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Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments.

McInroy GR, Beraldi D, Raiber EA, Modrzynska K, van Delft P, Billker O, Balasubramanian S - PLoS ONE (2016)

Bottom Line: However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification.The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis.Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom.

ABSTRACT
Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available.

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Cytosine methylation in P. berghei.(a) The log2 fold change of 5mC contexts from cytosine contexts within the P. berghei genome. Each bar is annotated with the percentage of 5mC loci within the context. The sequence context varies greatly between PCR-free and amplified samples. ReBuilT data reveals an enrichment in the asymmetric CAH context, and CHG and CC contexts are strongly disfavoured. PCR-BS methylation occurs more often in CG and CHG contexts (H = A, T, G). (b) The distribution of methylation across genomic regions, shown as log2 fold change from the distribution of cytosines. The percentage of 5mC loci in the regions is given above each bar. Methylation in the AT-rich intergenic regions is underrepresented in the PCR-BS dataset. (c) The profile of 5mC levels over exons. Traditional PCR-BS gives a similar profile to ReBuilT, but greatly over-estimates the 5mC levels.
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pone.0152322.g004: Cytosine methylation in P. berghei.(a) The log2 fold change of 5mC contexts from cytosine contexts within the P. berghei genome. Each bar is annotated with the percentage of 5mC loci within the context. The sequence context varies greatly between PCR-free and amplified samples. ReBuilT data reveals an enrichment in the asymmetric CAH context, and CHG and CC contexts are strongly disfavoured. PCR-BS methylation occurs more often in CG and CHG contexts (H = A, T, G). (b) The distribution of methylation across genomic regions, shown as log2 fold change from the distribution of cytosines. The percentage of 5mC loci in the regions is given above each bar. Methylation in the AT-rich intergenic regions is underrepresented in the PCR-BS dataset. (c) The profile of 5mC levels over exons. Traditional PCR-BS gives a similar profile to ReBuilT, but greatly over-estimates the 5mC levels.

Mentions: From the ReBuilT dataset we found the context of methylated loci to be primarily CAH (68.62%) and CTH (23.46%), with the remaining sites being found in CG (3.6%), CHG (2.2%), and CC (2.1%) contexts. While the genomic context of all cytosines shows a preference for adenine in the +1 position, this preference is significantly increased for methylated loci. Fig 4a demonstrates this point by showing the log2 fold change of methylated loci context from the genomic cytosines. All contexts are underrepresented against the P. berghei background with the exception of CAH, which is overrepresented. This asymmetric context contrasts with the strong preference for CG methylation in mammalian genomes. Furthermore, cytosine and guanine bases are generally depleted around methylated loci, in agreement with previously reported data from P. falciparum[22]. While the majority of methylation from the PCR-BS dataset also occurs within the CAH context, a defining feature is a preference for nearby guanines. This context distribution may be affected by the read count bias towards GC rich regions, such as the exons described in Fig 3c.


Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments.

McInroy GR, Beraldi D, Raiber EA, Modrzynska K, van Delft P, Billker O, Balasubramanian S - PLoS ONE (2016)

Cytosine methylation in P. berghei.(a) The log2 fold change of 5mC contexts from cytosine contexts within the P. berghei genome. Each bar is annotated with the percentage of 5mC loci within the context. The sequence context varies greatly between PCR-free and amplified samples. ReBuilT data reveals an enrichment in the asymmetric CAH context, and CHG and CC contexts are strongly disfavoured. PCR-BS methylation occurs more often in CG and CHG contexts (H = A, T, G). (b) The distribution of methylation across genomic regions, shown as log2 fold change from the distribution of cytosines. The percentage of 5mC loci in the regions is given above each bar. Methylation in the AT-rich intergenic regions is underrepresented in the PCR-BS dataset. (c) The profile of 5mC levels over exons. Traditional PCR-BS gives a similar profile to ReBuilT, but greatly over-estimates the 5mC levels.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4816320&req=5

pone.0152322.g004: Cytosine methylation in P. berghei.(a) The log2 fold change of 5mC contexts from cytosine contexts within the P. berghei genome. Each bar is annotated with the percentage of 5mC loci within the context. The sequence context varies greatly between PCR-free and amplified samples. ReBuilT data reveals an enrichment in the asymmetric CAH context, and CHG and CC contexts are strongly disfavoured. PCR-BS methylation occurs more often in CG and CHG contexts (H = A, T, G). (b) The distribution of methylation across genomic regions, shown as log2 fold change from the distribution of cytosines. The percentage of 5mC loci in the regions is given above each bar. Methylation in the AT-rich intergenic regions is underrepresented in the PCR-BS dataset. (c) The profile of 5mC levels over exons. Traditional PCR-BS gives a similar profile to ReBuilT, but greatly over-estimates the 5mC levels.
Mentions: From the ReBuilT dataset we found the context of methylated loci to be primarily CAH (68.62%) and CTH (23.46%), with the remaining sites being found in CG (3.6%), CHG (2.2%), and CC (2.1%) contexts. While the genomic context of all cytosines shows a preference for adenine in the +1 position, this preference is significantly increased for methylated loci. Fig 4a demonstrates this point by showing the log2 fold change of methylated loci context from the genomic cytosines. All contexts are underrepresented against the P. berghei background with the exception of CAH, which is overrepresented. This asymmetric context contrasts with the strong preference for CG methylation in mammalian genomes. Furthermore, cytosine and guanine bases are generally depleted around methylated loci, in agreement with previously reported data from P. falciparum[22]. While the majority of methylation from the PCR-BS dataset also occurs within the CAH context, a defining feature is a preference for nearby guanines. This context distribution may be affected by the read count bias towards GC rich regions, such as the exons described in Fig 3c.

Bottom Line: However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification.The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis.Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom.

ABSTRACT
Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available.

Show MeSH
Related in: MedlinePlus