Open Access

Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation

  • Authors:
    • Jennifer Dang
    • Pedro Mendez
    • Sharon Lee
    • James W. Kim
    • Jun-Hee Yoon
    • Thomas W. Kim
    • Charles J. Sailey
    • David M. Jablons
    • Il-Jin Kim
  • View Affiliations

  • Published online on: August 11, 2016     https://doi.org/10.3892/ijo.2016.3654
  • Pages: 1755-1765
  • Copyright: © Dang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control. We found that SYBR-Green-based qPCR assay provides a consistent and accurate DNA quantification while keeping its cost relatively low and the throughput high. We designed a dual-probe SYBR-Green qPCR assay for DNA quantity and quality assessment for targeted NGS library preparation. This assay provides a Dscore (degradation score) of the interrogated DNA by analyzing two different sizes of amplicons. We show an example of a clinical sample with a very high Dscore (high degradation). With a regular DNA quantification, without considering the degradation status, no correct NGS libraries were obtained. However, after optimizing the library condition by considering its poor DNA quality, a reasonably good library and sequencing results were obtained. In summary, we developed and presented a new DNA quantity and quality analysis qPCR assay for the targeted NGS library preparation. This assay may be mostly efficient for the clinical samples with high degradation and poor DNA quality.
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October-2016
Volume 49 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Spandidos Publications style
Dang J, Mendez P, Lee S, Kim JW, Yoon J, Kim TW, Sailey CJ, Jablons DM and Kim I: Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation. Int J Oncol 49: 1755-1765, 2016
APA
Dang, J., Mendez, P., Lee, S., Kim, J.W., Yoon, J., Kim, T.W. ... Kim, I. (2016). Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation. International Journal of Oncology, 49, 1755-1765. https://doi.org/10.3892/ijo.2016.3654
MLA
Dang, J., Mendez, P., Lee, S., Kim, J. W., Yoon, J., Kim, T. W., Sailey, C. J., Jablons, D. M., Kim, I."Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation". International Journal of Oncology 49.4 (2016): 1755-1765.
Chicago
Dang, J., Mendez, P., Lee, S., Kim, J. W., Yoon, J., Kim, T. W., Sailey, C. J., Jablons, D. M., Kim, I."Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation". International Journal of Oncology 49, no. 4 (2016): 1755-1765. https://doi.org/10.3892/ijo.2016.3654