title: Analysis options for high-throughput sequencing in miRNA expression profiling.
abstract: BACKGROUND
  Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling.Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression.
  
  
  METHODS
  We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors.
  
  
  RESULTS
  High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers.
  
  
  CONCLUSIONS
  Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Stokowy
authors.forename: Markus
authors.initials: M
authors.lastname: Eszlinger
authors.forename: Michał
authors.initials: M
authors.lastname: Świerniak
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Fujarewicz
authors.forename: Barbara
authors.initials: B
authors.lastname: Jarząb
authors.forename: Ralf
authors.initials: R
authors.lastname: Paschke
authors.affiliation: Interdisciplinary Center for Clinical Research (IZKF), University of Leipzig, Liebigstr, 21, 04103 Leipzig, Germany. krok@medizin.uni-leipzig.de.
authors.forename: Knut
authors.initials: K
authors.lastname: Krohn
mesh_terms: D018263:Adenocarcinoma, Follicular / Q000175:diagnosis / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000706:statistics & numerical data*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013964:Thyroid Neoplasms / Q000175:diagnosis / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D035683:MicroRNAs; C554293:MIRN144 microRNA, human; C546085:MIRN197 microRNA, human; C525709:MIR221, human; C525710:MIR222, human