Selecting the Best Tools for NGS Sample Prep

Nucleic Acid Extraction as the First Step

Extraction is the foundational step in which nucleic acids are liberated from their biological matrices, resulting in DNA/RNA samples devoid of proteins, inhibitory enzymes, and other contaminants. Using optimized nucleic acid extraction methods is important for achieving maximal yield as well as preserving the integrity of genetic material for accurate sequencing. The efficiency of nucleic acid extraction directly impacts the quantity of DNA or RNA obtained from biological samples; the minimum amount of starting material required varies with library preparation kit and target and may be a critical factor in success of NGS library preparation and sequencing.

Nucleic acid starting material must also be of sufficient purity (absence of contaminants) to reduce risk of failed reactions during library preparation. Purity is vital for successful downstream processing and reliable NGS data, highlighting the necessity of investing in optimal laboratory equipment and methodologies.
 

Impact of Sequencing Method and Goal on NGS Library Preparation

The chosen sequencing technology determines the library preparation process: short-read sequencing typically requires fragments between 50 to 300 bp in length, whereas long-read sequencing fragments may range from 8 to >20 kbp.  By controlling factors such as fragmentation method and incubation time, extraction protocols can be adjusted to achieve the desired fragment size to optimize NGS results.

In addition, the library preparation workflow depends on the area of application and the type of nucleic acid (DNA or RNA). Targeted NGS 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. Targeted sequencing generally involves 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. PCR-free methods have been shown to produce high-quality samples, but amplification is necessary for single-cell seq and other samples with extremely low amounts of nucleic acid.

Biometra thermal cyclers are perfectly adapted to the requirements of NGS library preparation. Offering high flexibility with various models and block models, the Biometra thermal cyclers are suitable for different sample throughputs and workflow needs. They are compatible with almost all commercially available PCR plastic consumables. Notably, the Biometra TAdvanced thermal cycler is the only PCR cycler on the market that is verified and recommended by 10x Genomics as compatible with all of their innovative methods at the single-cell level and in spatially resolved samples, making the Biometra TAdvanced a future-proof investment. By addressing these key aspects, the Biometra thermal cycler ensures reproducibility, minimizes bias, and optimizes yield, thereby enhancing the efficiency and reliability of NGS library preparation.

Sample Normalization and Pooling

Normalization is a critical step in NGS library preparation that ensures every library is evenly represented and sequenced at sufficient depth. It equalizes concentrations for multiplexing, avoiding over- or underrepresentation of samples. Overrepresentation wastes capacity, while underrepresentation results in poor read depth and loss of data, potentially wasting precious samples. Therefore, normalization of library concentrations helps to generate consistent and reliable NGS data in a cost-efficient manner.

To save resources, multiple libraries can be pooled and sequenced in the same run. Combining sequencing libraries from several samples into one run ensures the optimal use of the flow cell, resulting in a more economical process.