Accel-NGS® Methyl-Seq DNA Library Kit
The Accel-NGS Methyl-Seq DNA Library Kit maximizes DNA recovery of bisulfite-converted samples and resultant libraries accurately represent sample base composition. The kit currently provides the most comprehensive coverage of the methylome and is an excellent choice for bisulfite sequencing applications, as well as targeted sequencing, such as RRBS and hybridization capture using the NimbleGen™ SeqCap™ Epi Enrichment Sysem. The Accel-NGS Methyl-Seq Kit is also compatible with bisulfite-converted DNA samples enriched by ChIP or other methods as well as ancient DNA samples that may contain uracil nucleotides as a result of damage.
- High recovery of input DNA
- Low bias library preparation
- Simple, 2-hour protocol
- Inputs from 100 pg to 100 ng
- Minimal PCR cycles required
- Maintain precious samples
- Reveals the complete methylome
- Processing more samples/day
- Use multiple sample types
- Reduced amplification errors
Improve Recovery of Library Molecules with Unbiased Capture of ssDNA
Each step is color-coded to make the protocol easy to follow. A simple, single tube “with bead” protocol is provided with the kit in order to streamline processing between reactions. As the clean-up beads are not provided with the kit, we recommend the SPRIselect™ Reagent Kit from Beckman Coulter.
Whole Genome Bisulfite Sequencing of Human and Arabidopsis DNA
Increased Yields and Even Sample Coverage from Low Input Amounts
High Complexity Library from 1 ng Input DNA
Cover More CpX Sites/CpG Islands
Detection of Cancer-Associated Genome-Wide Hypomethylation
Examine Genome-Wide Methylation Status from 5 ng of cfDNA
Percent hypomethylation of 8 cancer samples was calculated by comparing the methylation density (MD) of 1 Mb bins to the average of the 5 healthy control samples. Bins were assigned as hypomethylated if MD was >3 SD lower than the average MD.
This Circos plot represents the methylation status of 1 Mb bins across chromosomes 1-22 for Sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary).
Targeted Sequencing with Hybridization Capture Enrichment and RRBS
Methylation sequencing of an enriched target region is a cost-effective alternative to whole genome bisulfite sequencing (WGBS). For targeted bisulfite sequencing, the methods used for library preparation and hybridization capture impact library complexity, required sequencing depth, and associated costs. Swift’s Accel-NGS Methyl-Seq DNA Library Kit is compatible with the single-stranded DNA resulting from bisulfite conversion and avoids loss of complexity due to broken library molecules. Combining Accel-NGS Methyl-Seq with the NimbleGen SeqCap Epi Enrichment System (Roche NimbleGen) enables lower input DNA quantites while maintaining library complexity.
Accel-NGS Methyl-Seq and SeqCap Epi enrichment enable low input targeted bisulfite sequencing (left panel). Other techniques require a large amount of input DNA in order to maintain library complexity (center and right panels). These methods require a workflow that constructs library molecules prior to bisulfite conversion, and bisulfite treatment results in broken, non-functional library molecules. Further, hybridization capture that utilizes probes incompatible with bisulfite-converted DNA (Agilent SureSelect) must be performed on library molecules that have not undergone PCR amplification as amplification does not preserve methylation status (right panel).
Hybridization capture with SeqCap Epi CpGiant Enrichment captures 80.5 Mb of the human genome, which contains greater than 5.5 million CpG dinucleotide sites. Sequencing metrics for libraries prepared with the Accel-NGS Methyl-Seq DNA Library Kit from Swift Biosciences were compared to those from the Kapa library preparation kit currently recommended by Roche NimbleGen. Coverage metrics were analyzed for inputs of 1 μg and 100 ng, quantities that are within specification for the Kapa and Swift library preparation, respectively. Additionally, lower inputs of 10 ng (Kapa and Swift) and 1 ng (Swift only) were also analyzed. While the Kapa library preparation performs well with 1 μg of input DNA, a substantial increase in duplicate reads and decrease in genome coverage can be observed at 10 ng. However, the Swift kit performs well at 10 ng, with the performance metrics at 1 ng comparable to the 10 ng metrics from the Kapa kit.
Targeted Methylation Sequencing from 1 ng with SeqCap Epi CpGiant
DMRs Called from 10 ng Libraries with the Swift Methyl-Seq Kit
DMRs were identified from 10 ng libraries from an H1 ES cell line and a B-lymphocyte cell line (NA12878). Libraries created with the Accel-NGS Methl-Seq kit identified 294,130 DMRs (shown above). Libraries created with the Kapa kit identified only 464 DMRs (not shown).
Reduced Representation Bisulfite Sequencing (RRBS)
RRBS workflow comparison illustrating differences between a traditional method and Accel-NGS Methyl-Seq.
Protocols and Tools
- Accel-NGS Methyl-Seq DNA Library Kit
- Input DNA Quantification Assay
- Accel-NGS Methyl-Seq Master Mixing Volume Calculator
- A Novel Method for Achieving Single Cell Resolution of Epigenomic Status (ASHG 2016)
- Cost-Effective NGS Analysis of Circulating, Cell-Free DNA (Festival of Genomics 2016)
- Low Frequency Variant Detection and Tissue-of-Origin Exploration Using Liquid Biopsies (AACR 2017)
- NGS Library Methods to Achieve Comprehensive Coverage for WGS and Targeted Sequencing…(ICHG 2016)
- Techniques for Measuring Cancer Burden in Liquid Biopsy Samples (MMTC 2016, ESHG 2016)
The Accel-NGS Methyl-Seq Kit has been validated with the EZ DNA Methylation-Gold™ Kit (Zymo Research). While other bisulfite conversion kits may be compatible with the Swift kit, it is not recommended to use Accel-NGS Methyl-Seq with bisulfite conversion kits that utilize a single stranded nucleic acid carrier, as this carrier may act as substrate and result in creation of artifactual library molecules.
Yes. The unique chemistry of Accel-NGS Methyl-Seq easily works in an RRBS workflow. The process differs slightly from traditional methods, as illustrated below. To capture the fraction of DNA fragments that are enriched for CpG islands, RRBS protocols require that size selection of inserts occurs after MspI DNA digestion, but prior to bisulfite conversion. As Accel-NGS Methyl-Seq performs bisulfite conversion prior to library preparation, size selection must be performed on the DNA fragments immediately following MspI digestion (rather than on library molecules, in the traditional workflow).
It is not recommended. Fragmentation solely by bisulfite treatment results in a broad size distribution of larger DNA fragments. Such fragments may be converted into library molecules, however the wide distribution of library molecules will cluster poorly on the flow cell. If skipping fragmentation is unavoidable, we recommend examining the size of library molecules prior to library quantification, and, if necessary, performing a right side size selection to remove very large library molecules to improve clustering on the flow cell. This scenario will result in reduced complexity of sample representation. Please contact us at Tech Support for recommended modifications if relying on bisulfite treatment for fragmentation.
In addition to converting unmethylated cytosines, bisulfite treatment also results in damage which fragments DNA. Therefore, it is normal to observe bisulfite-converted libraries that are slightly smaller in size than their unconverted control libraries, as illustrated by the Bioanalyzer profile below.
The Accel-NGS Methyl-Seq Kit constructs libraries that are readily compatible with NimbleGen™ SeqCap™ Epi Target Enrichment Systems. Sequencing data from capture with the SeqCap Epi CpGiant panel can be found on the Accel-NGS Methyl-Seq Product Page. The Accel-NGS Methyl-Seq Kit is not compatible with the Agilent SureSelectXT Human Methyl-Seq Enrichment Systems, as this technology uses capture probes that are not compatible with bisulfite converted DNA.
The Accel-NGS 2S Hyb DNA Library Kit creates libraries that are readily compatible with SureSelectXT Human Methyl-Seq Enrichment. However, because this approach to targeted methylation sequencing results in decreased sample complexity and increased input requirements compared to the Methyl-Seq workflow, this is only recommended if the Methyl-Seq workflow cannot be performed. Please contact us at Tech Support for more information.
Yes. Many users are attracted to this technology because it can convert the short, single-stranded fragments common in ancient DNA into NGS library molecules. Additionally, Accel-NGS Methyl-Seq is capable of preserving uracil-containing DNA resulting from the damage inflicted on ancient DNA samples. Please contact us at Tech Support for recommended changes to the standard protocol.
Bisulfite treatment results in a low complexity library, which requires a high complexity spike-in to avoid low Q-scores, issues with cluster recognition and demultiplexing ability. The Accel-NGS Methyl-Seq Kit has been validated on a MiSeq and HiSeq 2500 with a 10% spike-in of a PhiX library. Please contact Illumina for questions concerning sequencing bisulfite converted samples on other instruments.
Yes. Bases added to the 3’ termini of DNA fragments during the Adaptase reaction may contain unmethylated cytosines, which adds both artifactual sequence and methylation information to the dataset. Therefore, trimming of Methyl-Seq libraries is required to obtain accurate mapping efficiency and precise methylation information. Please view our technical note titled Accel-NGS 1S Plus and Methyl-Seq: Tail Trimming for Better Data for more details on the tail and how to trim it.
First, select a product and quantity:
|30024||Accel-NGS Methyl-Seq DNA Library Kit (24 rxns)||$1750.00|
|30096||Accel-NGS Methyl-Seq DNA Library Kit (96 rxns)||$6370.00|
Next, an Indexing Kit is required for complete functionality. Please select from the following:
|36024||Methyl-Seq Set A Indexing Kit (12 indices, 24 rxns)||$120.00|
|38096||Methyl-Seq Dual Indexing Kit (96 unique combinations)||$480.00|