Accel-NGS® Methyl-Seq DNA Library KitOnobird2019-07-19T18:46:02+00:00
Accel-NGS® Methyl-Seq DNA Library Kit
For the Preparation of Bisulfite Converted Libraries
The Accel-NGS Methyl-Seq DNA Library Kit maximizes DNA recovery of bisulfite-converted samples and resultant libraries accurately represent sample base composition on Illumina® NGS Platforms. 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 System. 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.
“We have been using Swift’s Accel NGS Methyl-Seq DNA Library kit since 2017. It is our go-to kit to determine the methylation status of circulating cell-free DNA at the genome level. The Swift Methyl-Seq kit has consistently produced high-quality libraries for our technically challenging whole genome bisulfite sequencing. We are confident in the complexity and yield, with steady reliable performance that exceeds our stringent quality metrics for our lab and our global collaborators. I highly recommend this kit to anyone performing methylation sequencing. It is a great assay and more people should know about it!” -Renowned Philadelphia Institution
Which bisulfite conversion kits are compatible with the Accel-NGS Methyl-Seq Kit?
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.
Is the Accel-NGS Methyl-Seq Kit compatible with reduced representation bisulfite sequencing (RRBS)?
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).
Can bisulfite conversion replace the Fragmentation Step in the 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.
Why does a bisulfite-converted library contain slightly smaller inserts than an unconverted control library?
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.
Is the Accel-NGS Methyl-Seq Kit compatible with hybridization capture technologies?
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.
Can the Accel-NGS Methyl-Seq Kit be used with ancient DNA?
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.
Why is my sequencing run exhibiting low Q-scores?
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.
Does the Adaptase™ tail need to be bioinformatically trimmed?
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.
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
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 and Even Sample Coverage from Low Input Amounts
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
Cover More CpX Sites/CpG Islands
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(2013) 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
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 quantities 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
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).
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) 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 comparison illustrating differences between a traditional method and Accel-NGS Methyl-Seq.