4.10. UMI Analysis: The Geneglobe Data Analysis Center

The GeneGlobe data analysis center (accessed on 18 February 2020, www.qiagen.com/us/shop/genes-and-pathways/data-analysis-center-overview-page/) can align and report on the QIAseq miRNA spike-ins in addition to the aligned small/miRNA/piRNA from each sample. This QIAGEN analysis tool was used to assess the effectiveness of QIAseq’s UMIs. For the synthetic miRNA samples, the option ‘other’ was chosen for mapping, while ‘human’ was chosen for the human total RNA samples during the primary data analysis. The resulting count table included UMI and raw read counts for each miRNA in the samples. Before analyzing the correlation between UMI and raw read counts, the counts were rlog transformed.

Next-generation sequencing (NGS) allows not only the quantification of known miRNAs, but also the identification and quantification of novel miRNAs, isomiRs (miRNA variants), and other small RNA species that can be functionally relevant in diseases and is therefore used as potential disease biomarker (Figure 2). miRNAs are identified by aligning the reads to miRBase (version 21), and the reads are tallied to generate total counts for each miRNA. Statistical significance (p-Value) between two or more samples was calculated to generate differential expression profiles.