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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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Results of the five DNA quantification measurements.Distribution of DNA concentrations of samples 1–16 measured by each of the five quantification method.
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pone-0104566-g003: Results of the five DNA quantification measurements.Distribution of DNA concentrations of samples 1–16 measured by each of the five quantification method.

Mentions: The DNA of 16 previously extracted FFPE tissue blocks were analysed with five different quantification methods. Some of these extracts were prepared manually using the QIAamp DNA Mini Kit. According to previous studies DNA extracts from the QIAamp DNA Mini Kit are of good DNA quality and quantity [8]. The results of the five DNA quantification methods are shown in figure 3 and table S2.


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)

Results of the five DNA quantification measurements.Distribution of DNA concentrations of samples 1–16 measured by each of the five quantification method.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104566-g003: Results of the five DNA quantification measurements.Distribution of DNA concentrations of samples 1–16 measured by each of the five quantification method.
Mentions: The DNA of 16 previously extracted FFPE tissue blocks were analysed with five different quantification methods. Some of these extracts were prepared manually using the QIAamp DNA Mini Kit. According to previous studies DNA extracts from the QIAamp DNA Mini Kit are of good DNA quality and quantity [8]. The results of the five DNA quantification methods are shown in figure 3 and table S2.

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