DNA qualification workflow for next generation sequencing of histopathological samples

PLoS One. 2013 Jun 6;8(6):e62692. doi: 10.1371/journal.pone.0062692. Print 2013.


Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA / analysis*
  • DNA / genetics
  • DNA, Neoplasm / analysis*
  • DNA, Neoplasm / genetics
  • Formaldehyde
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Multiplex Polymerase Chain Reaction
  • Observer Variation
  • Paraffin Embedding
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods
  • Sequence Analysis, DNA / standards*
  • Tissue Fixation
  • Workflow


  • DNA, Neoplasm
  • Formaldehyde
  • DNA

Grant support

This research has been supported financially by Associazione Italiana Ricerca sul Cancro 5xmille grant n. 12182; and an Italian Cancer Genome Project grant from the Italian Ministry of Research (FIRB – RBAP10AHJB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.