Next-generation Sequencing (NGS) instrument requires preparation of your sample library into a suitable format for sequencing platform of interest. Celemics offers two different NGS library preparation kits tailored for sequencing platforms such as Illumina, MGI, and Ion Torrent.
In this post, we will introduce Celemics’ two library preparation kits: LP and EP.
- What is NGS Library Preparation?
NGS library preparation is a crucial process that converts the DNA or RNA from your sample into a format that can be read by sequencing instruments. This process involves isolating and purifying the genetic material from the sample, fragmenting it into suitable sizes for sequencing, and attaching short DNA sequences called adapters to both ends. The prepared library can then be read by the sequencing instrument, allowing researchers to perform genomic analysis.
Celemics provides both LP and EP kits for each of the three sequencing platforms mentioned above. While there are slight differences depending on the platform, the principle and process are generally consistent with those for LP kits and those for EP kits.
In this post, we will take a closer look at each method using the Illumina platform as a reference.
- Library Preparation (LP)
The LP method is the most commonly used library preparation method. This method requires a DNA fragmentation to make a suitable size for sequencing.
There are two main ways to cut DNA:- Mechanical fragmentation (Sonication)
- Enzymatic fragmentation
The LP kit processes pre-fragmented DNA through a series of enzymatic treatments to create a library suitable for sequencing platforms.
The steps involved are as follows:- End Repair/A-tailing (ER/A): During the fragmentation process (especially sonication-based shearing), sticky ends occur on both ends of DNA fragments. Enzymes convert those sticky ends to blunt ends and an A-bases are added to the 3′ end. Similar to many competitors’ kits, Celemics’ LP kit provides an ER/A enzyme mix that combines the End Repair and A-tailing steps into one.
- Adapter Ligation: Adapters suitable for the specific sequencing platform are attached to the DNA.
- Index Attachment PCR (Pre-PCR): Sequencing index are attached and amplified. If the index is already attached during the adapter ligation step, this PCR step serves to amplify the DNA library.
- Enzymatic Library Preparation (EP)
Celemics’ EP kit differs from the LP method by integrating DNA fragmentation into the ER/A step. This eliminates the need for a separate DNA fragmentation step, making the EP kit more labor-friendly. The EP kit also processes the DNA into a library format through a series of enzymatic treatments.
The steps involved are as follows:- Fragmentation/End Repair/A-tailing (FER/A): Large sample DNA is fragmented, the ends are converted to blunt ends, and an A-bases are added to the 3′ end.
- Adapter Ligation: Adapters suitable for the specific sequencing platform are attached to the DNA fragments.
- Index Attachment PCR (Pre-PCR): Sequencing index are attached and amplified. As with the LP kit, if the index is already attached during the adapter ligation step, this PCR step amplifies the DNA library.
Celemics offers both LP and EP Library Prep kits tailored for Illumina, MGI, and Thermo Fisher (Ion Torrent) platforms. To find out more or to request for more information in regards to Celemics Library Prep kits and how we optimize the libraries for each platform, please contact us.