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

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.

Figure 1. 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.

Figure 2. Assessment of DNA quality obtained from each extraction method and impact on downstream applications.

(A) Electrophoretic pattern of DNA extracts 1, 3, 4 and 5 of each extraction method on a 1% agarose gel. Ladder indicates a 1 kb DNA ladder as molecular weight size marker (B) 201 bp, 404 bp and 614 bp amplified DNA fragments of the GAPDH gene for sample 1, 3, 4, 5, of each extraction method. + indicates a positive control and – a negative control. (C) 125–175 bp multiplex PCR product for sample 1, 3, 4 and 5 of each extraction method. + indicates a positive control and – a negative control.

Figure 3. Results of the five DNA quantification measurements.

Distribution of DNA concentrations of samples 1–16 measured by each of the five quantification method.

Figure 4. 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.

Figure 5. 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.