title: Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation.
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.
authors: CureSeq Inc., Brisbane, CA 94005, USA.
authors: Jennifer
authors: J
authors: Dang
authors: Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
authors: Pedro
authors: P
authors: Mendez
authors: CureSeq Inc., Brisbane, CA 94005, USA.
authors: Sharon
authors: S
authors: Lee
authors: CureSeq Inc., Brisbane, CA 94005, USA.
authors: James W
authors: JW
authors: Kim
authors: Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
authors: Jun-Hee
authors: JH
authors: Yoon
authors: CureSeq Inc., Brisbane, CA 94005, USA.
authors: Thomas W
authors: TW
authors: Kim
authors: CureSeq Inc., Brisbane, CA 94005, USA.
authors: Charles J
authors: CJ
authors: Sailey
authors: Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
authors: David M
authors: DM
authors: Jablons
authors: Thoracic Oncology Laboratory, Department of Surgery, University of California San Francisco, San Francisco, CA 94115, USA.
authors: Il-Jin
authors: IJ
authors: Kim
mesh_terms: D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation
chemical_list: D004273:DNA, Neoplasm