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Ovation® Ultralow Methyl-Seq Library Systems
The Ovation Ultralow Methyl-Seq Library Systems provide a simple, fast and scalable solution for producing libraries used in conjunction with bisulfite sequencing to analyze DNA methylation. The system requires as little as 10 ng of DNA, thereby enabling methylation studies with a broad range of sample types. The protocol is compatible with whole genome bisulfite sequencing, and can be completed in approximately nine hours, including bisulfite conversion steps. As shown in Figure 1, the streamlined workflow consists of four main steps: fragmentation of genomic DNA, end repair to generate blunt ends, adaptor ligation, and fill-in with 5-methyl-cytosine dNTPs. Following adaptor ligation, bisulfite conversion is performed to yield libraries ready for PCR amplification and cluster formation.
The Ovation Ultralow Methyl-Seq Library Systems is available in two kit configurations to enable multiplexing on the Illumina sequencing platforms. The Ovation Ultralow Methyl-Seq DR Multiplex System 1-8 (Part No. 0335) and Ovation Ultralow Methyl-Seq DR Multiplex System 9-16 (Part No. 0336) each provide eight unique barcoded adaptors for multiplex sequencing. In combination these kits enable up to 16-plex sequencing using a dedicated read (DR) barcode design.
Figure 1. The Ovation Ultralow Methyl-Seq Library System Workflow
Figure 2. Coverage uniformity
Figure 2. Coverage uniformity. Whole genome bisulfite sequencing was performed on libraries generated from 100ng and 10ng of human genomic DNA. Sequencing was performed on the Illumina GAIIx platform. From each library, 225,000 reads that mapped to a 40 million bp region of Chromosome 5 were randomly selected. This region was divided into 3 kbp bins. The number of reads with a start position within that bin was recorded. Bins were then sorted by number of reads from high to low and plotted.
Figure 3. Methylated CpG quantification
Figure 3. Methylated CpG quantification. Libraries were made from 10ng Sample 1 (Normal Human gDNA), 10ng Sample 2 (in vitro CpG methylated Human gDNA), 10ng of a 25:75 mixture of Sample 1:Sample 2, and 10ng of a 75:25 mixture. Libraries were sequenced to determine the % Methyl CpG present on average across all reads uniquely mapping to the genome. Sample 1 and Sample 2 % Methyl CpG values were used to calculate the expected % Methyl CpG present in the mixtures.