StrandNGS MethylSeq supports Agilent’s SureSelect XT HS2 Target enrichment protocol

DNA methylation (DNAm) is an epigenetic mechanism that plays a vital role in regulating gene expression. It occurs predominantly on cytosines followed by guanine residues (CpG), a process referred to as CpG methylation. 

CpG islands (CGIs), regions in the genome with a high frequency of CpG sites, are usually present in the promoter regions of housekeeping genes in most vertebrates. These CGIs are usually unmethylated, allowing for binding by transcriptional activators and thus promoting transcription [1]. 

While DNAm at CpG sites is known to play critical roles in cellular development and differentiation [1], [2], the process also occurs at non-CpG contexts (CpH where H is A, C, or T)

However, the underlying role of non-CpG DNAm and its potential impact on disease and development are not fully understood.  Some studies indicate a role in neurological development and functioning [3], [4] but how it regulates gene expression and may contribute to disease remains a black box. 

To probe non-CPG DNAm further, Agilent has developed a modified version of their SureSelect XT HS2 Target Enrichment protocol. This hybrid capture protocol is used for the detection and analysis of methylation by next-generation sequencing (NGS) using bisulfite-converted libraries. 

Agilent recently published a detailed description of this protocol through a use case involving an animal model of autism spectrum disorder (ASD). This experimental workflow included using a custom panel for ASD, sample collection, library preparation, and sequencing using NextSeq550.  

The results of the experiment were analyzed using command line scripts as well as StrandNGS, Strand’s secondary analysis platform for NGS data. 

The analysis involved performing differential methylation evaluations to a narrowed down region of interest and picking the StrandNGS wizard-driven workflow, a built-in workflow in the software for methyl-Seq analyses, in its default settings.  

The results were annotated and visualized in various graphical data representations, an example of one such representation is included below.

Agilent’s team notes the ease of use of StrandNGS for non-bioinformatics experts and discusses the utility of the built-in analysis pipeline for the detection and evaluation of methylation differences between cytosines and genomic regions. They also acknowledge that the diverse database integration options serve as user-friendly ways for non-statisticians to explore and dive deeper into data.  

The modified Methyl-seq SureSelect targeted capture protocol results analyzed with the help of StrandNGS yield promising results. For more details on their findings, take a look at their recent app note.   

References

1. Reik, Wolf, Wendy Dean, and Jörn Walter. “Epigenetic Reprogramming in Mammalian Development.” Science 293, no. 5532 (August 10, 2001): 1089–93.
2. Reik, Wolf. “Stability and Flexibility of Epigenetic Gene Regulation in Mammalian Development.” Nature 447, no. 7143 (May 2007): 425–32.
3. Lister, Ryan, Eran A. Mukamel, Joseph R. Nery, Mark Urich, Clare A. Puddifoot, Nicholas D. Johnson, Jacinta Lucero, et al. “Global Epigenomic Reconfiguration During Mammalian Brain Development.” Science 341, no. 6146 (August 9, 2013): 1237905.
4. Jang, Hyun Sik, Woo Jung Shin, Jeong Eon Lee, and Jeong Tae Do. “CpG and Non-CpG Methylation in Epigenetic Gene Regulation and Brain Function.” Genes 8, no. 6 (June 2017): 148.