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DNA qualification workflow for next generation sequencing of histopathological samples.

Simbolo M, Gottardi M, Corbo V, Fassan M, Mafficini A, Malpeli G, Lawlor RT, Scarpa A - PLoS ONE (2013)

Bottom Line: Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results.NanoDrop UV-spectrum verified contamination of the unsuccessful sample.In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

View Article: PubMed Central - PubMed

Affiliation: ARC-NET Research Centre, University of Verona, Verona, Italy.

ABSTRACT
Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

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Cross-validation of DNA samples quantification by qPCR.Bland-Altman plots for inter-technology (NanoDrop or Qubit vs. qPCR) comparison of all samples (A), and according to the different sample sources, as indicated (B, C). A) Qubit measurements show high correlation (mean measured/expected ratio  = 0.92; SD  = 0.69; Wilcoxon signed rank test p = 0.07) with the measurements obtained by qPCR (x-axis), whereas NanoDrop measurements tend to overestimate samples concentration (mean measured/expected ratio  = 3.8; SD  = 6.4; Wilcoxon signed rank test p<0.0001). B) Fresh frozen sample quantification by NanoDrop overestimates (mean measured/expected ratio  = 1.48; SD  = 0.57; Wilcoxon signed rank test p<0.01) the DNA concentration detected by quantitative PCR, while Qubit underestimates (mean measured/expected ratio  = 0.78; SD  = 0.32; Wilcoxon signed rank test p<0.001) the value. C) In formalin-fixed paraffin-embedded samples a better concentration estimation is obtained by Qubit (mean measured/expected ratio  = 1.23; SD  = 1.15; Wilcoxon signed rank test p = 0.91) than by NanoDrop (mean measured/expected ratio  = 9.21; SD  = 9.95; Wilcoxon signed rank test p<0.004).
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pone-0062692-g003: Cross-validation of DNA samples quantification by qPCR.Bland-Altman plots for inter-technology (NanoDrop or Qubit vs. qPCR) comparison of all samples (A), and according to the different sample sources, as indicated (B, C). A) Qubit measurements show high correlation (mean measured/expected ratio  = 0.92; SD  = 0.69; Wilcoxon signed rank test p = 0.07) with the measurements obtained by qPCR (x-axis), whereas NanoDrop measurements tend to overestimate samples concentration (mean measured/expected ratio  = 3.8; SD  = 6.4; Wilcoxon signed rank test p<0.0001). B) Fresh frozen sample quantification by NanoDrop overestimates (mean measured/expected ratio  = 1.48; SD  = 0.57; Wilcoxon signed rank test p<0.01) the DNA concentration detected by quantitative PCR, while Qubit underestimates (mean measured/expected ratio  = 0.78; SD  = 0.32; Wilcoxon signed rank test p<0.001) the value. C) In formalin-fixed paraffin-embedded samples a better concentration estimation is obtained by Qubit (mean measured/expected ratio  = 1.23; SD  = 1.15; Wilcoxon signed rank test p = 0.91) than by NanoDrop (mean measured/expected ratio  = 9.21; SD  = 9.95; Wilcoxon signed rank test p<0.004).

Mentions: To compare the accuracy of the two methods in quantifying DNA, a PCR-based assay (PrimerDesign) was used as reference, as it both quantitates DNA and assesses its suitability for PCR amplification. NanoDrop and Qubit measurements of three dilutions of samples (1∶1, 1∶5, 1∶10) were plotted against the concentrations as detected by the qPCR assay. From the Bland-Altman analysis for inter-technology comparison, NanoDrop data showed a high dispersion and a higher content of DNA compared to qPCR (mean measured/expected ratio  = 3.8; SD  = 6.4, Figure 3); Qubit measurements showed high concordance with qPCR data (mean measured/expected ratio  = 0.92; SD  = 0.69).


DNA qualification workflow for next generation sequencing of histopathological samples.

Simbolo M, Gottardi M, Corbo V, Fassan M, Mafficini A, Malpeli G, Lawlor RT, Scarpa A - PLoS ONE (2013)

Cross-validation of DNA samples quantification by qPCR.Bland-Altman plots for inter-technology (NanoDrop or Qubit vs. qPCR) comparison of all samples (A), and according to the different sample sources, as indicated (B, C). A) Qubit measurements show high correlation (mean measured/expected ratio  = 0.92; SD  = 0.69; Wilcoxon signed rank test p = 0.07) with the measurements obtained by qPCR (x-axis), whereas NanoDrop measurements tend to overestimate samples concentration (mean measured/expected ratio  = 3.8; SD  = 6.4; Wilcoxon signed rank test p<0.0001). B) Fresh frozen sample quantification by NanoDrop overestimates (mean measured/expected ratio  = 1.48; SD  = 0.57; Wilcoxon signed rank test p<0.01) the DNA concentration detected by quantitative PCR, while Qubit underestimates (mean measured/expected ratio  = 0.78; SD  = 0.32; Wilcoxon signed rank test p<0.001) the value. C) In formalin-fixed paraffin-embedded samples a better concentration estimation is obtained by Qubit (mean measured/expected ratio  = 1.23; SD  = 1.15; Wilcoxon signed rank test p = 0.91) than by NanoDrop (mean measured/expected ratio  = 9.21; SD  = 9.95; Wilcoxon signed rank test p<0.004).
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Related In: Results  -  Collection

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pone-0062692-g003: Cross-validation of DNA samples quantification by qPCR.Bland-Altman plots for inter-technology (NanoDrop or Qubit vs. qPCR) comparison of all samples (A), and according to the different sample sources, as indicated (B, C). A) Qubit measurements show high correlation (mean measured/expected ratio  = 0.92; SD  = 0.69; Wilcoxon signed rank test p = 0.07) with the measurements obtained by qPCR (x-axis), whereas NanoDrop measurements tend to overestimate samples concentration (mean measured/expected ratio  = 3.8; SD  = 6.4; Wilcoxon signed rank test p<0.0001). B) Fresh frozen sample quantification by NanoDrop overestimates (mean measured/expected ratio  = 1.48; SD  = 0.57; Wilcoxon signed rank test p<0.01) the DNA concentration detected by quantitative PCR, while Qubit underestimates (mean measured/expected ratio  = 0.78; SD  = 0.32; Wilcoxon signed rank test p<0.001) the value. C) In formalin-fixed paraffin-embedded samples a better concentration estimation is obtained by Qubit (mean measured/expected ratio  = 1.23; SD  = 1.15; Wilcoxon signed rank test p = 0.91) than by NanoDrop (mean measured/expected ratio  = 9.21; SD  = 9.95; Wilcoxon signed rank test p<0.004).
Mentions: To compare the accuracy of the two methods in quantifying DNA, a PCR-based assay (PrimerDesign) was used as reference, as it both quantitates DNA and assesses its suitability for PCR amplification. NanoDrop and Qubit measurements of three dilutions of samples (1∶1, 1∶5, 1∶10) were plotted against the concentrations as detected by the qPCR assay. From the Bland-Altman analysis for inter-technology comparison, NanoDrop data showed a high dispersion and a higher content of DNA compared to qPCR (mean measured/expected ratio  = 3.8; SD  = 6.4, Figure 3); Qubit measurements showed high concordance with qPCR data (mean measured/expected ratio  = 0.92; SD  = 0.69).

Bottom Line: Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results.NanoDrop UV-spectrum verified contamination of the unsuccessful sample.In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

View Article: PubMed Central - PubMed

Affiliation: ARC-NET Research Centre, University of Verona, Verona, Italy.

ABSTRACT
Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

Show MeSH
Related in: MedlinePlus