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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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Impact of DNA quantification methods on downstream applications.(A) Electrophoretic pattern of 13 DNA extracts on a 1% agarose gel. (B) Amplified 275 bp fragment of the EGFR gene. 20 ng of sample DNA determined by each quantification method was used for PCR amplification. + indicates a positive control, - a negative control.
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pone-0104566-g004: Impact of DNA quantification methods on downstream applications.(A) Electrophoretic pattern of 13 DNA extracts on a 1% agarose gel. (B) Amplified 275 bp fragment of the EGFR gene. 20 ng of sample DNA determined by each quantification method was used for PCR amplification. + indicates a positive control, - a negative control.

Mentions: Concerning the level of DNA concentration there was no uniformity in comparing the five methods. In samples 4, 5, 6 and 7 the Qubit 2.0 fluorometer measured the highest DNA concentrations, in sample 10, 15 and 16 the Quant-iT PicoGreen dsDNA reagent measurement gave the highest values, in sample 2 the qPCR and in all other samples the NanoDrop 2000c spectrophotometer estimated the highest DNA concentrations. This is in contrast to other studies where the NanoDrop 2000c spectrophotometer always overestimated the DNA concentration [15]. In general, DNA concentrations determined by the different quantification methods were more diverse at a concentration higher than 10 ng/µl, especially in samples 6 and 11. In samples 6 and 11 we saw differences of almost 50 ng/µl and 70 ng/µl between the lowest and the highest measured concentrations. Relating these findings to a 1% agarose gel of the DNA extracts (Figure 4 A) it could be seen that sample 6 and 11 have a high level of degraded DNA. In degraded DNA samples the DNA is fragmented into smaller DNA fragments. Sedlackova et al. [15] showed that fragmentation does not affect the concentration measured with the NanoDrop 2000c spectrophotometer and that this method might even overestimate the DNA concentration due to the fact that the NanoDrop 2000c spectrophotometer also measures single-stranded DNA and oligonucleotides present. Further they state that fluorescent-dye based quantification methods and qPCR are significantly affected by DNA fragmentation: The higher the fragmentation the lower the DNA concentration. This effect can also be seen in our study.


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)

Impact of DNA quantification methods on downstream applications.(A) Electrophoretic pattern of 13 DNA extracts on a 1% agarose gel. (B) Amplified 275 bp fragment of the EGFR gene. 20 ng of sample DNA determined by each quantification method was used for PCR amplification. + indicates a positive control, - a negative control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104566-g004: Impact of DNA quantification methods on downstream applications.(A) Electrophoretic pattern of 13 DNA extracts on a 1% agarose gel. (B) Amplified 275 bp fragment of the EGFR gene. 20 ng of sample DNA determined by each quantification method was used for PCR amplification. + indicates a positive control, - a negative control.
Mentions: Concerning the level of DNA concentration there was no uniformity in comparing the five methods. In samples 4, 5, 6 and 7 the Qubit 2.0 fluorometer measured the highest DNA concentrations, in sample 10, 15 and 16 the Quant-iT PicoGreen dsDNA reagent measurement gave the highest values, in sample 2 the qPCR and in all other samples the NanoDrop 2000c spectrophotometer estimated the highest DNA concentrations. This is in contrast to other studies where the NanoDrop 2000c spectrophotometer always overestimated the DNA concentration [15]. In general, DNA concentrations determined by the different quantification methods were more diverse at a concentration higher than 10 ng/µl, especially in samples 6 and 11. In samples 6 and 11 we saw differences of almost 50 ng/µl and 70 ng/µl between the lowest and the highest measured concentrations. Relating these findings to a 1% agarose gel of the DNA extracts (Figure 4 A) it could be seen that sample 6 and 11 have a high level of degraded DNA. In degraded DNA samples the DNA is fragmented into smaller DNA fragments. Sedlackova et al. [15] showed that fragmentation does not affect the concentration measured with the NanoDrop 2000c spectrophotometer and that this method might even overestimate the DNA concentration due to the fact that the NanoDrop 2000c spectrophotometer also measures single-stranded DNA and oligonucleotides present. Further they state that fluorescent-dye based quantification methods and qPCR are significantly affected by DNA fragmentation: The higher the fragmentation the lower the DNA concentration. This effect can also be seen in our study.

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