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

Background Next 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. Findings NGS (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 1000 vs. 500 vs. 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. Conclusion Absolute 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.The current work was supported by a pilot grant from the American Cancer Society (AB and LKC), the American College of Gastroenterology Junior Faculty Development Award (AB) and Hall Family Foundation (LKC)

MicroRNA Expression can be a Promising Strategy for the Detection of Barrett's Esophagus: A Pilot Study

Clinical and Translational Gastroenterology is an open-access journal published by Nature Publishing Group.Patient outcomes for esophageal adenocarcinoma (EAC) have not improved despite huge advances in endoscopic therapy because cancers are being diagnosed late. Barrett's esophagus (BE) is the primary precursor lesion for EAC, and thus the non-endoscopic molecular diagnosis of BE can be an important approach to improve EAC outcomes if robust biomarkers for timely diagnosis are identified. MicroRNAs (miRNAs) are tissue-specific novel biomarkers that regulate gene expression and may satisfy this requirement.The current work was supported by a pilot grant from the American Cancer Society (A.B. and L.K.C.), the American College of Gastroenterology Junior Faculty Development Award (A.B.) and grants from Hall Family Foundation (L.K.C.) and Kansas IDeA Network of Biomedical Research Excellence (A.B., L.K.C.). None of the funding bodies had any role in design, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication