Next-generation sequencing (NGS) is a massively parallel sequencing technology that provides various applications, including thorough analysis of entire genomes or select target sections.1
Targeted sequencing focuses time, cost, and data analysis on specific areas of interest in a genome, allowing for significantly higher levels of coverage.
Targeted sequencing is the enrichment of a gene fragment or a group of genes from a genome. Polymerase chain reaction (PCR) rapidly and cost-effectively performs the enrichment required for targeted sequencing.
Amplicon sequencing is a low-cost, robust, well-established NGS technique that provides high throughput capacity in numerous applications, including oncology, evolutionary biology, and microbiome research.
In all NGS applications, the quality of DNA sequencing libraries significantly impacts the preparation processes and, consequently, the result.2 It is, therefore, critical to undertake DNA library preparation with maximum precision and reproducibility.
DNA library preparation for amplicon sequencing consists of several processes, including enzymatic incubations, adapter ligation, barcoding using unique index sequences during library enrichment, and quality control measurements using thermal cyclers.3
The thermal cycler’s properties are crucial for producing high-quality libraries. The precise temperature control, efficient heating and cooling rates, and great temperature homogeneity over the entire block result in the repeatable synthesis of preparation products and amplicons in all sample wells, free of non-specific side reactions and PCR bias.4
The Biometra TAdvanced thermal cycler (Analytik Jena GmbH+Co. KG) is popular among scientists for NGS library preparation because it meets all necessary standards.
This article presents the preparation of DNA amplicon sequencing libraries for NGS using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs GmbH) on the Biometra TAdvanced 96 SG. It also describes library quantification using the real-time thermal cycler qTOWERiris (Analytik Jena GmbH+Co. KG).
Figure 1. Workflow procedure for amplicon sequencing library preparation and quality control. Image Credit: Analytik Jena US
Materials and methods
Samples and reagents
- Promega Cooperation’s Human Genomic DNA (cat: G3041)
- Commercial PCR master mix
- Commercial PCR purification kit
- New England Biolabs Inc.’s NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® with sample purification beads (cat: #E7103).
- NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) - New England Biolabs Inc., cat. #E7335
- NEBNext® Library Quant Kit for Illumina® - New England Biolabs Inc., cat. #E7630.
Sample preparation
Four genes from human genomic DNA (Promega Cooperation) were targeted via PCR (Table 1) to prepare the library. A commercial master mix was used in a 50 µL volume, with six replicate reactions for each gene. The PCR was then cleaned up using a commercial PCR purification kit.
Agarose gel electrophoresis was used to assess product integrity and fragment size using a Biometra Compact M (Figure 2A). The DNA concentrations of PCR amplicons were measured using the ScanDrop2 microvolume spectrophotometer (Analytik Jena GmbH+Co KG).
Table 1. Four gene targets for amplicon library preparation were chosen derived from human genomic DNA (Promega Cooperation, G3041) representing varying amplicon lengths and GC content. Source: Analytik Jena US
| Gene targets |
Amplicon length (bp) |
GC content (%) |
Concentration (ng/μL) |
| ACTB |
250 |
61.2 |
11.09 |
| MYC |
480 |
36.0 |
16.84 |
| B2M short |
203 |
41.4 |
16.37 |
| B2M long |
473 |
43.3 |
18.95 |
Instrumentation
- Biometra TAdvanced 96 SG thermal cycler (Analytik Jena GmbH+Co. KG), part number 846-2-070-241.
- Biometra Compact Line gel electrophoresis system (Analytik Jena GmbH+Co. KG, cat: 846-025-xxx*).
- ScanDrop2 microvolume spectrophotometer (Analytik Jena GmbH+Co. KG), model 844-00203-x*.
- qTOWERiris real-time PCR cycler (Analytik Jena GmbH+Co. KG), model 844-0085x-x*.
- Standard vortex mixer
- Benchtop centrifuge for 2 mL tubes
- Centrifuge for PCR plates
Library preparation workflow
The NEB Next Ultra™ II DNA Library Prep Kit for Illumina® was used to prepare a library from purified PCR products ranging from 300 ng to 1 µg. All procedures were followed from the handbook.
Samples were spread across the block of the Biometra TAdvanced thermal cycler (Figure 2B) to assess the consistency of sample temperature control for library preparation processes. The NEBNext® adaptors were ligated without dilution, followed by a preliminary library clean-up without size selection.
The libraries were then analyzed using gel electrophoresis (Figure 2C). Adaptor-ligated DNA libraries were barcoded with the Index Primers Set1 from the NEBNext® Multiplex Oligos for Illumina® and enriched over four PCR cycles. Gel electrophoresis was used to assess the final library integrity (Figure 2D).
Figure 2. Validation of amplicon integrity by agarose gel electrophoresis. A) PCR purified amplicons before library preparation. B) Position of reaction tubes in the Biometra TAdvanced 96 SG thermal cycler block during the steps of library preparation. Triplicate samples of each gene were distributed at the corner and center of the thermal block. C) Adaptor-ligated amplicon libraries after first clean-up. D) Final amplicon library after barcoding, enrichment and second clean-up. Image Credit: Analytik Jena US
Library quantification
The resulting libraries were quantified using the real-time thermal cycler qTOWERiris using the sample configuration (Figure 3) and PCR software (Figure 4), as specified in the NEBNext® Library Quant Kit for Illumina® manual.
Sample libraries were diluted at 1:100,000 and evaluated in triplicate reactions with the color channel SYBR Green/FAM.
Figure 3. Plate layout for the quantification of amplicon libraries using the NEBNext® Library Quant Kit for Illumina® in a 20 µL qPCR reaction volume. The quantification assay consisted of six provided standards, the 24 gene libraries and no-template controls (NTC) in triplicate reactions. Image Credit: Analytik Jena US
Figure 4. PCR program for the library quantification, screenshot taken from the qPCRsoft 5.0.2.0 software. Image Credit: Analytik Jena US
Results and discussion
The sample libraries were processed consistently across all library preparation processes, regardless of their position on the Biometra TAdvanced 96 SG’s heat block, as seen by gel electrophoresis following adaptor ligation (Figure 2C) and PCR enrichment using Index Primers Set1 (Figure 2D).
No PCR bias or cross-contamination was detected between the libraries. Libraries of varied lengths and GC concentrations were successfully generated, demonstrating the appropriate performance of equipment and reagents throughout the preparation process.
Concentrations were measured to normalize the sequencing libraries using the NEB library quantification procedure. The amplification of libraries (Figure 5B) had an adequate PCR efficiency of 103 % as measured by linear regression of the standard curve (R2 > 0.99) (Figure 5A).
The system’s Ct values were highly homogeneous, with a standard variation of less than 0.3 across all replicates. The libraries were quantified at concentrations from 10 to 63 nM (Figure 5C), corresponding to the recommended library concentrations for Illumina® sequencing.
In conclusion, the workflow presented, utilizing PCR and real-time PCR thermal cyclers from Analytik Jena and a library preparation kit from NEB, proved to be a reliable and efficient method for NGS DNA library preparation for amplicon sequencing, regardless of gene target, amplicon length, or GC content.
Figure 5. Amplicon library quantification. Amplification of A) six standards generating a standard curve for quantification of library concentrations and B) of sample libraries from each gene using the NEBNext® Library Quant Kit for Illumina® and the qTOWERiris. C) Calculated library concentrations of each gene determined by qPCR. Image Credit: Analytik Jena US
Summary
This article highlights the need to produce high-quality sequencing libraries in NGS applications, emphasizing amplicon sequencing of genes from human genomic DNA.
The library preparation procedure includes targeted PCR, end repair, adaptor ligation, barcoding, library enrichment and purification, and agarose gel electrophoresis to verify fragment integrity.
The Biometra TAdvanced thermal cycler (Figure 6) works well with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® to produce consistent DNA amplicon libraries. The resultant libraries had variable lengths and GC content, demonstrating the equipment and reagents’ appropriateness for this procedure.
The qTOWERiris real-time PCR thermal cycler produced accurate results for library quantification and subsequent normalization. The library amplification achieved excellent PCR efficiency, and quantification produced quantities within the specified range for Illumina® sequencing.
Figure 6. Biometra TAdvanced 96 SG. Image Credit: Analytik Jena US
Recommended devices
Table 2. Devices used for library preparation. Source: Analytik Jena US
| Article |
Article number |
Description |
| Biometra TAdvanced 96 SG thermal cycler |
846-x-070-241* |
The flexible high-quality thermal cycler with 96-well silver block with gradient function and block quick-change system for 12 different block modules.
It is available as 100 V, 115 V and 230 V version. |
| qTOWER iris real-time PCR cycler |
844-00853-x* |
Real-time PCR system designed in the standard 96-well format, operable via PC, customizable with up to 6 color modules. Available as 100 V, 115 V, and 230 V version. |
| Biometra Compact Line gel electrophoresis system |
846-025-xxx** |
The Biometra Compact series a robust and durable system for agarose gel electrophoresis with various chamber sizes for different sample throughputs. |
* x=2 for 230 V, x=4 for 115 V and x=5 for 100 V
** xxx… indicates the chamber variant number for different sizes
References
- Hu, T., Chitnis, N., Monos, D. & Dinh, A. Next-generation sequencing technologies: An overview. Human Immunology 82, 801–811 (2021).
- Vincent, A. T., Derome, N., Boyle, B., Culley, A. I. & Charette, S. J. Next-generation sequencing (NGS) in the microbiological world: How to make the most of your money. Journal of Microbiological Methods 138, 60–71 (2017).
- van Dijk, E. L., Jaszczyszyn, Y. & Thermes, C. Library preparation methods for next-generation sequencing: Tone down the bias. Experimental Cell Research 322, 12–20 (2014).
- Head, S.R., Komori, H.K., LaMere, S.A., Whisenant, T., Van Nieuwerburgh, F., Salomon, D.R., Ordoukhanian, P. Library construction for next-generation sequencing: Overviews and challenges. BioTechniques 56, 61–77 (2014).
About Analytik Jena US
Analytik Jena is a provider of instruments and products in the areas of analytical measuring technology and life science. Its portfolio includes the most modern analytical technology and complete systems for bioanalytical applications in the life science area.
Comprehensive laboratory software management and information systems (LIMS), service offerings, as well as device-specific consumables and disposables, such as reagents or plastic articles, complete the Group’s extensive range of products.
About Life Science
The Life Science product area demonstrates the biotechnological competence of Analytik Jena AG. We provide a wide product spectrum for automated total, as well as individual solutions for molecular diagnostics. Our products are focused to offer you a quality and the reproducibility of your laboratory results. This will surely ease your daily work and speed up your work processes in a certain way.
All together we support you through the complete process of the lab work. Besides we offer customized solutions and are able to adapt our products to your needs. Automated high-throughput screening systems for the pharmaceutical sector are also part of this segment’s extensive portfolio.
About Analytical Instrumentation
Analytik Jena has a long tradition in developing high-performance precision analytical systems that date back to the inventions made by Ernst Abbe and Carl Zeiss. We have grown to become one of the most innovative manufacturers of analytical measuring technology worldwide.
Our business unit Analytical Instrumentation offers excellent competencies in the fields of optical spectroscopy, sum parameters, and elemental analysis. Being proud of our core competency we grant all our customers a long-term warranty of 10 years for our high-performance optics.
About Lab Automation
With more than 25 years of market experience, Analytik Jena with its CyBio® Product Line is a leading provider for high-quality liquid handling and automation technologies. In the pharmaceutical and life science industries, our products enjoy the highest reputation for precision, reliability, robustness, and simplicity.
Moreover, the Automation Team designs produce, and install fully automated systems tailored to our clients' application, throughput, and capacity requirements. From stand-alone CyBio® Well up to fully customized robotic systems, we handle your compounds, biomolecules, and cells with great care.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.