The Foundation for Your Next-Generation Sequencing (NGS)

Over the last decade, Next-Generation Sequencing (NGS) technologies have enhanced various fields of biology and medicine including genomics, personalized medicine, and evolutionary biology. NGS allows for the comprehensive study of entire genomes (DNA) or transcriptomes (RNA), the high-throughput identification of sequence variations, and single cell analysis, providing novel insights into genome structure, function, and variability.

The preparation of sequencing libraries forms the basis of a successful NGS application, involving the generation of modified nucleotide fragments from DNA or RNA samples in a format suitable for sequencing. The entire sequencing process and subsequent bioinformatic analysis are influenced by the quality of this library preparation. Through careful library preparation, the sequencing output can be optimized, technical biases or artifacts inherent in the sequencing process can be minimized, and sequencing coverage can be enhanced, especially for precious sample materials.

Preparation of NGS Sequencing Libraries - Step by Step

• Fragmentation: In addition to sonification or acoustic shearing, there are also more specific and gentler methods for the sample, such as enzymatic fragmentation.
• Reverse Transcription: Reverse transcription is an enzymatic process carried out in a thermal cycler. This process is crucial for converting RNA into complementary DNA (cDNA), which can then be used for sequencing. Regardless of the sequencing technology used, library preparation often involves several steps.
• End Repair: This process involves preparing smooth ends of DNA fragments to be compatible with the ligation of sequencing adapters.
• Adapter Ligation: Sequencing adapters are attached to the ends of the DNA fragments for complementary binding to the flow cell of the sequencing platform.
• Indexing/Barcoding: Unique short sequence indexes are added to each library in order to distinguish between all samples after sequencing for bioinformatic analysis. Those indexes can also be used for multiplex sequencing to fit more samples in one sequencing run by multiple combinations of different index sequences.
• PCR Amplification: Enrichment of libraries by PCR is used to amplify the sequencing libraries to a level suitable for sequencing.?
• Fragment Size Selection/Clean Up: The suitable DNA fragment sizes are selected and purified ensuring high-quality libraries.
• Quality Control: The prepared libraries are assessed for concentration and size distribution.
• Normalization & Pooling: Library concentrations are normalized and pooled to enable simultaneous sequencing of multiple samples.

Nucleic Acid Extraction as the First Step

Extraction represents the initial step wherein DNA or RNA samples are released from their biological matrices, thereby facilitating subsequent downstream processing. This crucial step not only impacts the yield and purity of extracted nucleic acids but also exerts a significant influence on fragment sizes, defining NGS applications and shaping the quality of sequencing libraries. The purity of these samples is critical for the reliability of sequencing results, underscoring the importance of investing in optimal laboratory equipment and methods.

The efficiency of nucleic acid extraction determines the yield of DNA or RNA obtained from biological samples. High yield ensures an ample supply of genetic material for downstream applications, while purity reflects the absence of contaminants that could skew sequencing results. Stringent extraction protocols are employed to maximize yield and purity, ensuring the integrity of genetic material for accurate sequencing. In addition, the choice between short-read and long-read sequencing methods demonstrates the importance of tailoring library preparation protocols for specific applications. During extraction, unintended fragmentation of DNA or RNA can occur, which affects the distribution of fragment sizes in the sequencing libraries.

Nucleic Acid Extraction Options

The solubility of nucleic acids influences their extraction efficiency, particularly in aqueous or organic phases during phase separation. Furthermore, nucleic acids can bind to various materials during extraction, such as solid-phase matrices or beads. Bead-based extraction methods have gained popularity due to their automation capabilities and compatibility with high-throughput workflows. These methods offer reliability, reproducibility, and scalability, making them indispensable tools in modern NGS library preparation.

By using an automated liquid handling system such as the CyBio FeliX, various protocols, including bead-based extraction and tip extraction, can be seamlessly integrated in NGS library preparation workflows. Automating repetitive pipetting tasks, CyBio FeliX minimizes human error and maximizes throughput, ensuring consistent extraction results across all samples accelerating the pace and quality of NGS library preparation.

Impact of the Sequencing Method on the Library Preparation

The chosen sequencing technology determines the library preparation process for either short-read sequencing of typically between 50 to 300 bp fragments or long-read sequencing for whole DNA molecules up to Mbp fragments.?

Fragment size plays a crucial role in NGS library preparation, as it determines the suitability of nucleic acids for sequencing.

By controlling factors such as fragmentation method and incubation time, extraction protocols can be adjusted to achieve the desired fragment size in order to optimize sequencing results.

In addition, the library preparation workflow and the necessary steps depend on the area of application and the type of nucleic acid (DNA or RNA). Targeted sequencing focuses on specific areas of the genome, thus limiting the scope of data analysis and at the same time reducing time and costs. This enables sequencing with a much higher level of coverage as the focus is on smaller and specific regions of the genome. Therefore, targeted sequencing requires the amplification of specific gene fragments or panels of genes from a genome. In contrast, Whole Genome Sequencing (WGS) provides a comprehensive view of a whole genome without potential bias from PCR amplicons and is suitable for a wider range of applications.

Important thermal cycler parameters ensuring optimal library preparation

The quality of the sequencing results depends directly on the quality of the library preparation. All steps of the library preparation require precise temperature accuracy for which PCR thermal cyclers are the perfect platform. Suitable thermal cyclers must fulfil several key parameters:

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