The EpiNext™ High-Sensitivity DNA Library Preparation Kit (Illumina) is a complete set of optimized reagents to prepare a DNA library from very small amount of samples for use in next-generation sequencing applications. This kit is suitable for preparing a DNA library using sub-nanogram amounts of DNA input for next generation sequencing applications using an Illumina sequencer. These applications include genomic DNA-seq, ChIP-seq, MeDIP/hMeDIP-seq, traditional bisulfite-seq, and targeted re-sequencing. The optimized protocol and components of the kit allow both non-barcoded (singleplexed) and barcoded (multiplexed) DNA libraries to be constructed quickly with reduced bias. The kit has the following advantages:
- High sensitivity and flexibility - Can be used for both non-barcoded (singleplexed) and barcoded (multiplexed) DNA library preparation. The amount of input DNA can be as low as 0.2 ng with a range from 0.2 to 100 ng. Various dsDNA can be used, which includes limited amounts of fragmented dsDNA isolated from various tissue or cell samples, dsDNA enriched from ChIP reactions, and dsDNA enriched from MeDIP/hMeDIP reactions or exon capture.
- Fast and streamlined procedure - The procedure from fragmented DNA to size selection is less than 1 hour and 30 minutes. No clean-up is required between each step and all reactions take place in the same tube, thereby saving time and preventing handling errors as well as loss of valuable samples. Gel-free size selection further reduces the preparation time.
- Highly convenient - The kit contains all required components for each step of DNA library preparation, which are sufficient for end polishing, ligation, clean-up, size selection, and library amplification, thereby allowing the library preparation to be streamlined with the most reliable and consistent results.
- Minimized bias - Ultra HiFi amplification and optional PCR-free step enable the user to achieve reproducibly achieve high yields of DNA library with minimal sequence bias and low error rates.
DNA library preparation is a critical step for next generation sequencing (NGS). To generate accurate sequencing data in NGS, the prepared library DNA should be sufficient in yield and of high quality. As NGS technology is continuously improving, DNA library preparation is required to be optimized accordingly. Most of the currently used methods are time-consuming, expensive, inconvenient, and specifically need large amounts of DNA. These factors result in a DNA library preparation which cannot be used for biological samples with limited amounts of starting material such as tumor biopsy, early embryos, and embryonic tissues and circulating DNA. In addition, the amount of DNA enriched by ChIP or MeDIP/hmeDIP is often at low or sub-nanogram levels which causes insufficient DNA library yields. To address this issue, Epigentek offers the EpiNext™ High-Sensitivity DNA Library Preparation Kit.
Principle & Procedure
This kit includes all reagents required at each step of the workflow to carry out a successful DNA library preparation. In the library preparation, DNA is first fragmented to an appropriate size (about 300 bps in peak size). The end repair/dA tailing (end polishing) of the DNA fragments are performed simultaneously. Adaptors are then ligated to both ends of the polished DNA fragments for amplification and sequencing. Ligated fragments are size selected and purified with MQ beads, which allows for quick and precise size selection of DNA. Size-selected DNA fragments are amplified with a high-fidelity PCR Mix that ensures maximum yields from minimum amounts of starting material and provides highly accurate amplification of library DNA with low error rates and minimum bias.
Starting materials can include fragmented dsDNA isolated from various tissue or cell samples, dsDNA enriched from a ChIP reaction, MeDIP/hMeDIP reaction, or exon capture. DNA should be relatively free of RNA because large fractions of RNA will impair end repair and dA-tailing, resulting in reduced ligation capabilities. The input amount of DNA can be from 0.2 ng to 100 ng. For optimal preparation, the input amount should be 10 ng to 50 ng.
Fig. 1. Workflow of the EpiNext™ High-Sensitivity DNA Library Preparation Kit.
Fig. 2. Size distribution of library fragments. Human placenta DNA was sheared to around 300 bps in peak size and 0.2 ng of DNA was used for DNA library preparation using EpiNext™ High-Sensitivity DNA Library Preparation Kit.