Limits...
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 libraries prepared using the custom bisulfite PCR multiplex assay. After optimizing primer concentration for the individual primer pairs as well as the overall pools, barcoding of the final libraries demonstrated that the assay performed well on both high-quality white blood cell DNA (A) and degraded clinical FFPE samples (B). In comparison, even substantially degraded FFPE DNA which completely failed in Fluidigm Access Array (lanes 8 to 10 in Figure 2C) performed well with this multiplex assay (panel B lanes 1 to 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Representative libraries prepared using the custom bisulfite PCR multiplex assay. After optimizing primer concentration for the individual primer pairs as well as the overall pools, barcoding of the final libraries demonstrated that the assay performed well on both high-quality white blood cell DNA (A) and degraded clinical FFPE samples (B). In comparison, even substantially degraded FFPE DNA which completely failed in Fluidigm Access Array (lanes 8 to 10 in Figure 2C) performed well with this multiplex assay (panel B lanes 1 to 3).

Mentions: To determine which bisulfite PCR primer pairs should be pooled together, a similar strategy was employed. In brief, real-time qPCR was performed for 84 different amplicons; Cts ranged from 25.5 to 37.1. Based on these results, primer pairs with proximal Ct values were pooled together, taking care to ensure that primers for overlapping regions were separated, which resulted in 59 amplicons spread across 8 pools (five pools of 8, two pools of 6, and one pool of 5). Initial screening of the pools evaluated different final primer pool concentrations (from 1 μM final to 125 nM final) as well as different cycling parameters, enhancers, and MgCl2 concentrations; two primer pairs were excluded due to their tendency to cause dimers. After optimizing primer concentration for both the individual primer pairs as well as the overall pools, the same 13 clinical FFPE samples previously used on the Fluidigm platform were subjected to the custom bisulfite multiplex assay. Barcoding of the final libraries demonstrated that all eight pools were giving high-quality libraries (Figure 3A,B) and even substantially degraded FFPE DNA which completely failed in Fluidigm Access Array performed robustly (lanes 8 to 10 in Figure 3B, as compared to Fluidigm libraries in Figure 2C). These clinical samples were also sequenced using the Illumina MiSeq (Illumina, San Diego, CA) to determine the proportionality of amplicon representation and overall methylation state; libraries for each clinical sample were prepared three times on different days to assess reproducibility, and methylated control samples were also included.Figure 3


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 libraries prepared using the custom bisulfite PCR multiplex assay. After optimizing primer concentration for the individual primer pairs as well as the overall pools, barcoding of the final libraries demonstrated that the assay performed well on both high-quality white blood cell DNA (A) and degraded clinical FFPE samples (B). In comparison, even substantially degraded FFPE DNA which completely failed in Fluidigm Access Array (lanes 8 to 10 in Figure 2C) performed well with this multiplex assay (panel B lanes 1 to 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Representative libraries prepared using the custom bisulfite PCR multiplex assay. After optimizing primer concentration for the individual primer pairs as well as the overall pools, barcoding of the final libraries demonstrated that the assay performed well on both high-quality white blood cell DNA (A) and degraded clinical FFPE samples (B). In comparison, even substantially degraded FFPE DNA which completely failed in Fluidigm Access Array (lanes 8 to 10 in Figure 2C) performed well with this multiplex assay (panel B lanes 1 to 3).
Mentions: To determine which bisulfite PCR primer pairs should be pooled together, a similar strategy was employed. In brief, real-time qPCR was performed for 84 different amplicons; Cts ranged from 25.5 to 37.1. Based on these results, primer pairs with proximal Ct values were pooled together, taking care to ensure that primers for overlapping regions were separated, which resulted in 59 amplicons spread across 8 pools (five pools of 8, two pools of 6, and one pool of 5). Initial screening of the pools evaluated different final primer pool concentrations (from 1 μM final to 125 nM final) as well as different cycling parameters, enhancers, and MgCl2 concentrations; two primer pairs were excluded due to their tendency to cause dimers. After optimizing primer concentration for both the individual primer pairs as well as the overall pools, the same 13 clinical FFPE samples previously used on the Fluidigm platform were subjected to the custom bisulfite multiplex assay. Barcoding of the final libraries demonstrated that all eight pools were giving high-quality libraries (Figure 3A,B) and even substantially degraded FFPE DNA which completely failed in Fluidigm Access Array performed robustly (lanes 8 to 10 in Figure 3B, as compared to Fluidigm libraries in Figure 2C). These clinical samples were also sequenced using the Illumina MiSeq (Illumina, San Diego, CA) to determine the proportionality of amplicon representation and overall methylation state; libraries for each clinical sample were prepared three times on different days to assess reproducibility, and methylated control samples were also included.Figure 3

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