5 Best Practices for NGS Library Prep Success

Library prep is central in next-generation sequencing (NGS) workflows. Manual NGS library prep has many bottlenecks and pain points, including increased hands-on time, pipetting errors, and risk of enzyme degradation. There is an increasing need for NGS to be scaled up in the fields of oncology, infectious disease, and genomics, which also means there is an equally growing need for precision and reproducibility in results to ensure regulatory compliance.

This blog explores five best practices for NGS library prep success and highlight how automated, hands-off systems can accelerate and enhance NGS library prep with consistency, reliability, and speed.

Best Practice #1: Optimize Adapter Ligation Conditions

Optimizing adapter ligation is crucial for efficient NGS library prep. One key implementation step is using freshly prepared or properly stored adapters. This will ensure adapters are not degraded or improperly annealed, which can significantly reduce ligation efficiency. Secondly, controlled ligation temperature and duration will ensure yields are maximized. Typically, blunt-end ligations are performed at room temperature with high enzyme concentrations for 15–30 minutes, while ligations with cohesive ends are usually performed at 12–16°C and left overnight¹. Lower temperatures and longer durations have been shown to enhance ligation efficiency for low-input samples². Ensuring correct molar ratios can also reduce the chance of forming adapter dimers³. 

Automated systems like the G.STATION NGS Workstation from DISPENDIX, which includes the I.DOT Liquid Handler and the G.PURE NGS Clean-Up Device  ensure optimized and consistent reaction setups across samples. This automated system enhances reproducibility and provides high-quality results (Fig. 1).

Figure 1. Workflow of how the I.DOT Liquid Handler and G.PURE NGS Clean-Up Device  can be implemented into optimal NGS library prep.

Best Practice #2: Handle Enzymes with Care

Proper handling of enzymes is essential for maintaining their activity and high-quality, reproducible NGS library prep. Enzyme stability and cold chain management must be maintained by keeping enzymes at recommended temperatures and avoiding repeated freeze-thaw cycles⁴. Accurate pipetting is also crucial to ensure consistent and reproducible results.

Utilizing automated systems like the I.DOT Liquid Handler from DISPENDIX significantly minimizes the chance of human error. The I.DOT Non-Contact Dispenser precisely dispenses on a nanoliter scale, increasing throughput and reproducibility. The G.STATION NGS Workstation from DISPENDIX includes the I.DOT liquid handler and G.PURE NGS Clean-Up Device, providing end-to-end automation and temperature control, enabling consistent, high-throughput NGS library prep with reduced risk of human error. 

Best Practice #3: Normalize Libraries Accurately

Library normalization is critical before pooling to ensure each library equally contributes to the final sequencing pool. This ensures libraries are not under- or overrepresented, which could introduce bias and compromise the quality and reproducibility of the results^5,6. Manual quantification and dilution are often time-consuming and introduce variability due to human error. 

Automated systems like the G.STATION NGS Workstation from DISPENDIX includes the G.PURE NGS Clean-Up Device, which can normalize libraries using integrated protocols and bead-based cleanup. This increases consistency across pooled samples and reduces biased sequencing depth during NGS library prep (Fig. 2). 

Figure 2. The G.STATION NGS Workstation from DISPENDIX, which includes the I.DOT Liquid Handler and G.PURE NGS Clean-Up Device is a hands-off automated system that ensures consistency, accuracy, and reproducibility in NGS library prep workflows. 

Best Practice #4: Minimize Human Error with Automation

Manual pipetting steps in NGS library prep are not only time-consuming but also introduce the chance of human error and variability, particularly across large sample batches or between users^7,8. Inconsistencies in results lead to batch effects, which can lead to low-quality, non-comparable data.

Automation can solve these issues by standardizing workflows, reducing variability, and improving reproducibility. The G.STATION NGS Workstation from DISPENDIX significantly minimizes hands-on time, allowing researchers to dedicate time to other tasks. It can standardize protocols, ensuring each step is consistent across runs, and reduce the chances of human errors. The G.STATION NGS Workstation includes the I.DOT Liquid Handler, capable of dispensing 10 nanolitres across a 96-well plate in just 10 seconds and across a 384-well plate in only 20 seconds, ensuring accurate and reliable results are obtained with speed.

Best Practice #5: Validate Every Step to Ensure Quality

Several recommended quality control checkpoints within the NGS library prep workflow help ensure the integrity, accuracy, and reproducibility of sequencing results. Key checkpoints include post-ligation, post-polymerase chain reaction (PCR), and post-normalization. Validation methods like fragment analysis, qPCR, and fluorometry can assess the quality of the library, allowing for early detection of any issues to avoid costly repeat experiments^9–11. 

The G.STATION NGS Workstation from DISPENDIX enhances quality control by automating and integrating validation into workflows. This system can log every step of the workflow, allowing for audit trails and traceability. The G.STATION NGS Workstation also ensures protocol validation, which is vital for regulatory compliance and data reliability.

Conclusion: Build a Stronger NGS Workflow with Best Practices & Automation

Small optimization steps of NGS library prep can significantly improve sequencing results. By implementing these five key best practices, including ensuring adapter ligation conditions are optimized, enzymes are handled correctly, libraries are normalized, human error is reduced with automation, and every step is quality control checked, you can achieve optimal NGS library prep, ensuring confidence in your results. Adopting automation as part of your best practices toolkit with instruments from DISPENDIX, like the I.DOT Liquid Handler and G.PURE NGS Clean-Up Device, which are included in the G.STATION NGS Workstation, will enhance your NGS library prep workflows, allowing reproducible, accurate, and reliable results to be obtained at speed, all whilst being regulatory ready. 

Download the I.DOT Liquid Handler brochure or the G.STATION NGS Workstation brochure to find out how automation can be key to optimizing your NGS library prep workflows!

References

1. Suzuki M, Hayashi H, Mizuki T, Maekawa T, Morimoto H. Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field. Biochem Biophys Rep. 2016;8:360-364. doi:10.1016/j.bbrep.2016.10.006
2. Kurschat WC, Müller J, Wombacher R, Helm M. Optimizing splinted ligation of highly structured small RNAs. RNA. 2005;11(12):1909-1914. doi:10.1261/rna.2170705
3. Shore S, Henderson JM, Lebedev A, et al. Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation. Mahalingam R, ed. PLOS ONE. 2016;11(11):e0167009. doi:10.1371/journal.pone.0167009
4. Piszkiewicz S, Pielak GJ. Protecting Enzymes from Stress-Induced Inactivation. Biochemistry. 2019;58(37):3825-3833. doi:10.1021/acs.biochem.9b00675
5. Brennan C, Salido RA, Belda-Ferre P, et al. Maximizing the potential of high-throughput next-generation sequencing through precise normalization based on read count distribution. Sachdeva N, ed. mSystems. Published online June 23, 2023:e00006-23. doi:10.1128/msystems.00006-23
6. Robin JD, Ludlow AT, LaRanger R, Wright WE, Shay JW. Comparison of DNA Quantification Methods for Next Generation Sequencing. Sci Rep. 2016;6(1):24067. doi:10.1038/srep24067
7. Guan XL, Chang DPS, Mok ZX, Lee B. Assessing variations in manual pipetting: An under-investigated requirement of good laboratory practice. J Mass Spectrom Adv Clin Lab. 2023;30:25-29. doi:10.1016/j.jmsacl.2023.09.001
8. Lippi G, Lima-Oliveira G, Brocco G, Bassi A, Salvagno GL. Estimating the intra- and inter-individual imprecision of manual pipetting. Clin Chem Lab Med CCLM. 2017;55(7). doi:10.1515/cclm-2016-0810
9. Endrullat C, Glökler J, Franke P, Frohme M. Standardization and quality management in next-generation sequencing. Appl Transl Genomics. 2016;10:2-9. doi:10.1016/j.atg.2016.06.001
10. Jennings LJ, Arcila ME, Corless C, et al. Guidelines for Validation of Next-Generation Sequencing–Based Oncology Panels. J Mol Diagn. 2017;19(3):341-365. doi:10.1016/j.jmoldx.2017.01.011
11. De Abreu FB, Peterson JD, Amos CI, Wells WA, Tsongalis GJ. Effective quality management practices in routine clinical next-generation sequencing. Clin Chem Lab Med CCLM. 2016;54(5). doi:10.1515/cclm-2015-1190