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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

Bisulfite libraries prepared using the Fluidigm Access Array system. (A) Low-throughput libraries prepared manually were observed to produce strong dominant bands of the expected size with minimal visible dimer product, when visualized by agarose gel. Size in base pairs is indicated to the left (B) Sequencing results for the percent global methylation of the control libraries prepared manually. (C) Preliminary results with the Fluidigm Access Array platform resulted in weakly amplifying sequencing libraries with prominent dimer products. (D) After extensive optimization to identify the critical parameters, pre-amplification under ideal conditions still gave variable library performance using the Access Array system, with minor differences in pre-amplification primer concentration or the number of cycles of pre-amplification leading to failed libraries (that is, lane 1 vs lane 2). Lane 1, 200 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 2, 50 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 3, 200 nM primer, 15 cycles pre-amplification, Roche HF buffer; lane 4, 200 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 5, 50 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 6, 200 nM primer, 20 cycles pre-amplification, Roche HF buffer.
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Fig2: Bisulfite libraries prepared using the Fluidigm Access Array system. (A) Low-throughput libraries prepared manually were observed to produce strong dominant bands of the expected size with minimal visible dimer product, when visualized by agarose gel. Size in base pairs is indicated to the left (B) Sequencing results for the percent global methylation of the control libraries prepared manually. (C) Preliminary results with the Fluidigm Access Array platform resulted in weakly amplifying sequencing libraries with prominent dimer products. (D) After extensive optimization to identify the critical parameters, pre-amplification under ideal conditions still gave variable library performance using the Access Array system, with minor differences in pre-amplification primer concentration or the number of cycles of pre-amplification leading to failed libraries (that is, lane 1 vs lane 2). Lane 1, 200 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 2, 50 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 3, 200 nM primer, 15 cycles pre-amplification, Roche HF buffer; lane 4, 200 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 5, 50 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 6, 200 nM primer, 20 cycles pre-amplification, Roche HF buffer.

Mentions: The above control experiments with high-quality cell line DNA indicated good overall performance of the bisulfite PCR pre-amplification assay in producing a final library, and therefore the utility of the assay using clinical FFPE DNA was then assessed. To this end, 13 FFPE breast tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%), were evaluated in the pre-amplification strategy outlined above, which involved 44 well-performing primer pairs divided between two 20-plex and 24-plex pre-amplification pools which underwent 15 cycles of pre-amplification. An aliquot of this mix was then used in 44 individual singleplex PCR reactions (one for each primer pair) for another 35 cycles, using a standard PCR thermocycler. After checking all products by gel, an aliquot of each amplicon was pooled together followed by 15 rounds of barcoding PCR amplification and finally gel purification; these libraries were observed to give prominent single bands with no visible dimer product (Figure 2A) and were successfully sequenced. Sequenced libraries demonstrated high mappability (that is, over 90% of reads mapped to the reference index); however, it was noted that the methylated controls included in the analysis were below the methylation percentages expected, suggesting potential bias towards unmethylated transcripts (Figure 2B), possibly as a result the total number of cycles used (15 pre-amp + 35 secondary + 15 barcoding = 60 cycles of amplification).Figure 2


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)

Bisulfite libraries prepared using the Fluidigm Access Array system. (A) Low-throughput libraries prepared manually were observed to produce strong dominant bands of the expected size with minimal visible dimer product, when visualized by agarose gel. Size in base pairs is indicated to the left (B) Sequencing results for the percent global methylation of the control libraries prepared manually. (C) Preliminary results with the Fluidigm Access Array platform resulted in weakly amplifying sequencing libraries with prominent dimer products. (D) After extensive optimization to identify the critical parameters, pre-amplification under ideal conditions still gave variable library performance using the Access Array system, with minor differences in pre-amplification primer concentration or the number of cycles of pre-amplification leading to failed libraries (that is, lane 1 vs lane 2). Lane 1, 200 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 2, 50 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 3, 200 nM primer, 15 cycles pre-amplification, Roche HF buffer; lane 4, 200 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 5, 50 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 6, 200 nM primer, 20 cycles pre-amplification, Roche HF buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Bisulfite libraries prepared using the Fluidigm Access Array system. (A) Low-throughput libraries prepared manually were observed to produce strong dominant bands of the expected size with minimal visible dimer product, when visualized by agarose gel. Size in base pairs is indicated to the left (B) Sequencing results for the percent global methylation of the control libraries prepared manually. (C) Preliminary results with the Fluidigm Access Array platform resulted in weakly amplifying sequencing libraries with prominent dimer products. (D) After extensive optimization to identify the critical parameters, pre-amplification under ideal conditions still gave variable library performance using the Access Array system, with minor differences in pre-amplification primer concentration or the number of cycles of pre-amplification leading to failed libraries (that is, lane 1 vs lane 2). Lane 1, 200 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 2, 50 nM primer, 15 cycles pre-amplification, GoTaq Flexi buffer; lane 3, 200 nM primer, 15 cycles pre-amplification, Roche HF buffer; lane 4, 200 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 5, 50 nM primer, 20 cycles pre-amplification, GoTaq Flexi buffer; lane 6, 200 nM primer, 20 cycles pre-amplification, Roche HF buffer.
Mentions: The above control experiments with high-quality cell line DNA indicated good overall performance of the bisulfite PCR pre-amplification assay in producing a final library, and therefore the utility of the assay using clinical FFPE DNA was then assessed. To this end, 13 FFPE breast tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%), were evaluated in the pre-amplification strategy outlined above, which involved 44 well-performing primer pairs divided between two 20-plex and 24-plex pre-amplification pools which underwent 15 cycles of pre-amplification. An aliquot of this mix was then used in 44 individual singleplex PCR reactions (one for each primer pair) for another 35 cycles, using a standard PCR thermocycler. After checking all products by gel, an aliquot of each amplicon was pooled together followed by 15 rounds of barcoding PCR amplification and finally gel purification; these libraries were observed to give prominent single bands with no visible dimer product (Figure 2A) and were successfully sequenced. Sequenced libraries demonstrated high mappability (that is, over 90% of reads mapped to the reference index); however, it was noted that the methylated controls included in the analysis were below the methylation percentages expected, suggesting potential bias towards unmethylated transcripts (Figure 2B), possibly as a result the total number of cycles used (15 pre-amp + 35 secondary + 15 barcoding = 60 cycles of amplification).Figure 2

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