GSE65116
Enhanced methylome sequencing by recovery of unsequenceable fragments
Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemistry renders the majority of DNA fragments unsequenceable, thus necessitating PCR amplification. Furthermore, bisulfite conversion generates an A,T-rich DNA library that leads to major PCR biases that may confound methylation analysis. Here we report a method that enables accurate methylation analysis, by rebuilding the damaged DNA library after bisulfite treatment. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We applied the ReBuilT method for whole methylome analysis of the A,T rich genome of Plasmodium berghei. We demonstrate substantial improvements in coverage and the reduction of sequence-context biases as compared to classical methylome analysis. Our method will be widely applicable for accurate, quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available. Overall design: From the same DNA sample we prepared 3 PCR-free Bisulfite-Seq replicates (ReBuilT) and 2 standard Bisulfite-Seq replicates (PCR-BS).