GeneAb™ LMO2

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.

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.

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.

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.

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.

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. Index information can be downloaded here.

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. Index information can be downloaded here.

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

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.

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.

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.

1 kb DNA Ladder in 1% agarose gel

• For sizing and quantification of double strand DNA fragments.
• Composed of ten bands as shown on right.
• The 4 kb band with higher concentration is easily distinguishable from the others.
• Premixed with 6X DNA loading buffer for direct gel loading.

1 kb DNA Ladder in 1% agarose gel

• For sizing and quantification of double strand DNA fragments.
• Composed of ten bands as shown on right.
• The 4 kb band with higher concentration is easily distinguishable from the others.
• Premixed with 6X DNA loading buffer for direct gel loading.

Overview

• Collect from nasal, buccal, saliva, fecal, skin, surfaces, and more
• Completely stabilize microbiota profiles from the point-of-collection
• Minimize bias in data analysis: NGS (Metagenomics/Microbiome), PCR and microarrays
• No need to immediately process samples: ship and store at ambient temperatures
• DNA preservation at room temperature over 2 years
• Renders sample non-infectious for safe and easy shipping
• Compatible with DNA isolation kits and protocols: Optimized isolation procedure using Norgen’s Microbiome DNA Isolation Kit

Norgen’s Swab Collection and DNA Preservation System is designed for collection, ambient storage and transport of DNA from samples collected using a swab, including nasal, buccal, saliva, fecal, skin, surfaces, etc. The Swab Collection and DNA Preservation System contains Norgen’s Swab Preservative in a liquid format. The user simply collects the specimen using the provided swab, and then transfers the swab into the Swab Preservative. The Swab Preservative prevents the growth of Gram-negative and Gram-positive bacteria and fungi, and also inactivates viruses allowing the resulting non-infectious samples to be handled and shipped safely. In addition, the Swab Preservative eliminates the need to immediately process or freeze samples and allows the samples to be shipped to centralized testing facilities at ambient temperatures. The components of the Swab Preservative allow DNA samples to be stored at room temperature for over 2 years.

DNA Isolation from Preservative

Swab samples collected and preserved using Norgen’s Swab Collection and DNA Preservation System are compatible with most DNA isolation methods. Samples stored in the tubes have been used successfully with most of Norgen Biotek’s DNA isolation kits and reagents. An optimized isolation procedure is available as part of Norgen’s Microbiome DNA Isolation Kit (Cat. 64100).

Details

Supporting Data

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Kit Specifications
Volume of Preservative	1.2 mL
Preservation Temperature	Room Temperature
Preservation time	2 years at room temperature

Shelf Life and Handling

When stored at room temperature, unused Norgen Swab Preservative Tubes are stable through to the collect-before date without any reduction in performance. Please see the device insert or tube label for collect-before date.

Kit Components	Cat. 45690 (50 units)
Swab Collection and DNA Preservation Kit	50
Individually Wrapped Swabs	50
Product Insert	1