Methyl Sequencing

Methyl-seq, also termed bisulfite sequencing, is a powerful tool for understanding genome-wide methylation with single-nucleotide resolution. In order to identify and quantify methylation patterns, bisulfite treatment of DNA is employed to convert cytosine residues into uracil. These residues will read as thymine during sequencing. Methylated
(5-methylcytosine) residues are left unmodified by the treatment. A comparison of bisulfite-treated to untreated DNA enables the identification of methylated loci.

Several strategies have been developed to understand the methylation status of a genome, including whole genome bisulfite sequencing (WGBS); reduced representation bisulfite sequencing (RRBS), which enriches for CpG islands; and methylated-DNA immunoprecipitation sequencing (MeDIP-seq), which utilizes antibodies against 5-methylcytosine to enrich for methylated DNA.

Bisulfite treatment results in a significant decrease in DNA input and quality; therefore, the construction of high-quality libraries and efficient downstream amplification is critical. Kapa Biosystems offers library preparation kits that provide increased yields of adapter-ligated library molecules and reduced amplification bias, which translates to higher library diversity, lower duplication rates, and more uniform coverage. KAPA HiFi Uracil+, a high-fidelity DNA polymerase engineered to be tolerant to uracil residues, is particularly well suited for the amplification of bisulfite-converted DNA libraries which typically produce low concentrations of AT-rich DNA.

For Research Use Only. Not for use in diagnostic procedures.

Are you interested in bisulfite sequencing of low-input DNA?

Read our new application note, “KAPA Hyper Prep and HiFi Uracil+ Kits allow for reduced representation bisulfite sequencing of ultra-low amounts of DNA in B cells

Download our App Note

Workflow

Bisulfite conversion after library preparation:

  • Sample Preparation

    Sample

  • Library Construction

    Library
     Construction

  • Bisulfite conversion

    Bisulfite Conversion

  • Library Amplification

    Library Amplification

  • Library Quantification

    Library Quantification

Bisulfite conversion prior to library preparation:

KAPA Library Preparation Kits do not support library construction from single-stranded, bisulfite-converted DNA.

  • Sample Preparation

    Sample

  • Bisulfite conversion

    Bisulfite Conversion

  • Library Construction

    Library
    Construction

  • Library Amplification

    Library Amplification

  • Library Quantification

    Library Quantification

Available Products by Workflow

Library Preparation

DNA Library Preparation

KAPA Hyper Prep Kits (Illumina®)

  • Construct high yield, low-bias libraries prior to bisulfite-conversion in less than 3 hours

KAPA Library Preparation Kits (Illumina)

  • Construct high yield, low-bias libraries prior to bisfulfite-conversion

KAPA Library Preparation Kits (Ion Torrent™)

  • Construct libraries with increased yields of adapter-ligated library molecules prior to
    bisulfite-conversion
Library Amplification

Library Amplification

KAPA HiFi Uracil+

  • Improve yields and reduce PCR-induced bias when amplifying bisulfite-converted DNA libraries
Library Quantification

Library Quantification

KAPA Library Quantification Kits

  • Accurately quantify libraries for downstream sequencing