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Zymo-Seq WGBS Library Kit
Zymo-Seq WGBS Library Kit
- Whole Genome Bisulfite (WGBS) library preparation in less than 4 hours and in one tube.
- Reduced library preparation bias for accurate methylation calling
- Reproducible genome coverage from any species (>90% of CpG sites detected in mammalian samples)
Zymo-Seq WGBS Library Kit is the only kit available for whole genome bisulfite (WGBS) library preparation in a single tube. Zymo-Seq WGBS Library Kit incorporates tagmentation technology to eliminate the tedious fragmentation, enzymatic, and clean-up steps required by conventional, ligation-based library preparation methods. This streamlined workflow minimizes hands-on time, making it an ideal choice for higher throughput applications. To prepare Zymo-Seq WGBS libraries, intact genomic DNA is first bisulfite converted. Then, the following library preparation procedures are completed in a single tube: (1) second-strand synthesis, (2) adapterization via tagmentation, and (3) library amplification and indexing. After purification, libraries are ready for sequencing on Illumina instruments.
WGBS is the gold standard for DNA methylation studies as it provides base-level methylation quantification for all cytosines. Zymo-Seq WGBS Library Kit is ideal for any species for whole-genome methylation calling of CG, CHG, and CHH sites. Over 90% of human and mouse CpG sites can be detected from Zymo-Seq libraries.
|DNA Input||For optimal results, use 100 ng of high quality, intact genomic DNA as input. Protocol is compatible with inputs between 10 ng to 100 ng. DNA should be free of enzymatic inhibitors and RNA contamination. DNA can be resuspended in water, TE, or a low salt buffer.|
|Processing Time||~ 4 hours|
|Required Equipment||Microcentrifuge, thermal cycler with heated lid, magnetic separator, multichannel pipette (suggested)|
|Required material not provided||Illumina DNA Prep, (M) Tagmentation Kit (Illumina, Cat. No. 20060059 or 20060060)|
|Sequencing Platform||Libraries are compatible with all Illumina sequencing platforms (MiSeq, MiniSeq, HiSeq, NextSeq, NovaSeq)|
Q1: Does the Zymo-Seq WGBS Library Kit offer all the necessary reagents to generate libraries for sequencing?
No. The Illumina DNA Prep Kit (Cat. No. 20018704 or 20018705) is required and must be purchased directly from Illumina.
Q2: Do I need to purchase indexing primers separately?
Q3: Can I use degraded samples, such as FFPE DNA or cell-free DNA to generate WGBS libraries from this kit?
The size of the input genomic DNA is important for high-quality and high-yield library. DNA from FFPE samples are often highly degraded, so the quality of the library will be affected. Cell-free DNA are not compatible with the kit because the fragment size is too short for efficient tagmentation.
Q4: What type of inputs is the Zymo-Seq WGBS Library Kit compatible with?
Zymo-Seq WGBS Library Kit is compatible with genomic DNA extracted from any species, whether it is mammalian, bacteria, or plants. It is best to use high-quality, intact genomic DNA that is RNA-free.
Q5: What are the suggested read length and sequencing depth needed for WGBS libraries?
Libraries are suitable for any cycling number, but increased cycling numbers will require greater amount of adapter trimming. For most, 100 bp paired-end reads are suitable. The sequencing depth will be dependent on the genome size and coverage desired. Generally, aiming for 10x coverage per CpG site is recommended.
The number of reads (N) required for the desired coverage can be estimated by using the following equation: N = (Coverage)*(Diploid Genome Length)/Read Length. Bisulfite conversion reduces the complexity of the genome and have lower alignment rates compared to DNA-seq, so it is recommended to increase the number of reads by 25%. Therefore, for human WGBS at 10X coverage using 100 bp PE sequencing, at least 400 million reads are recommended.
Q6: What is the difference between Zymo-Seq WGBS Library Kit and the Pico Methyl-Seq kit? Which one should I use?
Zymo-Seq WGBS kit is ideal for those working with higher inputs (10-100ng) of genomic DNA. It has minimal handling and clean-up steps, which makes it ideal for high-throughput library generation.
Pico Methyl-Seq would be a better option for those working with small DNA inputs (< 10ng) or highly fragmented DNA, such as cfDNA or FFPE DNA.
|D5016||E. coli Non-Methylated Genomic DNA||5 µg/20 µl|
|D5030-1||Lightning Conversion Reagent||1.5 ml|
|D5001-3||M-Binding Buffer||20 ml|
|D5001-4||M-Wash Buffer||6 ml|
|D5030-5||L-Desulphonation Buffer||10 ml|
|D3004-4-1||DNA Elution Buffer||1 ml|
|W1001-1||DNase/RNase-Free Water||1 ml|
|C1004-50||Zymo-Spin IC Columns||50 Pack|
|C1001-50||Collection Tubes||50 Pack|