Limits...
Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.

Wong EM, Joo JE, McLean CA, Baglietto L, English DR, Severi G, Hopper JL, Milne RL, FitzGerald LM, Giles GG, Southey MC - BMC Res Notes (2015)

Bottom Line: Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3.The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples.This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. emwong@unimelb.edu.au.

ABSTRACT

Background: Large population-based translational epigenetic studies are emerging due to recent technological advances that have made molecular analyses possible. For example, the Infinium HumanMethylation450 Beadchip (HM450K) has enabled studies of genome-wide methylation on a scale not previously possible. However, application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood. To facilitate the application of this assay consistently across a large number of FFPE tumour-enriched DNA samples we have devised a modification to the HM450K protocol for FFPE that includes an additional quality control (QC) checkpoint.

Results: QC checkpoint 3 was designed to assess the presence of DNA after bisulfite conversion and restoration, just prior to application of the HM450K assay. DNA was extracted from 474 archival FFPE breast tumour material. Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3. Genome-wide methylation was measured for the remaining 428 tumour-enriched DNA. Of these, only 4 samples failed our stringent HM450K data criteria thus representing a 99% success rate. Using prior knowledge about methylation marks associated with breast cancer we further explored the quality of the data. Twenty probes in the BRCA1 promoter region showed increased methylation in triple-negative breast cancers compared to Luminal A, Luminal B and HER2-positive breast cancer subtypes. Validation of this observation in published data from The Cancer Genome Atlas (TCGA) Network (obtained from DNA extracted from fresh frozen tumour samples) confirms the quality of the data obtained from the improved protocol.

Conclusions: The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples. This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

No MeSH data available.


Related in: MedlinePlus

Post HM450K assay data quality checks of FFPE tumour-enriched DNA. a Average probe detection p-values across all probes for each sample. Each black dot represents a single sample. The average detection p–value across all probes for all samples (n = 423) was 4.54 × 10−4. b Scatterplots of replicates assayed on different beadchips (n = 11). The average correlation coefficient (rave) across all replicates was 0.993
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4595238&req=5

Fig2: Post HM450K assay data quality checks of FFPE tumour-enriched DNA. a Average probe detection p-values across all probes for each sample. Each black dot represents a single sample. The average detection p–value across all probes for all samples (n = 423) was 4.54 × 10−4. b Scatterplots of replicates assayed on different beadchips (n = 11). The average correlation coefficient (rave) across all replicates was 0.993

Mentions: DNA was macrodissected from marked up areas of 474 FFPE breast tumours. Five samples did not have a detectable amount of DNA at QC checkpoint 1. Of the remaining 469 FFPE tumour-enriched DNA samples that were progressed past QC checkpoint 2, 42 (8.93 %) samples failed to progress past QC checkpoint 3 with ∆Cq values ranging from 0 (no amplification) to 3.77. The methylome of 427 FFPE tumour-enriched DNA were subsequently evaluated on the HM450K platform (Fig. 1). Of these, 4 (<1 %) had an average detection p-value across all probes of ≥0.01 and were removed from further analysis. Based on our criteria, 5 % (24,816) of probes were removed, with 460,696 common probes remaining across 423 FFPE tumour-enriched DNA for downstream data analysis. The average detection p-value across these probes for all remaining samples (n = 423) was 4.54 × 10−4 (Fig. 2a) with high correlations observed between sample replicates (correlation coefficients r > 0.98) (Fig. 2b) thereby confirming the capacity of our protocol to generate reproducible, high quality data.Fig. 2


Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.

Wong EM, Joo JE, McLean CA, Baglietto L, English DR, Severi G, Hopper JL, Milne RL, FitzGerald LM, Giles GG, Southey MC - BMC Res Notes (2015)

Post HM450K assay data quality checks of FFPE tumour-enriched DNA. a Average probe detection p-values across all probes for each sample. Each black dot represents a single sample. The average detection p–value across all probes for all samples (n = 423) was 4.54 × 10−4. b Scatterplots of replicates assayed on different beadchips (n = 11). The average correlation coefficient (rave) across all replicates was 0.993
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595238&req=5

Fig2: Post HM450K assay data quality checks of FFPE tumour-enriched DNA. a Average probe detection p-values across all probes for each sample. Each black dot represents a single sample. The average detection p–value across all probes for all samples (n = 423) was 4.54 × 10−4. b Scatterplots of replicates assayed on different beadchips (n = 11). The average correlation coefficient (rave) across all replicates was 0.993
Mentions: DNA was macrodissected from marked up areas of 474 FFPE breast tumours. Five samples did not have a detectable amount of DNA at QC checkpoint 1. Of the remaining 469 FFPE tumour-enriched DNA samples that were progressed past QC checkpoint 2, 42 (8.93 %) samples failed to progress past QC checkpoint 3 with ∆Cq values ranging from 0 (no amplification) to 3.77. The methylome of 427 FFPE tumour-enriched DNA were subsequently evaluated on the HM450K platform (Fig. 1). Of these, 4 (<1 %) had an average detection p-value across all probes of ≥0.01 and were removed from further analysis. Based on our criteria, 5 % (24,816) of probes were removed, with 460,696 common probes remaining across 423 FFPE tumour-enriched DNA for downstream data analysis. The average detection p-value across these probes for all remaining samples (n = 423) was 4.54 × 10−4 (Fig. 2a) with high correlations observed between sample replicates (correlation coefficients r > 0.98) (Fig. 2b) thereby confirming the capacity of our protocol to generate reproducible, high quality data.Fig. 2

Bottom Line: Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3.The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples.This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. emwong@unimelb.edu.au.

ABSTRACT

Background: Large population-based translational epigenetic studies are emerging due to recent technological advances that have made molecular analyses possible. For example, the Infinium HumanMethylation450 Beadchip (HM450K) has enabled studies of genome-wide methylation on a scale not previously possible. However, application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood. To facilitate the application of this assay consistently across a large number of FFPE tumour-enriched DNA samples we have devised a modification to the HM450K protocol for FFPE that includes an additional quality control (QC) checkpoint.

Results: QC checkpoint 3 was designed to assess the presence of DNA after bisulfite conversion and restoration, just prior to application of the HM450K assay. DNA was extracted from 474 archival FFPE breast tumour material. Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3. Genome-wide methylation was measured for the remaining 428 tumour-enriched DNA. Of these, only 4 samples failed our stringent HM450K data criteria thus representing a 99% success rate. Using prior knowledge about methylation marks associated with breast cancer we further explored the quality of the data. Twenty probes in the BRCA1 promoter region showed increased methylation in triple-negative breast cancers compared to Luminal A, Luminal B and HER2-positive breast cancer subtypes. Validation of this observation in published data from The Cancer Genome Atlas (TCGA) Network (obtained from DNA extracted from fresh frozen tumour samples) confirms the quality of the data obtained from the improved protocol.

Conclusions: The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples. This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.

No MeSH data available.


Related in: MedlinePlus