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DNA qualification workflow for next generation sequencing of histopathological samples
Michele Simbolo1, Marisa Gottardi, Vincenzo Corbo
1ARC-NET Research Centre, University of Verona, Verona, Italy.
Plos One
|June 14, 2013
Summary
A standardized workflow using NanoDrop and Qubit is essential for qualifying DNA from histopathological samples. This combination accurately assesses DNA purity and quantity for reproducible clinical research and next-generation sequencing.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Histopathological samples are a valuable source of DNA for clinical research.
- DNA quality and quantity can vary significantly based on sample type and extraction methods.
- Standardized DNA qualification is crucial for reproducible interlaboratory results.
Purpose of the Study:
- To evaluate NanoDrop and Qubit for DNA qualification in clinical research.
- To establish a cost-effective and standardized workflow for DNA assessment.
- To determine the suitability of DNA for downstream applications like next-generation sequencing.
Main Methods:
- Comparison of NanoDrop (UV spectroscopy) and Qubit 2.0 (fluorometry) for DNA quantification.
- Quantitative PCR (qPCR) used as a reference for DNA concentration and PCR suitability.
- Testing on diverse DNA samples: fresh-frozen tissues, FFPE tissues, cell lines, and commercial preparations.
- Downstream application: multiplex PCR for next-generation sequencing library preparation.
Main Results:
- NanoDrop consistently overestimated DNA concentrations compared to Qubit.
- Qubit demonstrated high reproducibility and consistency with qPCR for degraded DNA (FFPE).
- NanoDrop's accuracy was limited to high molecular weight DNA from fresh-frozen samples and cell lines.
- A workflow using NanoDrop for purity and Qubit for dsDNA quantity enabled successful NGS library preparation for most samples.
Conclusions:
- A combined NanoDrop and Qubit workflow offers an effective alternative to costly and labor-intensive qPCR for DNA qualification.
- This sequential approach ensures accurate assessment of DNA purity and double-strand DNA quantity.
- The proposed workflow enhances the reliability of DNA preparations for downstream genomic applications.
