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Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.

Heydt C, Fassunke J, Künstlinger H, Ihle MA, König K, Heukamp LC, Schildhaus HU, Odenthal M, Büttner R, Merkelbach-Bruse S - PLoS ONE (2014)

Bottom Line: The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material.Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).No difference could be detected in mutation analysis based on the results of the quantification methods.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.

ABSTRACT
Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3-24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing.

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Comparison of five automated DNA extraction systems.Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.
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pone-0104566-g001: Comparison of five automated DNA extraction systems.Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.

Mentions: For the comparison of the five automated DNA extraction systems 10 FFPE tissue samples were extracted with each system (Table S1). The relative DNA concentrations (percentage of the highest concentration obtained by the Maxwell 16 extracts) for each method and sample is shown in figure 1. The extracted DNA was quantified by the NanoDrop 2000c spectrophotometer (Figure 1 A) and the Qubit 2.0 fluorometer (Figure 1 B). Additionally, the purity of each sample was determined by measuring the absorbance ratio at wavelength 260/280 nm (Table S1). As illustrated in figure 1, the amount of DNA extracted by the Maxwell 16 system was the highest in all samples, independent of tumour size. The DNA quantity obtained from the BioRobot M48 was the lowest in 9 samples when measured with the NanoDrop 2000c spectrophotometer and in 8 samples when measured with the Qubit 2.0 fluorometer. A 1.3–24.6-fold difference between the concentration of the Maxwell 16 extracts and the extracts of the other systems could be seen. The second highest DNA concentrations were obtained by the QIAcube and the QIAsymphony SP followed by the InnuPure C16 and the BioRobot M48. The DNA concentrations determined by the Qubit 2.0 fluorometer showed in all samples an even higher difference in DNA quantity between the extracts of the Maxwell 16 and the extracts of the other instruments whereas the order of the instruments from highest concentration to lowest stayed more or less the same.


Comparison of pre-analytical FFPE sample preparation methods and their impact on massively parallel sequencing in routine diagnostics.

Heydt C, Fassunke J, Künstlinger H, Ihle MA, König K, Heukamp LC, Schildhaus HU, Odenthal M, Büttner R, Merkelbach-Bruse S - PLoS ONE (2014)

Comparison of five automated DNA extraction systems.Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126727&req=5

pone-0104566-g001: Comparison of five automated DNA extraction systems.Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.
Mentions: For the comparison of the five automated DNA extraction systems 10 FFPE tissue samples were extracted with each system (Table S1). The relative DNA concentrations (percentage of the highest concentration obtained by the Maxwell 16 extracts) for each method and sample is shown in figure 1. The extracted DNA was quantified by the NanoDrop 2000c spectrophotometer (Figure 1 A) and the Qubit 2.0 fluorometer (Figure 1 B). Additionally, the purity of each sample was determined by measuring the absorbance ratio at wavelength 260/280 nm (Table S1). As illustrated in figure 1, the amount of DNA extracted by the Maxwell 16 system was the highest in all samples, independent of tumour size. The DNA quantity obtained from the BioRobot M48 was the lowest in 9 samples when measured with the NanoDrop 2000c spectrophotometer and in 8 samples when measured with the Qubit 2.0 fluorometer. A 1.3–24.6-fold difference between the concentration of the Maxwell 16 extracts and the extracts of the other systems could be seen. The second highest DNA concentrations were obtained by the QIAcube and the QIAsymphony SP followed by the InnuPure C16 and the BioRobot M48. The DNA concentrations determined by the Qubit 2.0 fluorometer showed in all samples an even higher difference in DNA quantity between the extracts of the Maxwell 16 and the extracts of the other instruments whereas the order of the instruments from highest concentration to lowest stayed more or less the same.

Bottom Line: The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material.Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).No difference could be detected in mutation analysis based on the results of the quantification methods.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany.

ABSTRACT
Over the last years, massively parallel sequencing has rapidly evolved and has now transitioned into molecular pathology routine laboratories. It is an attractive platform for analysing multiple genes at the same time with very little input material. Therefore, the need for high quality DNA obtained from automated DNA extraction systems has increased, especially to those laboratories which are dealing with formalin-fixed paraffin-embedded (FFPE) material and high sample throughput. This study evaluated five automated FFPE DNA extraction systems as well as five DNA quantification systems using the three most common techniques, UV spectrophotometry, fluorescent dye-based quantification and quantitative PCR, on 26 FFPE tissue samples. Additionally, the effects on downstream applications were analysed to find the most suitable pre-analytical methods for massively parallel sequencing in routine diagnostics. The results revealed that the Maxwell 16 from Promega (Mannheim, Germany) seems to be the superior system for DNA extraction from FFPE material. The extracts had a 1.3-24.6-fold higher DNA concentration in comparison to the other extraction systems, a higher quality and were most suitable for downstream applications. The comparison of the five quantification methods showed intermethod variations but all methods could be used to estimate the right amount for PCR amplification and for massively parallel sequencing. Interestingly, the best results in massively parallel sequencing were obtained with a DNA input of 15 ng determined by the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). No difference could be detected in mutation analysis based on the results of the quantification methods. These findings emphasise, that it is particularly important to choose the most reliable and constant DNA extraction system, especially when using small biopsies and low elution volumes, and that all common DNA quantification techniques can be used for downstream applications like massively parallel sequencing.

Show MeSH