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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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ReBuilT produces superior quality sequence data.(a) Retention of raw data through bioinformatic preprocessing. Trimming indicates removal of adapter or low-quality sequences; alignment was to a chimeric P. berghei/M. musculus genome to remove host contamination; Map to P. berghei shows the fraction of reads aligning to the parasitic genome. ReBuilT retained almost twice percentage of raw data. (b) The distribution of mapped reads in 50 bp windows across the genome. The Poisson distribution (dashed line) describes the ideal distribution in the absence of external biases. REBUiLT approximates this distribution, while PCR-BS does not.
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pone.0152322.g002: ReBuilT produces superior quality sequence data.(a) Retention of raw data through bioinformatic preprocessing. Trimming indicates removal of adapter or low-quality sequences; alignment was to a chimeric P. berghei/M. musculus genome to remove host contamination; Map to P. berghei shows the fraction of reads aligning to the parasitic genome. ReBuilT retained almost twice percentage of raw data. (b) The distribution of mapped reads in 50 bp windows across the genome. The Poisson distribution (dashed line) describes the ideal distribution in the absence of external biases. REBUiLT approximates this distribution, while PCR-BS does not.

Mentions: We first looked at how much raw sequencing data was retained following a bioinformatic pipeline including adapter trimming, quality trimming and read alignment (Fig 2a). Following all trimming steps the ReBuilT libraries retained on average 87.4% of the raw data, compared to 63.6% for the PCR-BS libraries. The majority of the PCR-BS loss was due to removal of dimeric adapter sequences, which cannot form in the initial ReBuilT ligation and are not retained during the second ligation. Surviving reads were aligned to a chimeric P. berghei and M. musculus reference genome, as extracted parasite DNA may be contaminated with some genomic material from the host. Average alignment rates to this chimeric reference were 80.5% and 72.1% for the ReBuilT and PCR-BS samples respectively. From this subset of aligned reads 90.9% of ReBuilT and 70.6% of PCR-BS reads were aligned to the P. berghei reference genome, with the remaining reads aligning to the host mouse genome. Following all data processing, 58.8% of the raw ReBuilT data could be used for methylation calling, while the PCR-BS libraries retained only 29.7% of the raw data. The ReBuilT method, therefore, yields considerably more useable data, which reduces the sequencing power required for methylation analysis.


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)

ReBuilT produces superior quality sequence data.(a) Retention of raw data through bioinformatic preprocessing. Trimming indicates removal of adapter or low-quality sequences; alignment was to a chimeric P. berghei/M. musculus genome to remove host contamination; Map to P. berghei shows the fraction of reads aligning to the parasitic genome. ReBuilT retained almost twice percentage of raw data. (b) The distribution of mapped reads in 50 bp windows across the genome. The Poisson distribution (dashed line) describes the ideal distribution in the absence of external biases. REBUiLT approximates this distribution, while PCR-BS does not.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152322.g002: ReBuilT produces superior quality sequence data.(a) Retention of raw data through bioinformatic preprocessing. Trimming indicates removal of adapter or low-quality sequences; alignment was to a chimeric P. berghei/M. musculus genome to remove host contamination; Map to P. berghei shows the fraction of reads aligning to the parasitic genome. ReBuilT retained almost twice percentage of raw data. (b) The distribution of mapped reads in 50 bp windows across the genome. The Poisson distribution (dashed line) describes the ideal distribution in the absence of external biases. REBUiLT approximates this distribution, while PCR-BS does not.
Mentions: We first looked at how much raw sequencing data was retained following a bioinformatic pipeline including adapter trimming, quality trimming and read alignment (Fig 2a). Following all trimming steps the ReBuilT libraries retained on average 87.4% of the raw data, compared to 63.6% for the PCR-BS libraries. The majority of the PCR-BS loss was due to removal of dimeric adapter sequences, which cannot form in the initial ReBuilT ligation and are not retained during the second ligation. Surviving reads were aligned to a chimeric P. berghei and M. musculus reference genome, as extracted parasite DNA may be contaminated with some genomic material from the host. Average alignment rates to this chimeric reference were 80.5% and 72.1% for the ReBuilT and PCR-BS samples respectively. From this subset of aligned reads 90.9% of ReBuilT and 70.6% of PCR-BS reads were aligned to the P. berghei reference genome, with the remaining reads aligning to the host mouse genome. Following all data processing, 58.8% of the raw ReBuilT data could be used for methylation calling, while the PCR-BS libraries retained only 29.7% of the raw data. The ReBuilT method, therefore, yields considerably more useable data, which reduces the sequencing power required for methylation analysis.

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