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Multiplex bisulfite PCR resequencing of clinical FFPE DNA.

Korbie D, Lin E, Wall D, Nair SS, Stirzaker C, Clark SJ, Trau M - Clin Epigenetics (2015)

Bottom Line: Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fixed, paraffin-embedded (FFPE) blocks.Moreover, the library construction process detailed here can be rapidly optimized and implemented with a minimal amount of work, can be performed using the standard equipment found in any molecular biology laboratory, and can be easily adapted for use on both genomic DNA and bisulfite DNA applications.However, in preparing bisulfite libraries for sequencing, the use of ExoSAP-IT is not recommended due to potential off-target nuclease effects which may impact downstream methylation analysis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Personalised Nanomedicine, The University of Queensland, St Lucia, 4072 QLD Australia ; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Corner College and Cooper Rds (Bldg 75), St Lucia, 4072 QLD Australia.

ABSTRACT

Background: The clinical utility of DNA methylation as a predictive or prognostic biomarker requires scalable resequencing protocols for bisulfite-converted DNA. Key features of any validation method should be adaptability for low- or high-throughput needs and high reproducibility, and should only require minimal amounts of precious clinical sample as input material. Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fixed, paraffin-embedded (FFPE) blocks.

Results: We report here for the first time on comparison studies between the Fluidigm Access Array system and multiplex assays for multiplex bisulfite PCR resequencing. The requirement of the Fluidigm Access Array system for high template amounts and its sensitivity to variations in template quality rendered it unsuitable for bisulfite PCR applications utilizing FFPE DNA. In response to this limitation, we established a multiplex bisulfite PCR assay capable of delivering robust methylation data using minimal amounts of FFPE clinical DNA. To evaluate the parameters and reproducibility of this assay, 57 amplicons were used to prepare sequencing libraries in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%). Analysis of this data demonstrated that this multiplex assay had high reproducibility (mean standard deviation of 1.4% for methylation values), was low cost, required low sample input (50 ng of DNA or less), and could be scaled for both low- and high-throughput needs. Notably, ExoSAP-IT (exonuclease I) treatment to remove residual primers in bisulfite resequencing libraries appeared to degrade the library and generate a high-molecular weight smear which may impact on the degree of methylation assessed.

Conclusions: Multiplex bisulfite PCR assays represent a convenient and scalable method for validation and screening of methylated DNA regions from archival FFPE DNA. Moreover, the library construction process detailed here can be rapidly optimized and implemented with a minimal amount of work, can be performed using the standard equipment found in any molecular biology laboratory, and can be easily adapted for use on both genomic DNA and bisulfite DNA applications. However, in preparing bisulfite libraries for sequencing, the use of ExoSAP-IT is not recommended due to potential off-target nuclease effects which may impact downstream methylation analysis.

No MeSH data available.


Related in: MedlinePlus

Representative methylation results. Representative data of 4 out of the 13 clinical FFPE samples assayed are shown. FFPE breast tumour samples were assayed in triplicate, along with a set of methylation controls (0%, 25%, 50%, 75%, and 100% methylation controls). Whereas the methylation values for the control samples were observed to be maintained at consistent levels across the region of interest, the breast cancer samples were observed to give unique patterns of methylation.
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Fig5: Representative methylation results. Representative data of 4 out of the 13 clinical FFPE samples assayed are shown. FFPE breast tumour samples were assayed in triplicate, along with a set of methylation controls (0%, 25%, 50%, 75%, and 100% methylation controls). Whereas the methylation values for the control samples were observed to be maintained at consistent levels across the region of interest, the breast cancer samples were observed to give unique patterns of methylation.

Mentions: To assess assay reproducibility in determining the methylation state, 293 CpGs were examined in triplicate across a separate set of 13 different tumour samples. Overall assay performance based on methylated controls and clinical FFPE tumour samples indicated consistent assay performance. A representative region along with methylated controls is shown in Figure 5 for 13 CpGs; a larger bias plot showing representative data across 68 CpGs is also shown in Additional file 4: Figure S4. The lowest SD observed across 293 CpGs was 0.0043%, with a mean SD of 1.5%. The maximum SD was calculated to be 23.8%; however, this high value was due to one erratic amplicon which gave consistently high SDs across its length, and after this amplicon was removed, the maximal SD observed across 281 CpGs dropped to 8.04% (Additional file 5: Table S1). Moreover, although standard deviation values are present in Figure 5, they were observed to be so small that the standard deviation spread is not readily visible, and for this reason, the values have been included in a separate table (Additional file 6: Table S2).Figure 5


Multiplex bisulfite PCR resequencing of clinical FFPE DNA.

Korbie D, Lin E, Wall D, Nair SS, Stirzaker C, Clark SJ, Trau M - Clin Epigenetics (2015)

Representative methylation results. Representative data of 4 out of the 13 clinical FFPE samples assayed are shown. FFPE breast tumour samples were assayed in triplicate, along with a set of methylation controls (0%, 25%, 50%, 75%, and 100% methylation controls). Whereas the methylation values for the control samples were observed to be maintained at consistent levels across the region of interest, the breast cancer samples were observed to give unique patterns of methylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389706&req=5

Fig5: Representative methylation results. Representative data of 4 out of the 13 clinical FFPE samples assayed are shown. FFPE breast tumour samples were assayed in triplicate, along with a set of methylation controls (0%, 25%, 50%, 75%, and 100% methylation controls). Whereas the methylation values for the control samples were observed to be maintained at consistent levels across the region of interest, the breast cancer samples were observed to give unique patterns of methylation.
Mentions: To assess assay reproducibility in determining the methylation state, 293 CpGs were examined in triplicate across a separate set of 13 different tumour samples. Overall assay performance based on methylated controls and clinical FFPE tumour samples indicated consistent assay performance. A representative region along with methylated controls is shown in Figure 5 for 13 CpGs; a larger bias plot showing representative data across 68 CpGs is also shown in Additional file 4: Figure S4. The lowest SD observed across 293 CpGs was 0.0043%, with a mean SD of 1.5%. The maximum SD was calculated to be 23.8%; however, this high value was due to one erratic amplicon which gave consistently high SDs across its length, and after this amplicon was removed, the maximal SD observed across 281 CpGs dropped to 8.04% (Additional file 5: Table S1). Moreover, although standard deviation values are present in Figure 5, they were observed to be so small that the standard deviation spread is not readily visible, and for this reason, the values have been included in a separate table (Additional file 6: Table S2).Figure 5

Bottom Line: Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fixed, paraffin-embedded (FFPE) blocks.Moreover, the library construction process detailed here can be rapidly optimized and implemented with a minimal amount of work, can be performed using the standard equipment found in any molecular biology laboratory, and can be easily adapted for use on both genomic DNA and bisulfite DNA applications.However, in preparing bisulfite libraries for sequencing, the use of ExoSAP-IT is not recommended due to potential off-target nuclease effects which may impact downstream methylation analysis.

View Article: PubMed Central - PubMed

Affiliation: Centre for Personalised Nanomedicine, The University of Queensland, St Lucia, 4072 QLD Australia ; Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Corner College and Cooper Rds (Bldg 75), St Lucia, 4072 QLD Australia.

ABSTRACT

Background: The clinical utility of DNA methylation as a predictive or prognostic biomarker requires scalable resequencing protocols for bisulfite-converted DNA. Key features of any validation method should be adaptability for low- or high-throughput needs and high reproducibility, and should only require minimal amounts of precious clinical sample as input material. Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fixed, paraffin-embedded (FFPE) blocks.

Results: We report here for the first time on comparison studies between the Fluidigm Access Array system and multiplex assays for multiplex bisulfite PCR resequencing. The requirement of the Fluidigm Access Array system for high template amounts and its sensitivity to variations in template quality rendered it unsuitable for bisulfite PCR applications utilizing FFPE DNA. In response to this limitation, we established a multiplex bisulfite PCR assay capable of delivering robust methylation data using minimal amounts of FFPE clinical DNA. To evaluate the parameters and reproducibility of this assay, 57 amplicons were used to prepare sequencing libraries in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%). Analysis of this data demonstrated that this multiplex assay had high reproducibility (mean standard deviation of 1.4% for methylation values), was low cost, required low sample input (50 ng of DNA or less), and could be scaled for both low- and high-throughput needs. Notably, ExoSAP-IT (exonuclease I) treatment to remove residual primers in bisulfite resequencing libraries appeared to degrade the library and generate a high-molecular weight smear which may impact on the degree of methylation assessed.

Conclusions: Multiplex bisulfite PCR assays represent a convenient and scalable method for validation and screening of methylated DNA regions from archival FFPE DNA. Moreover, the library construction process detailed here can be rapidly optimized and implemented with a minimal amount of work, can be performed using the standard equipment found in any molecular biology laboratory, and can be easily adapted for use on both genomic DNA and bisulfite DNA applications. However, in preparing bisulfite libraries for sequencing, the use of ExoSAP-IT is not recommended due to potential off-target nuclease effects which may impact downstream methylation analysis.

No MeSH data available.


Related in: MedlinePlus