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Bisulfite Sequencing

Bisulfite sequencing permits analysis of epigenetic status at a single nucleotide resolution. Through bisulfite or oxidative bisulfite sequencing, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) can be identified in the genome, respectively. As environmental epigenetic modifications profoundly impact gene expression1 during development, the epigenetic status of cytosines within the genome can provide clues about the specifics of cellular differentiation2 and disease processes. For example, a recent study3 utilizing cell-free DNA samples has uncovered a link between genome-wide hypomethylation and cancer status.

Bisulfite treatment can be used in conjunction with next-generation sequencing library preparation methods in order to perform high throughput epigenetic analysis. However, traditional library preparation methods are vulnerable to sample loss due to the DNA damage resulting from bisulfite treatment, and are, therefore, limited to relatively large DNA input quantities. Swift Biosciences has developed a sample preparation solution which defends against sample loss. The Accel-NGS® Methyl-Seq DNA Library Kit maximizes recovery of input DNA, enabling library construction from as little as 100 pg of DNA while achieving comprehensive coverage of the epigenome.

SWIFT PRODUCT LINES COMPATIBLE WITH BISULFITE SEQUENCING:

Accel-NGS Methyl-Seq DNA Library Kit

The Swift advantage:
  • High recovery of input DNA maximizes use of precious samples.
  • Low bias library preparation reveals the complete methylome.
  • Simple, 2-hour protocol allows you to process more samples per day.
  • Inputs from 100 pg to 100 ng enable compatibility with multiple sample types.
  • Minimal PCR cycles reduce amplification errors.

Retain More Sample with the Methyl-Seq Workflow

The Accel-NGS Methyl-Seq Kit is based on Swift’s Adaptase™ technology which enables unbiased adapter attachment to single-stranded bisulfite-converted DNA. This preferable workflow eliminates significant library loss associated with workflows where the NGS library is constructed prior to bisulfite conversion. The template-independent adapter attachment of the Accel-NGS Methyl-Seq Kit also provides significant improvements in methylome coverage uniformity compared to methods that incorporate adapters by random priming of single-stranded DNA.

16-1282_methylseq-comparison-workflow-v2

For both Accel-NGS Methyl-Seq and random priming kits, bisulfite conversion is performed prior to library construction. With the traditional library kit, bisulfite conversion is performed on the completed library. The lightning bolts represent bisulfite-induced fragmentation, NGS adapters are depicted in green and blue, and non-uracil containing library products are shown in yellow.

Superior Whole Genome Bisulfite Sequencing Coverage

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 Libraries from 1 ng Arabidopsis DNA

ap_bisulfite_table_high-complexity-libraries

Cover More CpX Sites

CpXSitesA

cpxsitesb

Human CpG Coverage from as Low as 100 pg

WGBS coverage metrics were analyzed for NA12878 sample inputs of 10 ng, 1 ng and 100 pg. Approximately 500M reads were used for each sample. Sequencing was performed on a HiSeq® 2500 v4 (2×100 PE).

Detection of Cancer-Associated Genome-Wide Hypomethylation

Liquid biopsies, the collecting of 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 cfDNA3. We performed whole genome bisulfite sequencing (WGBS) on cfDNA from eight cancer patient samples and five normal controls. Five nanograms 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

AP_Bisulfite_Table_Examine Genome-wide

Percent hypomethylation of eight cancer samples was calculated by comparing the methylation density (MD) of 1 Mb bins to the average of the five healthy control samples. Bins were assigned as hypomethylated if MD was > 3 SD lower than the average MD.

Meth-FIG-circos-plot

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

Hybridization capture is a cost-effective method for methylation analysis as a small portion of the genome is captured requiring less overall sequencing. The SeqCap™ Epi CpGiant Enrichment panel captures 80.5 Mb of the human genome, which contains greater than 5.5 million CpG dinucleotide sites. The Accel-NGS Methyl-Seq DNA Library Kit was used prior to the enrichment and compared sequencing metrics for libraries prepared with 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

AP_Bisulfite_Table_Targeted Methylation Seq

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 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 Accel-NGS 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).
References:

1Lister and Ecker. Genome Res., vol 19, no 6(2009) pp959-966

2 Brunner et al. Genome Res., vol 19, no 6(2009) pp1044-1056

3Chan et al. PNAS, vol 110, no 47(2103) pp18761-18768