A detailed analysis of next generation sequencing reads of microRNA expression in Barrett’s Esophagus: absolute versus relative quantification

Abstract

BackgroundNext generation sequencing (NGS) is a state of the art technology for microRNA (miRNA) analysis. The quantitative interpretation of the primary output of NGS i.e. the read counts for a miRNA sequence that can vary by several orders of magnitude (1 to 107) remains incompletely understood.FindingsNGS (SOLiD 3 technology) was performed on biopsies from 6 Barrett’s esophagus (BE) and 5 Gastroesophageal Reflux Disease (GERD) patients. Read sequences were aligned to miRBase 18.0. Differential expression analysis was adjusted for false discovery rate of 5%. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for 36 miRNA in a validation cohort of 47 patients (27 BE and 20 GERD). Correlation coefficients, accuracy, precision and recall of NGS compared to qRT-PCR were calculated. Increase in NGS reads was associated with progressively lower Cq values, p < 0.05. Although absolute quantification between NGS reads and Cq values correlated modestly: -0.38, p = 0.01 for BE and -0.32, p = 0.05 for GERD, relative quantification (fold changes) of miRNA expression between BE &GERD by NGS correlated highly with qRT-PCR 0.86, p = 2.45E-11. Fold change correlations were unaffected when different thresholds of NGS read counts were compared (>1000 vs. <1000, >500 vs. <500 and >100 vs. <100). The accuracy, precision and recall of NGS to label a miRNA as differentially expressed were 0.71, 0.88 and 0.74 respectively.ConclusionAbsolute NGS reads correlated modestly with qRT-PCR but fold changes correlated highly. NGS is robust at relative but not absolute quantification of miRNA levels and accurate for high-throughput identification of differentially expressed miRNA.