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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 massively parallel sequencing.(A) Mean amplicon coverage determined by in-house software for each sample, quantification method and amount of DNA used for multiplex PCR amplification. (B) Number of all variants called by in-house software for each sample, quantification method and starting material used.
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pone-0104566-g005: Impact of DNA quantification methods on massively parallel sequencing.(A) Mean amplicon coverage determined by in-house software for each sample, quantification method and amount of DNA used for multiplex PCR amplification. (B) Number of all variants called by in-house software for each sample, quantification method and starting material used.

Mentions: The usefulness of each DNA quantification method for downstream applications was further assessed by massively parallel sequencing to see if different quantification methods have an impact on mutation calling, the number of variants and the mean coverage. Therefore two additional samples were extracted with the Maxwell 16 and quantified. The results for sample 1 were 155 ng/µl (NanoDrop 2000c spectrophotometer), 84.2 ng/µl (Qubit 2.0 fluorometer) and 20.9 ng/µl (qPCR). The results for sample 2 were 86.7 ng/µl (NanoDrop 2000c spectrophotometer), 46.8 ng/µl (Qubit 2.0 fluorometer) and 10.7 ng/µl (qPCR). PCR products could be seen for each sample, quantification method and concentration. The libraries were sequenced on a MiSeq benchtop sequencer with a cluster density of 1154 K/mm2, a Q30 score of 96.5% and a cluster passing filter value of 94.8%. These results are in the upper range for massively parallel sequencing on a MiSeq benchtop sequencer according to the Illumina specifications [26]. The data analyses revealed that independently from the quantification method and the amount of DNA used the massively parallel sequencing worked well when using an extensively validated routine workflow (Figure 5). Until now 2000 samples were routinely analysed in our laboratory with the Ion AmpliSeq custom DNA panel used in this study. This workflow was also used in the study of Ihle et al., where 72 samples were analysed with the same Ion AmpliSeq custom DNA panel [16].


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 massively parallel sequencing.(A) Mean amplicon coverage determined by in-house software for each sample, quantification method and amount of DNA used for multiplex PCR amplification. (B) Number of all variants called by in-house software for each sample, quantification method and starting material used.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104566-g005: Impact of DNA quantification methods on massively parallel sequencing.(A) Mean amplicon coverage determined by in-house software for each sample, quantification method and amount of DNA used for multiplex PCR amplification. (B) Number of all variants called by in-house software for each sample, quantification method and starting material used.
Mentions: The usefulness of each DNA quantification method for downstream applications was further assessed by massively parallel sequencing to see if different quantification methods have an impact on mutation calling, the number of variants and the mean coverage. Therefore two additional samples were extracted with the Maxwell 16 and quantified. The results for sample 1 were 155 ng/µl (NanoDrop 2000c spectrophotometer), 84.2 ng/µl (Qubit 2.0 fluorometer) and 20.9 ng/µl (qPCR). The results for sample 2 were 86.7 ng/µl (NanoDrop 2000c spectrophotometer), 46.8 ng/µl (Qubit 2.0 fluorometer) and 10.7 ng/µl (qPCR). PCR products could be seen for each sample, quantification method and concentration. The libraries were sequenced on a MiSeq benchtop sequencer with a cluster density of 1154 K/mm2, a Q30 score of 96.5% and a cluster passing filter value of 94.8%. These results are in the upper range for massively parallel sequencing on a MiSeq benchtop sequencer according to the Illumina specifications [26]. The data analyses revealed that independently from the quantification method and the amount of DNA used the massively parallel sequencing worked well when using an extensively validated routine workflow (Figure 5). Until now 2000 samples were routinely analysed in our laboratory with the Ion AmpliSeq custom DNA panel used in this study. This workflow was also used in the study of Ihle et al., where 72 samples were analysed with the same Ion AmpliSeq custom DNA panel [16].

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