The Foundation for Your Next-Generation Sequencing (NGS)
Tue 10 Sep, 2024
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.
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Sample Preparation for NGS
Our eBook “Sample Preparation for Next-Generation Sequencing” offers selected applications of our products regarding the manual library construction and the partial automation of library preparation. Get all the necessary information for your sequencing challenges.
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:
- Precise Temperature Control Systems: Precise temperature control is essential to minimize PCR bias and the risk of non-specific amplifications.
- Fine-Tuning Reaction Conditions Temperature overshoots and undershoots to reach the target temperature should be avoided, as they can also lead to undesirable amplifications via undefined temperatures. This ensures optimal yield by maintaining enzyme activity.
- High-Performance Lid Pressure: Regardless of the height of the plastic ware used, the thermal cycler should be able to exert a reproducible lid contact pressure. This minimizes the risk of sample evaporation, ensures consistent reaction conditions and prevents cross-contamination.
- Temperature Uniformity: Temperature homogeneity across the entire sample block is essential in order to apply the same conditions in all sample positions and thus generate reliable sequencing libraries.
- Compatibility: The thermal cycler should be compatible with all common PCR consumables, including special adaptors, which are used in some cases in single-cell sequencing.
- Flexibility: Interchangeability of sample blocks is advantageous in order to be able to change to a different thermal cycler block format easily and cost-effectively, even with changing sample numbers and volumes.
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.
"Essential for our HLA diagnostics"
“The Biometra TAdvanced Thermocycler, in combination with a kit from GenDx, is essential for our HLA diagnostics. The exceptional precision of the TAdvanced is excellent for our sensitive applications in stem cell and organ donation. This solution provides us with the robustness, reproducibility, and durability we need.”
Dr. Volker Oberle, EFI Director Transplantation Immunology, Jena University Hospital, Germany
Fragment Size Selection and Clean Up
Size selection and clean-up are critical steps to eliminate suboptimal nucleic acid fragments. The optimal fragment size for sequencing devices and precise bioinformatic analysis varies depending on the chosen instrument. For instance, instruments, such as Illumina™ systems, typically work best with fragments between 200 – 500 base pairs (bp). Sequencing efficiency can be affected by the fragment size. Fragments below 150 bp can lead to adaptor and primer dimers which have a higher preference to cluster on a flow cell than larger fragments, resulting in biased data from short fragments. Size selection and clean-up is also important from a cost perspective. Efficient processes can help to reduce the overall cost of sequencing.
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.
Ensuring Reproducibility and Optimization
Maintaining consistency in NGS library preparation is crucial for reliable sequencing results. Library preparation poses significant challenges, from labor-intensive manual pipetting to ensuring consistency and scalability. Manual pipetting not only consumes valuable time but also introduces the risk of errors.
The CyBio FeliX pipetting robot eliminates variability by automating the workflow, ensuring reproducible results and enhancing reliability and efficiency. CyBio FeliX offers unparalleled advantages in tasks such as size selection and library normalization. In manual handling, achieving uniformity in these parameters across many samples can be challenging, leading to size selection biases and skewed results. With CyBio FeliX, you can eliminate the need for repetitive manual pipetting tasks, allowing for "walk-away time" – enabling focusing on high-value analyses and tasks that require your expertise.
Workflow automation with the flexible CyBio FeliX reduces manual work in pipetting-intensive steps in a compact footprint. The ratio of footprint to possible applications, beginning from nucleic acid extraction to library pooling, is optimal. With varying sample numbers from day to day, some days manual processing may not be manageable. On other days, there may be just a small number of samples. CyBio FeliX offers maximum flexibility - 1 to 96, up to 384 samples can be processed.
As sample volumes increase, scalability becomes essential. FeliX's modular design allows seamless scalability, accommodating growing demands without sacrificing efficiency or quality, future-proofing NGS workflows.
"CyBio FeliX has significantly contributed to optimizing our workflow"
“FeliX automates the size selection of DNA fragments. This automation not only increases efficiency in handling rising sample numbers but also improves the quality of size selection. We are thrilled with this performance.”
Eugen Bauer, Department of Transfusion Medicine, University of Cologne
"CyBio FeliX boosted our consistency and saved us time"
“The CyBio FeliX is a great tool for us to standardize protocols for bead purification and multiplexing libraries into one sequencing pool. It is helping us to increase the consistency and to optimize our time by making walk-away possible.”
Julie Bitz-Thorsen, Geo Genetics Centre, Lundbeck Foundation, University of Copenhagen
Important Quality Control Assessments
In NGS library preparation, quality control plays a pivotal role to ensure the success of downstream sequencing processes. By leveraging qPCR for accurate quantification and gel electrophoresis for fragment size validation, you can confidently validate the quality and quantity of DNA fragments and optimize NGS workflows for reliable and reproducible results.
qPCR as the Gold Standard for Library Quantification
qPCR is widely recognized as the gold standard for quantifying DNA fragments in NGS library preparation. It’s not only endorsed by NGS kit reagent providers but also acknowledged as the method of choice by researchers worldwide. Determining the library concentrations by qPCR benefits from precise quantification for the optimal normalization factors between multiple samples, leading to optimal results achieved through optimal flow cell loading. Furthermore, qPCR is a cost-efficient technology, which minimizes the need for excess sample material and prevents unnecessary experiment repetitions, resulting in reduced costs and optimal utilization of resources.
Gel Electrophoresis for Fragment Size Validation
Gel electrophoresis serves as an effective method for checking DNA fragment sizes. Analytik Jena offers solutions for every throughput: Choose gel electrophoresis systems of the Compact Line in sizes S, M, Multi-Wide, and L according to your needs. Gel electrophoresis is a cost-efficient method that enables you to use your resources economically.
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Automated Purification of Plasmid DNA using Promega Wizard MagneSil Tfx™ System on the Analytik Jena CyBio FeliX Liquid Handler (EN)
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