Spotlight 59 DNA Input Quantification Assay: Instructions for Use
For next-generation sequencing (NGS) library preparation, input quantification by spectrophotometric-based (NanoDrop®) or fluorometric-based (Qubit®) methods may not provide an accurate assessment of the usable DNA within the sample. Quantification by spectrophotometric-based methods commonly overestimate DNA concentration and are limited to relatively high-concentration samples. Quantification by fluorometric-based methods provides accurate DNA concentrations for samples with high quality DNA (e.g., whole blood, fresh frozen samples, cultured cells), but performs poorly with low quality samples and cannot distinguish between circulating, cell- free DNA (cfDNA) and high molecular weight cellular gDNA. Therefore, for low quality samples [e.g., formalin-fixed, paraffin-embedded (FFPE) samples] and cfDNA samples, we recommend quantification by a qPCR-method, using both short and long amplicons to accurately determine the concentration and quality of sample DNA [Simbolo M. et al. PLoS ONE (2013) 8(6): e62692].