Abstract 2098: Twist Fast Hybridization targeted methylation sequencing: a tunable target enrichment solution for methylation detection
Authors: Graham Brenton I. , Arbiza Leonardo , Butcher Kristin , Chen Siyuan , Gantt Richard , Gude Sabina , Nugent Rebecca , Thompson Christina , Zeitoun Ramsey 
Published: July 2021

DNA methylation is a genetic occurrence that plays a principal role in the epigenetic regulation of gene expression. Genome wide association studies (GWAS) have linked differential methylation patterns, especially at cytosines in CpG context, to disease states like cancer and obesity. Well-known chemical and enzymatic techniques are available to detect the methylation state of cytosines throughout the genome. With Next-Generation Sequencing (NGS) becoming increasingly affordable and high-throughput, methylation sequencing has become a popular and accessible approach for disease detection. Correspondingly, there is an increasing demand for targeted methylation sequencing techniques that enrich for CpG-rich genomic regions of interest. Such techniques have historically been inefficient due to the reduced sequence complexity caused by bisulfite or enzymatic library preparation methods. These methods involve the conversion of unmethylated cytosines to uracils, subsequently resulting in thymines after PCR amplification, and inherently increase similarities between library sequences in a given population. This effect causes issues with specificity during hybrid capture since baits and targets with repetitive sub-sequences can incidentally hybridize to non-targeted molecules during capture.

In this poster, we present the Twist Targeted Methylation Sequencing Product, an end-to-end solution for preparing methyl-seq libraries and performing in-solution hybrid capture. We demonstrate benefits of using the NEBNext Enzymatic Methyl-seq Methylation Library Preparation protocol in our system, compared to traditional bisulfite treatment, and discuss approaches that were taken to maximize target enrichment specificity and uniformity. First, we introduce Twist's custom panel design approach, which leverages Twist's oligo manufacturing capabilities and uses predictive methods to remove probes most likely to cause poor capture performance. Second, we present methylation-specific Twist Fast Hybridization Target Enrichment protocol tunability, showing how FastHyb Wash Buffer 1 temperature and hybridization time can be used to manipulate performance metrics. Third, we introduce Twist's Methylation Enhancer, a novel blocker set that has shown to alleviate capture specificity issues across a wide range of methylation panels. Together, the data in this study showcase the Twist Targeted Methylation Sequencing Product as an efficient and flexible solution for custom methylation detection.

Citation Format: Brenton I. Graham, Leonardo Arbiza, Kristin Butcher, Siyuan Chen, Richard Gantt, Sabina Gude, Rebecca Nugent, Christina Thompson, Ramsey Zeitoun. Twist Fast Hybridization targeted methylation sequencing: a tunable target enrichment solution for methylation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2098.