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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

Sequencing results. Sequencing results for libraries prepared in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%) using the custom bisulfite PCR multiplex assay. (A) The proportions of each pool across 54 libraries, determined as a total amount of each library. Y-axis: Percentage of total library = Total number of reads for a pool ÷ Total number of reads for the library. Although pool 7 was observed to dominate all libraries, the proportion of each pool across 54 samples was maintained at consistent levels. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (B) The average standard deviation in 8-plex pool proportions observed across all libraries. Average pool standard deviation = (The sum of all standard deviations for a single pool ÷ The total number of entries, that is, the mean value). Across 13 FFPE samples amplified in triplicate, pool proportionality was maintained within similar values across all samples and libraries. (C) The proportion of each amplicon in each of the libraries, calculated as a percentage of its original 8-plex pool. Percentage pool proportion = Total number of reads for an amplicon ÷ Total number of reads for its pool. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (D) Histogram showing the distribution of the average standard deviations for all 57 amplicons in the assay. Across 13 FFPE samples amplified in triplicate, the proportion of each of the 56 amplicons was maintained at consistent levels. Average standard deviation = The sum of all standard deviation values for a single amplicon (as a percentage of its original 8-plex pool as outlined in C) ÷ The total number of entries.
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Fig4: Sequencing results. Sequencing results for libraries prepared in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%) using the custom bisulfite PCR multiplex assay. (A) The proportions of each pool across 54 libraries, determined as a total amount of each library. Y-axis: Percentage of total library = Total number of reads for a pool ÷ Total number of reads for the library. Although pool 7 was observed to dominate all libraries, the proportion of each pool across 54 samples was maintained at consistent levels. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (B) The average standard deviation in 8-plex pool proportions observed across all libraries. Average pool standard deviation = (The sum of all standard deviations for a single pool ÷ The total number of entries, that is, the mean value). Across 13 FFPE samples amplified in triplicate, pool proportionality was maintained within similar values across all samples and libraries. (C) The proportion of each amplicon in each of the libraries, calculated as a percentage of its original 8-plex pool. Percentage pool proportion = Total number of reads for an amplicon ÷ Total number of reads for its pool. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (D) Histogram showing the distribution of the average standard deviations for all 57 amplicons in the assay. Across 13 FFPE samples amplified in triplicate, the proportion of each of the 56 amplicons was maintained at consistent levels. Average standard deviation = The sum of all standard deviation values for a single amplicon (as a percentage of its original 8-plex pool as outlined in C) ÷ The total number of entries.

Mentions: Of the 57 amplicons assayed, only one amplicon was observed to consistently fail across all samples, and the remaining 56 amplicons were all present in the assay. Of the 56 amplicons remaining, the total reads for most amplicons were typically observed to be within an order of magnitude of each other, with the difference in total read numbers between the amplicons with the lowest and highest counts typically showing no more than two orders of magnitude difference (Additional file 3: Figure S3). The variability and reproducibility in the proportions of each pool as a total amount of each library were also determined, and the amount of each pool as a percentage of each library was calculated. Across 13 samples amplified in triplicate, pool proportionality was maintained within similar values across all samples and libraries (Figure 4A), with standard deviations of pool proportions less than 3% (Figure 4B). Although pool 7 was observed to dominate all sequencing libraries, subsequent experiments using a reduced concentration of primers for pool 7 reduced the amount present to levels proportional with the other pools (data not shown).Figure 4


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)

Sequencing results. Sequencing results for libraries prepared in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%) using the custom bisulfite PCR multiplex assay. (A) The proportions of each pool across 54 libraries, determined as a total amount of each library. Y-axis: Percentage of total library = Total number of reads for a pool ÷ Total number of reads for the library. Although pool 7 was observed to dominate all libraries, the proportion of each pool across 54 samples was maintained at consistent levels. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (B) The average standard deviation in 8-plex pool proportions observed across all libraries. Average pool standard deviation = (The sum of all standard deviations for a single pool ÷ The total number of entries, that is, the mean value). Across 13 FFPE samples amplified in triplicate, pool proportionality was maintained within similar values across all samples and libraries. (C) The proportion of each amplicon in each of the libraries, calculated as a percentage of its original 8-plex pool. Percentage pool proportion = Total number of reads for an amplicon ÷ Total number of reads for its pool. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (D) Histogram showing the distribution of the average standard deviations for all 57 amplicons in the assay. Across 13 FFPE samples amplified in triplicate, the proportion of each of the 56 amplicons was maintained at consistent levels. Average standard deviation = The sum of all standard deviation values for a single amplicon (as a percentage of its original 8-plex pool as outlined in C) ÷ The total number of entries.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389706&req=5

Fig4: Sequencing results. Sequencing results for libraries prepared in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%) using the custom bisulfite PCR multiplex assay. (A) The proportions of each pool across 54 libraries, determined as a total amount of each library. Y-axis: Percentage of total library = Total number of reads for a pool ÷ Total number of reads for the library. Although pool 7 was observed to dominate all libraries, the proportion of each pool across 54 samples was maintained at consistent levels. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (B) The average standard deviation in 8-plex pool proportions observed across all libraries. Average pool standard deviation = (The sum of all standard deviations for a single pool ÷ The total number of entries, that is, the mean value). Across 13 FFPE samples amplified in triplicate, pool proportionality was maintained within similar values across all samples and libraries. (C) The proportion of each amplicon in each of the libraries, calculated as a percentage of its original 8-plex pool. Percentage pool proportion = Total number of reads for an amplicon ÷ Total number of reads for its pool. Whiskers: 10th to 90th percentiles; black circles: 5th and 95th percentiles. (D) Histogram showing the distribution of the average standard deviations for all 57 amplicons in the assay. Across 13 FFPE samples amplified in triplicate, the proportion of each of the 56 amplicons was maintained at consistent levels. Average standard deviation = The sum of all standard deviation values for a single amplicon (as a percentage of its original 8-plex pool as outlined in C) ÷ The total number of entries.
Mentions: Of the 57 amplicons assayed, only one amplicon was observed to consistently fail across all samples, and the remaining 56 amplicons were all present in the assay. Of the 56 amplicons remaining, the total reads for most amplicons were typically observed to be within an order of magnitude of each other, with the difference in total read numbers between the amplicons with the lowest and highest counts typically showing no more than two orders of magnitude difference (Additional file 3: Figure S3). The variability and reproducibility in the proportions of each pool as a total amount of each library were also determined, and the amount of each pool as a percentage of each library was calculated. Across 13 samples amplified in triplicate, pool proportionality was maintained within similar values across all samples and libraries (Figure 4A), with standard deviations of pool proportions less than 3% (Figure 4B). Although pool 7 was observed to dominate all sequencing libraries, subsequent experiments using a reduced concentration of primers for pool 7 reduced the amount present to levels proportional with the other pools (data not shown).Figure 4

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