[Illumina platforms] Methylation Specific Bisulfite Seq Library Prep Kit [30101, 30102, 30103]_BioDynami - 코아사이언스

 

Methylation Specific Bisulfite Seq Library Prep Kit

Protocol download / MSDS download

Cat.#	30101S	30101L	30102S	30102L	30103S	30103L
Index type	non-index	non-index	index	index	unique dual index	unique dual index
Reactions	24	48	24	48	96	2 x 96

 

 

Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.

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Whole genome bisulfite sequencing (WGBS) is the most effective method of DNA methylation analysis. The only limitation is the sequencing cost is very high because the whole genome is sequenced including all the non-methylated regions.

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Reduced Representation Bisulfite Sequencing (RRBS) is the reduced representation of a smaller fraction of the methylated CpG sites. RRBS combines restriction enzyme digestion and bisulfite sequencing, and enriches the sequencing for methylated CpG sites. It is an efficient technology for estimate the whole genome methylation patterns at the single base level. Although this allows a higher coverage depth and reduces the sequencing cost, the limitation is only 10% of the methylated CpG sites are covered.

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Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) is another whole genome enrichment technique used for selection of methylated DNA. Using antibodies against 5-methylcytosine, methylated DNA is enriched from whole genomic DNA via immunoprecipitation. 5-methylcytosine antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing. There are several drawbacks of MeDIP-Seq: 1. Low resolution (150~200 bp) as opposed to the single base resolution; 2. Non-specific interaction due to antibody specificity and selectivity. 3. Bias towards hypermethylated regions.

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The Methylation Specific Bisulfite Seq (MSBS) Library Prep Kit (illumina platform) was developed for construction of NGS libraries for methylated CpG sites using bisulfite treated DNA (20 ng – 500 ng) as input. The kit enriches methylated CpG regions, thus significantly reduce the sequencing cost. The kit estimates the whole genome methylation patterns at the single base level since it is based on a bisulfite-seq technology.

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It is known that bisulfite treatment of completed NGS libraries causes tremendous damage to the libraries. By using bisulfite treated DNA as input, the kit overcomes the significant library loss due to the bisulfite conversion. The kit contains a mixture of PCR polymerases that have high-fidelity amplification and uracil tolerance which is ideal for bisulfite treated DNA.

Methylation Specific Bisulfite Seq Library Prep Kit Workflow

 

Three index types are available for the kit:

Non-index (Cat.# 30101): Libraries do not have index.

Index (Cat.# 30102): Each primer contains a unique barcode sequence of 6 bases to identify the individual library. Library multiplexing capacity is up to 48 samples.

Unique dual index (Cat.# 30103): The multiplexing of bisulfite sequencing library is up to 96 samples with unique dual indexes. We used a Four-Base Difference Index System to generate indexes that have at least 4 bases different from each other in the 8-base index. The index primers remove NGS errors including index cross-contamination, index hopping, reads mis-assignment etc.

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Methylation Specific Bisulfite Seq advantages

• Enrichment of methylated CpG sites
• Single-base resolution
• Low cost for sequencing
• Fast
• Total time: 1.5 hours
• Hands-on time: 10 minutes
• Simple workflow
• Bisulfite treated DNA as input: From 20 ng to 500 ng

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MSBS Library Prep Kit enriches CpG sites

High methylation regions and low methylation regions in human genome.

High methylation region in human genome.

Low methylation region in human genome.

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Sequencing setting: Single-end 35 cycles (Read 1, 35 bases) recommended

To maximize the methylated CpG enrichment, we recommend to sequence the MSBS libraries with single end 35 cycles (read1, 35 bases). This is because the enriched methylated CpG sites are mainly located around the beginning of read 1 sequences. Shorter single end reads tend to have better methylated CpG enrichment.

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Related Products

• NGS DNA Library Prep Kit
• NGS DNA Library Prep Customization Kit
• PCR-free NGS DNA Library Prep Kit
• NGS Low Input DNA Library Prep Kit
• ChIP-Seq Library Prep Kit
• NGS Cell-free DNA Library Prep Kit
• NGS FFPE DNA Library Prep Kit
• Bisulfite Sequencing Library Prep Kit
• Methylation Specific Bisulfite-Seq Library Prep Kit
• NGS Single Stranded DNA Library Prep Kit
• NGS Ancient DNA Library Prep Kit
• NGS DNA Fragmentation & Library Prep Kit
• RNA Seq Library Prep Kit
• NGS DNA Library Prep Kit (Ion Torrent Platform)
• NGS DNA Fragmentation & Library Prep Kit (Ion Torrent Platform)
• Multiplexing Index Primers (Illumina Platform)
• Multiplexing Unique Dual Index Primers (Illumina Platform)

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