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Matsubara, Takehiro Okayama University Hospital Biobank, Okayama University Hospital
Soh, Junichi Department of Surgery, Division of Thoracic Surgery, Kindai University Faculty of Medicine
Morita, Mizuki Department of Biomedical Informatics,Okayama University Graduate School of Interdisciplinary Science and Engineering in Health Systems
Uwabo, Takahiro Department of Biomedical Informatics,Okayama University Graduate School of Interdisciplinary Science and Engineering in Health Systems
Tomida, Shuta Department of Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kaken ID researchmap
Fujiwara, Toshiyoshi Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences ORCID Kaken ID publons researchmap
Kanazawa, Susumu Department of Radiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kaken ID publons
Toyooka, Shinichi Department of General Thoracic Surgery, Breast and Endocrinological Surgery, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences ORCID Kaken ID publons researchmap
Hirasawa, Akira Department of Clinical Genomic Medicine, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences
Poor quality of biological samples will result in an inaccurate analysis of next-generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy-one RNA samples were extracted from formalin-fixed paraffin-embedded tissue samples (n=30), fresh-frozen samples (n=25), or cell lines (n=16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1(st) PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe (R2=0.8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1(st) PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value>66.1% exhibit greater sensitivity and specificity than those prepared with the RINe values>2.3. These findings suggest that the DV200 is superior to the RINe, especially for low-quality RNA, because it is a more consistent assessment of the amount of the 1(st) NGS library product per input.
BioMed Research International
© 2020 Takehiro Matsubara et al.
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