Identifying transcriptional miRNA biomarkers by integrating high-throughput sequencing and real-time PCR data

Using both high-throughput sequencing and real-time PCR, the miRNA transcriptome can be analyzed in complementary ways. We describe the necessary bioinformatics pipeline, including software tools, and key methodological steps in the process, such as adapter removal, read mapping, normalization, and multiple testing issues for biomarker identification. The methods are exemplified by the analysis of five favorable (event-free survival) vs. five unfavorable (died of disease) neuroblastoma tumor samples with a total of over 188 million reads.