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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

Pre-amplification multiplex results for bisulfite DNA samples. (A) A flow diagram outlining the different conditions examined with respect to multiplexability, pooling, and exonuclease treatment. (B) The results of different conditions after 15 cycles of multiplex pre-amplification. Forty-eight primer pairs were assessed for the multiplexability in a pre-amplification reaction. All samples had the same amount of input DNA. Lane 1: A positive control involving singleplex PCR reaction of an individual primer pair, with the same DNA template amount as used in the pre-amplification. Lane 2: Eight-plex pre-amplification reaction and ExoSAP-IT treatment of individual pre-amp reactions, followed by pooling and singleplex amplification (as illustrated in the upper panel of A). Lane 3: Results of three different 8-plex reactions pooled together first then ExoSAP-IT treatment of the combined pool, for a total of 24 amplicons in the ExoSAP-IT treatment, followed by singleplex amplification of a primer pair (as illustrated in the lower panel of A). Lane 4: Twenty-four-plex pre-amplification results. (C) The effect of ExoSAP-IT treatment on bisulfite libraries, as compared to gDNA amplicons. The arrow indicates where primers migrate on the gel. Lane 1: Pool of 48 amplicons prior to barcoding PCR. Lane 2: Sample library after barcoding PCR. Primers are visible at the bottom of the lane. Lane 3: Sample library after ExoSAP-IT treatment at 37°C. Lane 4: Sample library after ExoSAP-IT treatment at 37°C, followed by heat inactivation of the ExoSAP-IT at 80°C. Note the higher molecular weight smear in the methylation library, which is not observed with gDNA amplicons. Lane 5: Sample library cycled at 37°C, followed by 80°C heat denaturation step, but with no ExoSAP-IT.
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Fig1: Pre-amplification multiplex results for bisulfite DNA samples. (A) A flow diagram outlining the different conditions examined with respect to multiplexability, pooling, and exonuclease treatment. (B) The results of different conditions after 15 cycles of multiplex pre-amplification. Forty-eight primer pairs were assessed for the multiplexability in a pre-amplification reaction. All samples had the same amount of input DNA. Lane 1: A positive control involving singleplex PCR reaction of an individual primer pair, with the same DNA template amount as used in the pre-amplification. Lane 2: Eight-plex pre-amplification reaction and ExoSAP-IT treatment of individual pre-amp reactions, followed by pooling and singleplex amplification (as illustrated in the upper panel of A). Lane 3: Results of three different 8-plex reactions pooled together first then ExoSAP-IT treatment of the combined pool, for a total of 24 amplicons in the ExoSAP-IT treatment, followed by singleplex amplification of a primer pair (as illustrated in the lower panel of A). Lane 4: Twenty-four-plex pre-amplification results. (C) The effect of ExoSAP-IT treatment on bisulfite libraries, as compared to gDNA amplicons. The arrow indicates where primers migrate on the gel. Lane 1: Pool of 48 amplicons prior to barcoding PCR. Lane 2: Sample library after barcoding PCR. Primers are visible at the bottom of the lane. Lane 3: Sample library after ExoSAP-IT treatment at 37°C. Lane 4: Sample library after ExoSAP-IT treatment at 37°C, followed by heat inactivation of the ExoSAP-IT at 80°C. Note the higher molecular weight smear in the methylation library, which is not observed with gDNA amplicons. Lane 5: Sample library cycled at 37°C, followed by 80°C heat denaturation step, but with no ExoSAP-IT.

Mentions: Based on the pre-amplification pools and ExoSAP-IT parameters outlined above, four different pre-amplification screening conditions were assessed, as outlined in Figure 1A:


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)

Pre-amplification multiplex results for bisulfite DNA samples. (A) A flow diagram outlining the different conditions examined with respect to multiplexability, pooling, and exonuclease treatment. (B) The results of different conditions after 15 cycles of multiplex pre-amplification. Forty-eight primer pairs were assessed for the multiplexability in a pre-amplification reaction. All samples had the same amount of input DNA. Lane 1: A positive control involving singleplex PCR reaction of an individual primer pair, with the same DNA template amount as used in the pre-amplification. Lane 2: Eight-plex pre-amplification reaction and ExoSAP-IT treatment of individual pre-amp reactions, followed by pooling and singleplex amplification (as illustrated in the upper panel of A). Lane 3: Results of three different 8-plex reactions pooled together first then ExoSAP-IT treatment of the combined pool, for a total of 24 amplicons in the ExoSAP-IT treatment, followed by singleplex amplification of a primer pair (as illustrated in the lower panel of A). Lane 4: Twenty-four-plex pre-amplification results. (C) The effect of ExoSAP-IT treatment on bisulfite libraries, as compared to gDNA amplicons. The arrow indicates where primers migrate on the gel. Lane 1: Pool of 48 amplicons prior to barcoding PCR. Lane 2: Sample library after barcoding PCR. Primers are visible at the bottom of the lane. Lane 3: Sample library after ExoSAP-IT treatment at 37°C. Lane 4: Sample library after ExoSAP-IT treatment at 37°C, followed by heat inactivation of the ExoSAP-IT at 80°C. Note the higher molecular weight smear in the methylation library, which is not observed with gDNA amplicons. Lane 5: Sample library cycled at 37°C, followed by 80°C heat denaturation step, but with no ExoSAP-IT.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Pre-amplification multiplex results for bisulfite DNA samples. (A) A flow diagram outlining the different conditions examined with respect to multiplexability, pooling, and exonuclease treatment. (B) The results of different conditions after 15 cycles of multiplex pre-amplification. Forty-eight primer pairs were assessed for the multiplexability in a pre-amplification reaction. All samples had the same amount of input DNA. Lane 1: A positive control involving singleplex PCR reaction of an individual primer pair, with the same DNA template amount as used in the pre-amplification. Lane 2: Eight-plex pre-amplification reaction and ExoSAP-IT treatment of individual pre-amp reactions, followed by pooling and singleplex amplification (as illustrated in the upper panel of A). Lane 3: Results of three different 8-plex reactions pooled together first then ExoSAP-IT treatment of the combined pool, for a total of 24 amplicons in the ExoSAP-IT treatment, followed by singleplex amplification of a primer pair (as illustrated in the lower panel of A). Lane 4: Twenty-four-plex pre-amplification results. (C) The effect of ExoSAP-IT treatment on bisulfite libraries, as compared to gDNA amplicons. The arrow indicates where primers migrate on the gel. Lane 1: Pool of 48 amplicons prior to barcoding PCR. Lane 2: Sample library after barcoding PCR. Primers are visible at the bottom of the lane. Lane 3: Sample library after ExoSAP-IT treatment at 37°C. Lane 4: Sample library after ExoSAP-IT treatment at 37°C, followed by heat inactivation of the ExoSAP-IT at 80°C. Note the higher molecular weight smear in the methylation library, which is not observed with gDNA amplicons. Lane 5: Sample library cycled at 37°C, followed by 80°C heat denaturation step, but with no ExoSAP-IT.
Mentions: Based on the pre-amplification pools and ExoSAP-IT parameters outlined above, four different pre-amplification screening conditions were assessed, as outlined in Figure 1A:

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