Accel-NGS™ Methyl-Seq DNA Library Kit

For the Preparation of Bisulfite Sequencing Libraries on Illumina Next Generation Sequencing (NGS) Platforms

The Accel-NGS Methyl-Seq DNA Library Kit maximizes DNA recovery of bisulfite converted samples and constructs libraries that accurately represent sample base composition.  The kit currently provides the most comprehensive coverage of the methylome.

Catalog No Library Kit Reactions
30024  Accel-NGS Methyl-Seq DNA Library Kit for Illumina  24
30096 Accel-NGS Methyl-Seq DNA Library Kit for Illumina 96

An Indexing Kit is required for complete functionality. Please select one of the following:

Catalog No Indexing Adapter Kit Reactions
 36024 Methyl-Seq Indexing Kit for Illumina (7 indices x 12 combinations) 24
38096 Methyl-Seq Indexing Kit for Illumina (20 indices x 96 combinations) 96

Supported Applications

  • WGBS  (Whole Genome Bisulfite Sequencing)
  • RRBS (Reduced Representation Bisulfite Sequencing)
  • Bisulfite-converted DNA enriched by MeDIP, ChIP, or other methods
  • Hybridization capture: NimbleGen SeqCap Epi Enrichment System
  • Ancient DNA samples that may contain uracil nucleotides as a result of damage

Features

  • High recovery of input DNA
  • Low bias library preparation
  • Simple, 2 hour protocol
  • Inputs from 100 pg to 100 ng
  • Minimal PCR cycles required

Benefits

  • Maintain precious samples
  • Reveal complete methylome
  • Process more samples/day
  • Use multiple sample types
  • Reduced amplification errors

Improve Recovery of Library Molecules While Minimizing Fragment Bias

accel ngs methyl seq dna library kit
Fig 1.  Accel-NGS Methyl-Seq uses template-independent adapter attachment chemistry on post-bisulfite converted DNA, resulting in high recovery and low bias compared to the traditional pre-bisulfite method and the random-primed method.

Video

Click here to watch the new Accel-NGS Methyl-Seq Video


Webinar

Click here to view the Webinar from Swift Bio; Lowering the Limits for Epigenetic Methylation Analysis

Whole Genome Bisulfite Sequencing of Human and Arabidopsis DNA

Methylation sequencing combines bisulfite treatment with NGS, and allows for examination of methylation status at a single nucleotide resolution. Bisulfite conversion of DNA enables analysis of methylation status by converting unmethylated cytosines to uracils. However, bisulfite treatment also denatures and damages DNA. As traditional library preparations require double-stranded DNA as template for library construction, bisulfite conversion must be performed after library preparation. This results in damage and loss of library molecules. The Accel-NGS Methyl-Seq DNA library workflow maximizes DNA recovery through post-bisulfite library preparation, utilizing a highly efficient adapter attachment chemistry compatible with single-stranded, bisulfite-converted DNA. This process eliminates up to 95% library loss found in workflows with post-library construction bisulfite treatment.

Increased Yields from Low Input Amounts

accel ngs methyl seq dna library kit 2

High efficiency library preparation from Swift Biosciences enables methylation sequencing analysis of low input quantity samples. Additionally, high recovery of input DNA translates into high complexity libraries which contain more unique library molecules. These high complexity libraries require fewer PCR cycles, minimizing duplicate reads and maximizing fold coverage of the sample genome. For methylation studies, it is particularly important to cover sites that can experience methylation: CpG sites in the human genome, and CpX sites in the Arabidopsis genome (where CpX = CpG + CpH). There are more than 28 million CpG sites in the human genome, and there are more than 17 million CpX sites in the Arabidopsis genome.

High Complexity Library from 1 ng Input DNA

Arabidopsis Human Table

Cover More CpG Islands

cgx sites cpg islands 1

cgx sites cpg islands 2

cgx sites cpg islands 3

Detection of Cancer-Associated Genome-Wide Hypomethylation

Liquid biopsies, collecting circulating cell-free DNA (cfDNA) from blood plasma, can be used for the detection of cancer and monitoring of disease. This has been demonstrated recently by the detection of cancer-associated genome-wide hypomethlation in plasma circulating, cfDNA (Chan et al. PNAS, vol 110, no 47(2103) pp18761-18768). We performed WGBS on cfDNA from 8 cancer patient samples and 5 normal controls. Using 5 ng of input cfDNA and 10 million mapped reads per sample provided enough coverage to identify genome-wide hypomethylation status in cancer patient samples.

Examine Genome-Wide Methylation Status from 5 ng of cfDNA

8 Sample HypomethylationTable

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.

Cancer Sample 8 Circ Plot

The Circos plot represents the methylation status of 1Mb bins across chromosomes 1-22 for sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2cm 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.

Targeted Sequencing CpGiant vs SureSelect

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

target methylation sequencing

Analysis of differentially methylated regions (DMRs) was also performed for data from the 10 ng libraries from both Swift and Kapa, comparing DNA from an H1 ES cell line and a B-lymphocyte cell line (NA12878). The figure below illustrates the genomic distribution of the 294,130 DMRs called from the Swift Accel-NGS Methyl-Seq Library Kit (37,799 hypomethylated and 256,331 hypermethylated). In contrast, the 10 ng Kapa library resulted in only 464 total DMR calls (not shown).


DMR’s called from 10ng libraries with the Swift Methyl-Seq Kit

Meth-FIG-DMRs-2

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)

Reduced Representation Bisulfite Sequencing (RRBS) utilizes a MspI digestion, followed by selection of a specific range of fragment sizes, to enrich for library inserts containing CpG islands. Traditional methods require double-stranded DNA for library construction, so size selection must account for the approximately 120 bp of adapter sequence that is added during library construction (40-220 bp inserts corresponds to approximately 160-340 bp library molecules). As Accel-NGS Methyl-Seq library construction is compatible with single-stranded DNA, size selection of 100-220 bp fragments can be performed immediately following MspI digestion (the adapter length need not be considered). The above diagram illustrates the differences between RRBS workflows for traditional and Swift Methyl-Seq library preparation.

RRBS Workflow:

Methyl-Seq rbbs workflow

RRBS workflow comparison illustrating differences between a traditional method and Accel-NGS Methyl-Seq.

Brochure & Manual

Technical Resources

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