NGS (next-generation sequencing) library preparation is converting DNA samples into a form that can be sequenced on an NGS platform. This process involves several steps, including fragmentation, adapter ligation, amplification, and purification.
The resulting libraries can be used for various applications, including genome sequencing, gene expression analysis, and DNA methylation analysis. This blog will discuss the multiple steps involved in NGS library Prep and the importance of this process in modern genomics research.
How Does NGS Library Preparation Work?
The first step in NGS library Prep is fragmentation, which involves breaking the DNA sample into smaller pieces. Several methods for fragmenting DNA include mechanical shearing, sonication, and enzymatic digestion.
The choice of fragmentation method depends on the specific NGS platform and the size of the DNA fragments desired. For example, shorter DNA fragments are often preferred for RNA-seq, while longer pieces may be more suitable for whole genome sequencing.
After fragmentation, the DNA fragments are usually ligated to adapters. Adapters are short, synthetic oligonucleotides designed to bind specifically to the ends of the DNA fragments. These adapters serve several purposes.
First, they allow the DNA fragments to be captured and amplified during the next step of library preparation. Second, they provide the necessary sequences for the NGS platform to recognize and bind the DNA fragments during sequencing. Finally, they may also contain additional information, such as barcodes, which can identify the samples or distinguish between different libraries in a multiplexed sequencing experiment.
Once the DNA fragments have been ligated to adapters, the resulting libraries are usually amplified through PCR (polymerase chain reaction). It allows many copies of the libraries to be produced, which is necessary for the low-input nature of NGS platforms.
During PCR, the DNA fragments are amplified using primers that bind to the adapter sequences, allowing the polymerase enzyme to synthesize new DNA strands complementary to the templates.
After amplification, the libraries are usually purified to remove any excess primers, nucleotides, or enzymes that may be present. That is typically done using a purification kit or column, eliminating contaminants and leaving the purified libraries behind.
The purified libraries are then quantified using various methods, such as qPCR (quantitative PCR) or fluorimetry, to determine the concentration and size distribution of the DNA fragments.
NGS library preparation is a crucial step in modern genomics research, as it allows researchers to sequence large amounts of DNA in a relatively short amount of time.
The ability to rapidly and accurately sequence DNA has revolutionized our understanding of genetics and has led to numerous scientific and medical advances. It has also opened up new avenues of research, such as personalized medicine and gene editing, which have the potential to transform the way we treat and prevent diseases.
NGS library Prep is a complex and multi-step process that requires careful planning and execution to ensure high-quality data.
While the process has been streamlined and automated in recent years, it is still essential for researchers to understand the various steps involved and the factors that can impact library quality.
By following best practices and using high-quality reagents, researchers can maximize the accuracy and reproducibility of their NGS experiments, leading to more reliable and meaningful results.