Preparation / Purification of NGS Library

Next-generation sequencing requires careful preparation of the genetic material before/upstream of the sequencing reaction to ensure reliable results.

High throughput sequencing is performed on fragments of homogeneous size presenting at their extremities short sequences, called labels or tags, allowing to define the origin of the fragments when sequencing several samples simultaneously, the fixation on a solid support and the initiation of the sequencing reaction by incorporating marked dNTPs.

To generate these fragments which constitute what is called a library, there are different approaches such as:

The preparation of libraries by fragmentation: the genetic material is first cut into fragments of different sizes. Fragmentation can be mechanical, chemical or enzymatic. A selection of fragments is then made according to their size. The last step consists to add labels at the ends of the selected fragments.

Step to prepare libraries by fragmentation

The preparation of libraries by amplification: regions of interest are initially amplified by PCR with specific primers. Labels are added to the ends of the amplicons either by ligation or by PCR.

Steps to prepare libraries by amplification

The approach used and the order of the steps (selection of fragments and addition of labels) may vary according to the recommendations of the library preparation kit suppliers.

For the purification of libraries and the selection of fragments according to their size, BIOMNIGENE is equipped with PippinHT (Sage Science). This device allows DNA fragments from 24 different libraries to be selected simultaneously with an uncertainty of 5% of the expected size. For example, for a given size of 500 base pairs, the length of the preserved/conserved/isolated fragments will range from 475 to 525 bp.

This method of library purification allows us to homogenize all libraries running on our Illumina high throughput sequencing devices.

Whatever your project, Biomnigene offers to prepare your libraries.

PippinHT used for librairies purification

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