Targeted methyl sequencing

Overview

• Tried and true products—curated for targeted methylation sequencing
• Library preparation from low input—input as low as 5 ng^†
• Powered by xGen Adaptase™ technology—template independent adapter attachment reduces workflow time while generating a less biased library
• Cost effective and reliable—key targets are enriched and sequenced deeply
• Comprehensive sample representation—uniform methylome genome coverage by targeted bisulfite sequencing
• Customizable—select between a small, focused panel or a large panel depending on your research needs
• Workflow design for easy automation

^† Before bisulfate conversion.

What is targeted methyl-seq?

Methylation sequencing (methyl-seq), also referred to as bisulfite sequencing, is a next generation sequencing (NGS) approach that researchers use to study methylation patterns of DNA. These patterns can be used to identify different cell types in epigenetics and oncology research [1,2]. Researchers can study the methylation status of the entire genome using whole genome bisulfite sequencing (WGBS) or target specific regions of the genome using hybridization capture panels to enrich genomic regions of interest. More specifically, a targeted methyl-seq approach (also called targeted bisulfite sequencing) uses hybridization probes to capture and enrich biologically relevant regions of interest but with deeper sequencing depth and higher coverage than WGBS.

Targeted methyl-seq workflow

A targeted methyl-seq workflow provides researchers with a streamlined procedure for targeted methylation sequencing. The xGen Methyl-Seq DNA Library Prep Kit has a post-bisulfite library preparation workflow to maximize library complexity by converting bisulfite-induced single-stranded DNA fragments directly into library molecules. This kit is compatible with a range of input quantities and libraries can be generated in as little as 2 hours from bisulfite‑converted samples.

The addition of an xGen Custom Hyb Panel to this workflow enables researchers to capture biologically relevant regions of the genome for targeted methyl-seq. When used for targeted methyl-seq, the xGen Custom Hyb Panel results in sequences with a high on-target mapping percentage and efficiency (Figure 1), as well as methylation profiles that are highly correlated to those obtained from WGBS (Figure 2). Further details on the xGen Custom Hyb Panel for targeted methyl-seq can be found in the AppNote, Cost-effective targeted methyl-seq using an xGen Custom Hyb Capture Panel and the xGen Methyl-Seq DNA Library Prep Kit identifies DNA methylation in low input samples consistent with WGBS.

The recommended group of xGen methyl-seq product offerings also include the compatible core reagents the xGen Hybridization and Wash v2 Kit, xGen Universal Blockers TS, and xGen Library Amplification Primer Mix to accomplish methyl-seq with an easy and flexible workflow.

Together, these xGen NGS products offer you a comprehensive workflow for accelerating your epigenetic research using a targeted methylation sequencing approach.

Method data

xGen targeted methyl-seq workflow results in comprehensive coverage with low-input samples

High-quality sequencing results with the xGen Methyl-Seq DNA Library Prep Kit plus xGen Custom Hyb Panel workflow

To assess the xGen Methyl-Seq DNA Library Prep Kit plus xGen Custom Hyb Panel workflow, libraries were prepared and enriched from 5, 10, or 25 ng of cell-free DNA (cfDNA) reference material (RM) (Horizon HD833). The methylation‑specific xGen Custom Hyb Panel was designed to enrich for five genes associated with methylation biomarkers, covering a total region of 128 kb. Hybridization capture was performed on all libraries following the xGen Hybridization Capture protocol, hybridizing three libraries per capture for 16 hours. Sequencing was performed on a NextSeq™ 500 (Illumina^®) platform using 2 x 75 bp reads with 10% PhiX spike-in. The results of this workflow were assessed by generating common sequencing quality metrics outlined in Figure 1 using the Picard toolkit [3].

The xGen Methyl-Seq DNA Library Prep Kit, provides researchers with flexibility as it can prepare libraries from a wide range of sample inputs, which can then be enriched with a specially designed xGen Custom Hyb Panel to obtain high‑quality sequences from regions of interest. Together, the xGen Methyl-Seq Library Prep Kit and an xGen Custom Hyb Panel create a meaningful tool for accelerating focused epigenetic research.

Reliable methylation profiles from libraries enriched with the xGen Custom Hyb Panel

To determine if the xGen Custom Hyb Panel preserved methylation signatures, the methylation percent per CpG site for a whole-genome bisulfite sequencing (WGBS) library prepared using 10 ng of cfDNA was compared to the same metric obtained from the 10 ng cfDNA library enriched with the xGen Custom Hyb Panel. Across shared target spaces, the xGen Custom Hyb Panel and the WGBS library were highly correlated (Pearson, r ≥ 0.97; Figure 2).

The high correlation between libraries enriched using a methyl-specific xGen Custom Hyb Panel and the libraries obtained from WGBS shows that the xGen Custom Hyb Panel is a reliable, cost-effective way to assess methylation of key targets associated with different variants.

Ordering

Use our xGen Hyb Panel Design Tool to create your xGen custom Hyb panels.