From comprehensive genome analysis to disease gene identification, next generation sequencing (NGS) has emerged as an indispensable tool, propelling scientific research. The ongoing advancements in NGS library preparation workflows enhance sequencing accuracy, efficiency, and throughput, empowering scientists to uncover novel insights into disease mechanisms and advance precision medicine.
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Choosing the Right NGS Sample Preparation Partner
Support from expert industry partners enables the creation of more robust, versatile NGS workflows.
Next generation sequencing (NGS) is a complex, multistep process that requires unique expertise and resources to ensure sequencing success. Many clinical and diagnostic laboratories and research groups seek support from experienced NGS service providers. To achieve flexible, versatile, and scalable NGS sample preparation workflows, there are key questions and capabilities to ask and look for when choosing an NGS partner.
Does the partner have the expertise to develop adaptable workflows?
From sample collection to library construction and target enrichment, selecting the proper methods and optimizing each experimental protocol is crucial. Partnering with an NGS expert that has a deep understanding of different sequencing technologies can greatly reduce laborious trial and error. A good NGS partner helps develop highly robust, reliable workflows depending on specific sequencing needs, but also offers solutions that facilitate experimental scale-up for high-throughput production. NGS partners should be experts at utilizing NGS resources, including labor, consumables, reagents, and data, within budget to help research groups maximize sequencing cost-effectiveness.
Can the partner customize reagents for different experimental workflows?
Reagent functionality is the backbone of an NGS experiment but acquiring reagents from vendors does not always guarantee workflow compatibility. Relying on NGS experts to provide customized reagents for all sequencing steps offers ease and efficiency. An ideal NGS partner leads advanced enzyme engineering and buffer formulating technologies and specializes in developing high-performing enzymes and buffers tailored to each reaction. Additionally, extensive reagent customization capabilities that encompass specified fill volumes, kit configuration, labeling, and packaging can better meet various research and business needs from discovery to manufacturing.
Can the partner offer resourceful automation solutions?
Automating library preparation, target enrichment, and quantification steps increases operational efficiency and generates more consistent results. However, adopting automated systems usually requires highly technical expertise, including programming, software configuration, and computational calibration. The increasing number of automation products in the market further complicates this task. NGS partners that are knowledgeable in diverse automation platforms and experienced in creating and optimizing automation methods can help sequencing facilities make appropriate platform selections and accelerate automated workflow development. By streamlining the entire NGS sample preparation workflow, researchers can unlock more resources and gain time for innovations.
Does the partner provide robust quality assessment?
NGS requires accurate and reliable quality assessment for samples, reagents, and libraries. As an NGS service provider, having robust sample quantification methods can help researchers verify the integrity of nucleic acids, predict the yield and quality of libraries, and reduce repeated sample processing time and costs. Before sequencing, NGS users need to quantify prepared libraries to determine the amount of sequence-ready DNA. NGS partners that have sensitive and efficient quantitation methods for a wide range of library types, concentrations, fragment lengths, and GC content can allow researchers to accurately predict the probability of sequencing success and data quality. Additionally, producing reagents under the same quality standards improves experimental reproducibility.
What makes Roche a good NGS partner?
The Roche team has a long history of investing in NGS research since 2007. Their fully integrated NGS sample preparation workflow provides solutions across the entire NGS process. Experts at Roche develop suitable protocols, high-quality custom reagents, and effective automation methods. From whole genome to targeted sequencing and from single experiment to large scale manufacturing, Roche’s sequencing solutions provide extensive support to advance genomic research and personalized healthcare.
Maximizing the Value of NGS Data
Understanding sequencing metrics and how to improve them is key to reducing NGS costs.
The applications of NGS in numerous scientific fields allow researchers to investigate genomic variations, elucidate disease mechanisms, and develop novel diagnostics and therapies. However, NGS is expensive, particularly when sequencing large amounts of data or performing exploratory experiments. Making the most use of the sequencing data by choosing the most appropriate sequencing approach is vital to ensuring accurate downstream analysis while keeping NGS costs down.
Key quality control metrics
Understanding the process of NGS data generation and analyzing key sequencing metrics is essential to comprehensively measuring NGS data quality. After each sequencing run, researchers utilize sequence analysis tools to evaluate raw reads of nucleotide bases with several primary quality control metrics. These metrics include yield and error rate, which represent the number of bases generated and the percentage of erroneous base calls produced during the sequencing run (1). Using bioinformatic software, researchers then align their NGS reads to a reference sequence to map each nucleotide base to its corresponding location in the genome.
During sequence alignment, researchers examine the number of unique reads that map to a particular nucleotide base. This is known as coverage depth. This metric helps researchers to determine how confident they can be that the sequencing run captured the correct base for that position in the genome. Breadth of coverage refers to the percentage of target bases sequenced at a given depth.
This metric indicates what percentage of the genome or target is covered by sequencing data. Duplicate rate represents the number of reads that map to the same region of the reference genome. High duplicate rates reflect over sequencing and amplification bias during upstream steps.
Assessing unique NGS data needs
Additional quality metrics help researchers evaluate different types of NGS experiments, such as targeted NGS, a widely used method to sequence specific regions of the genome. Researchers capture genomic regions of interest using target-specific probes. During this process, sequence reads may distribute unevenly across target regions, resulting in varying genome coverage. To measure this, scientists commonly use a score, termed Fold-80 base penalty, which reflects how much additional sequencing is required to ensure that 80% of target bases achieve the desired average coverage. The Fold-80 base penalty score informs researchers about the capture efficiency of the probes, helping them choose well-designed probes to reduce additional costly sequencing runs.
A combination of factors, including experimental designs, library preparation methods, PCR protocols, instruments, and reagents affect NGS data quality. In one study, researchers from the University of Verona, Italy, explored the effect of using different DNA fragment lengths for target enrichment during exome sequencing (2). By calculating sequencing metrics such as duplicate rates and Fold-80 scores for various DNA fragment lengths, they determined that longer DNA fragments achieved a higher depth and uniformity of coverage. This allowed the researchers to identify increased numbers of clinically relevant variants. Likewise, a team of scientists from Fourth Military Medical University, China, discovered that different primer design methods can impact the uniformity and coverage depth of mitochondrial genome sequencing (3). By analyzing these metrics, they optimized a DNA enrichment strategy and improved the sequencing accuracy of mitochondrial DNA mutations.
Refining metrics to maximize NGS data value
The key to achieving maximum value with minimum sequencing is defining the necessary coverage depth and the number of required reads to answer different experimental questions. Prior to performing an NGS experiment, researchers can create a panel of test data sets with various quality metrics to check data parameters. This approach optimizes NGS metrics and helps scientists identify the minimum read count required for different experiments. Knowing the desired data quality further allows NGS users to compare experimental costs using different library preparation platforms, protocols, sequencers, and reagents, helping them to eliminate unnecessary expenses and make the most cost-effective decision.
References
1. Manley, L. J., Ma, D. & Levine, S. S. Monitoring Error Rates In Illumina Sequencing. J Biomol Tech 27, 125–128 (2016).
2. Iadarola, B. et al. Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length. Sci Rep 10, 9424 (2020).
3. Liu, Y. et al. Optimized PCR-Based Enrichment Improves Coverage Uniformity and Mutation Detection in Mitochondrial DNA Next-Generation Sequencing. The Journal of Molecular Diagnostics 22, 503–512 (2020).
Next Generation Sequencing (NGS) Reagent Customization: Ready to Sequence
One size does not fit all
To prepare NGS libraries, researchers often acquire reagents from different vendors, requiring them to spend extra time and cost validating reagent compatibility. A one-stop source for customizable NGS solutions enables researchers to seamlessly integrate library preparation reagents into unique workflows to improve sequencing success.
Novel enzymes and buffers
DNA and RNA modifying enzymes are the backbone of a robust NGS workflow. Using a directed evolution approach, experts at Roche screen enzyme variants and select the highest performing enzymes. They also optimize each buffer formulation to maximize reaction efficiency.
Specified bottles and volumes
Different NGS workflows and throughput scales require specific reagent volumes. To minimize reagent waste and save costs, researchers can customize bottle sizes and fill volumes for different projects.
Unique packages and labels
Packaging versatility ensures the safe transport, storage, and handling of unique reagents. Custom labels can also provide scientists with valuable instructions, help them identify hazards, and facilitate product distribution. Experts at Roche design packages and labels to customers’ specifications.
Building high throughput workflows
Experts at Roche collaborate with customers to produce high quality reagents for all the steps required to convert a sample to a sequencing-ready library. Scientists can readily streamline, automate, and scale up their workflows to optimize sequencing results.
Case Study: Improving the Understanding of Tumor Genomics and Behavior
The collaborative development of a single-tube total nucleic acid NGS sample prep workflow leads to the translation of sequencing data into clinical cancer research advancements.
In recent years, targeted NGS has become an instrumental tool in advancing clinical oncology, cancer diagnostics, and cancer genomics research. NGS extends the limits of tumor tissue characterization beyond morphological studies, allowing clinicians to detect cancer mutations and characterize gene expression changes across different cancer types and throughout disease progression.
Pathologists routinely formalin fix and paraffin embed (FFPE) tissues from patients for morphological or histological characterization. In a recent case study, a team of clinical oncologists sought to extract valuable nucleic acid data from FFPE samples. They hoped to detect cancer associated genes using NGS. To do this, they aimed to build a targeted NGS process to sequence FFPE tissue samples.
Converting FFPE tissue samples to sequencing-ready libraries, however, is challenging. Formalin fixation causes DNA fragmentation and adds chemical modifications to nucleic acids. This results in insufficient DNA yields and downstream sequence artifacts. To collect more genetic materials, the clinician team needed to develop sample preparation protocols for both DNA and RNA from FFPE tissues. Meanwhile, they also needed a specified targeted gene panel to detect a wide range of clinically relevant variants across the genome.
To overcome these obstacles, the clinical team partnered with industry experts at Roche. Formed with highly trained and experienced engineers and specialists, the Roche Support Network and Expert Design Team performed end-to-end support to develop a combined and automated workflow for both DNA and RNA extraction and NGS library preparation from FFPE samples. The NGS experts designed the workflow based on chemistry principles that allowed all sample preparation reaction steps in a single tube. This minimized sample loss and increased nucleic acid yield from FFPE tissue samples, ensuring sufficient input material for downstream sequencing.
After setting up the sample preparation framework, experts from Roche’s Custom Solutions team provided reagent optimization support. Since DNA and RNA from FFPE tissue samples are often partially damaged, it is important to preserve genome integrity with high sensitivity and fidelity enzymes during library preparation. Experts at Roche used directed evolution technology, a method that mimics the process of natural selection to select the highest-performing DNA Polymerases through iterative rounds of mutagenesis. They optimized enzyme and buffer formulation to maximize reaction efficiency. The customized reagents helped the clinical team to achieve high fidelity amplification and improved sequencing data quality.
The clinical team also looked into how to concurrently detect various cancer susceptibility genes in different target regions, including RNA targets, exome regions, and other custom targets. To comprehensively and efficiently enrich these regions, experts at Roche’s Expert Design Team designed a unique probe panel and a streamlined hybrid capture target enrichment workflow. By focusing sequencing reads on the genomic regions of greatest interest, the panel provided a sensitive, reproducible, and economical solution for studying gene expression dynamics and the underlying mechanisms in cancer.
In addition to protocol and reagent development, the team of Roche experts continued to help the clinical team throughout the NGS workflow from library preparation to target enrichment. Together, they periodically reviewed the sequencing results and further optimized the workflow to improve sequencing performance. The clinical team has since streamlined their NGS workflow, allowing them to tackle challenging FFPE tissue samples and decipher genetic changes to drive cancer research forward.
Reference
Do, H. & Dobrovic, A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem 61, 64–71 (2015).