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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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Intra- and inter-method accuracy and precision.Distribution of DNA sample concentration (dispersion chart) was estimated by both NanoDrop (black) and Qubit (gray) on repeated (n = 20) measurements of two commercial human genomic DNA preparations (Sample L 200 ng/µl; Sample G 5 ng/µl). For both samples, NanoDrop overestimated the DNA concentration (+8.8% for L and +24.0% for G, p<0.0003), while Qubit underestimated it (−5.0% for L and −7.3% for G, p<0.005).
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pone-0062692-g001: Intra- and inter-method accuracy and precision.Distribution of DNA sample concentration (dispersion chart) was estimated by both NanoDrop (black) and Qubit (gray) on repeated (n = 20) measurements of two commercial human genomic DNA preparations (Sample L 200 ng/µl; Sample G 5 ng/µl). For both samples, NanoDrop overestimated the DNA concentration (+8.8% for L and +24.0% for G, p<0.0003), while Qubit underestimated it (−5.0% for L and −7.3% for G, p<0.005).

Mentions: In order to first investigate the operator-dependent variability associated with both NanoDrop and Qubit measurements, the two commercial DNA preparations L [200 ng/µl] and G [5 ng/µl]) were tested. DNA concentration was estimated by both NanoDrop and Qubit on repeated (n = 20) measurements. Sample handling intra-operator variability, as measured by a coefficient of variation, was less than 6% for sample L and 14% for the more diluted sample G. Inter-operator consistency was assessed on repeated (n = 10) measurements and showed no significant differences (p = 0.154). For both samples, NanoDrop estimated a DNA concentration higher than Qubit (Mann-Whitney test p<0.0005 for both samples; Figure 1).


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)

Intra- and inter-method accuracy and precision.Distribution of DNA sample concentration (dispersion chart) was estimated by both NanoDrop (black) and Qubit (gray) on repeated (n = 20) measurements of two commercial human genomic DNA preparations (Sample L 200 ng/µl; Sample G 5 ng/µl). For both samples, NanoDrop overestimated the DNA concentration (+8.8% for L and +24.0% for G, p<0.0003), while Qubit underestimated it (−5.0% for L and −7.3% for G, p<0.005).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675123&req=5

pone-0062692-g001: Intra- and inter-method accuracy and precision.Distribution of DNA sample concentration (dispersion chart) was estimated by both NanoDrop (black) and Qubit (gray) on repeated (n = 20) measurements of two commercial human genomic DNA preparations (Sample L 200 ng/µl; Sample G 5 ng/µl). For both samples, NanoDrop overestimated the DNA concentration (+8.8% for L and +24.0% for G, p<0.0003), while Qubit underestimated it (−5.0% for L and −7.3% for G, p<0.005).
Mentions: In order to first investigate the operator-dependent variability associated with both NanoDrop and Qubit measurements, the two commercial DNA preparations L [200 ng/µl] and G [5 ng/µl]) were tested. DNA concentration was estimated by both NanoDrop and Qubit on repeated (n = 20) measurements. Sample handling intra-operator variability, as measured by a coefficient of variation, was less than 6% for sample L and 14% for the more diluted sample G. Inter-operator consistency was assessed on repeated (n = 10) measurements and showed no significant differences (p = 0.154). For both samples, NanoDrop estimated a DNA concentration higher than Qubit (Mann-Whitney test p<0.0005 for both samples; Figure 1).

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