles . ym500v2 provides the existing evidence of isomirs from enormous smrna - seq datasets for researchers whenever they discover significant isomirs in their studies . ngs has become the norm for large - scale cancer research , and cancer - related smrna - seq data will accumulate rapidly in the future . we have developed a pipeline to process new data and to incorporate them into the database semiautomatically . newly incorporated smrna - seq data will also be re - annotated for meta - analysis . ministry of science and technology ( most ) [ 101 - 2320-b-010 - 059-my3 , 102 - 2314-b-010 - 045 , 102 - 2321-b-010 - 010 , 103 - 2622-b-010 - 002 ] ; national health research institutes [ nhri - ex102 - 10254si ] ; veterans general hospitals university system of taiwan ( vghust ) joint research program ; tsou 's foundation [ vghust102-g7 - 3 - 2 ] ; ust - ucsd international center for excellence in advanced bioengineering sponsored by the taiwan nsc i - rice program [ 102 - 2911-i-009 - 101 ] ; national yang - ming university ( ministry of education , aim for the top university plan ) . funding for open access charge : most [ 101 - 2320-b-010 - 059-my3 , 102 - 2314-b-010 - 045 , 102 - 2321-b-010 - 010 , 103 - 2622-b-010 - 002 ] ; national health research institutes [ nhri - ex102 - 10254si ] ; vghust joint research program ; tsou 's foundation [ vghust102-g7 - 3 - 2 ] ; ust - ucsd international center for excellence in advanced bioengineering sponsored by the taiwan nsc i - rice program [ 102 - 2911-i-009 - 101 ] ; national yang - ming university ( ministry of education , aim for the top university plan ) . conflict of interest statement . none declared .
we previously presented ym500 , which is an integrated database for mirna quantification , isomir identification , arm switching discovery and novel mirna prediction from 468 human smrna - seq datasets . here in this updated ym500v2 database ( http://ngs.ym.edu.tw/ym500/ ) , we focus on the cancer mirnome to make the database more disease - orientated . 
 new mirna - related algorithms developed after ym500 were included in ym500v2 , and , more significantly , more than 8000 cancer - related smrna - seq datasets ( including those of primary tumors , paired normal tissues , pbmc , recurrent tumors , and metastatic tumors ) were incorporated into ym500v2 . 
 novel mirnas ( mirnas not included in the mirbase r21 ) were not only predicted by three independent algorithms but also cleaned by a new in silico filtration strategy and validated by wetlab data such as cross - linked immunoprecipitation sequencing ( clip - seq ) to reduce the false - positive rate . a new function 
 meta - analysis is additionally provided for allowing users to identify real - time differentially expressed mirnas and arm - switching events according to customer - defined sample groups and dozens of clinical criteria tidying up by proficient clinicians . 
 cancer mirnas identified hold the potential for both basic research and biotech applications .