A detailed analysis of next generation sequencing reads of microRNA expression in Barrett’s Esophagus: absolute versus relative quantification
dc.contributor.author | Lee, In-Hee | |
dc.contributor.author | Hong, Xiaoman | |
dc.contributor.author | Mathur, Sharad C. | |
dc.contributor.author | Sharma, Mukut | |
dc.contributor.author | Rastogi, Amit | |
dc.contributor.author | Sharma, Prateek | |
dc.contributor.author | Christenson, Lane K. | |
dc.contributor.author | Bansal, Ajay | |
dc.date.accessioned | 2014-05-01T18:41:05Z | |
dc.date.available | 2014-05-01T18:41:05Z | |
dc.date.issued | 2014-04-04 | |
dc.identifier.citation | Lee, In- Hee, Xiaoman Hong, Sharad C Mathur, Mukut Sharma, Amit Rastogi, Prateek Sharma, Lane K Christenson and Ajay Bansal (2014) “A detailed analysis of next generation sequencing reads of microRNA expression in Barrett’s Esophagus: absolute versus relative quantification.” BMC Research Notes 2014, 7:212 http://dx.doi.org/10.1186/1756-0500-7-212 | |
dc.identifier.uri | http://hdl.handle.net/1808/13615 | |
dc.description.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. | |
dc.description.sponsorship | 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). | |
dc.publisher | BioMed Central | |
dc.rights | This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. | |
dc.rights.uri | http://creativecommons.org/licenses/by/4.0 | |
dc.subject | Next generation sequencing | |
dc.subject | MicrioRNA | |
dc.subject | qRT-PCR | |
dc.subject | Correlation | |
dc.subject | Barrett's esophagus | |
dc.title | A detailed analysis of next generation sequencing reads of microRNA expression in Barrett’s Esophagus: absolute versus relative quantification | |
dc.type | Article | |
kusw.kuauthor | Lee, In-Hee | |
kusw.kudepartment | Bioinformatics Core Facility | |
kusw.oastatus | fullparticipation | |
dc.identifier.doi | 10.1186/1756-0500-7-212 | |
kusw.oaversion | Scholarly/refereed, publisher version | |
kusw.oapolicy | This item meets KU Open Access policy criteria. | |
dc.rights.accessrights | openAccess |
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