Water for Next-Generation Sequencing

Water is used throughout the next-generation sequencing (NGS) workflow, from DNA fragmentation to sequencing runs and instrument cleaning. Using the appropriate water quality for each step will not only eliminate the risk of water contaminants interfering with nucleic acids and the many enzymatic reactions performed in this process, but will also help prevent instrument contamination.

Section Overview

• Introduction to NGS
• Importance of Quality Water in the NGS Workflow
• DNA Fragmentation
• NGS Library Preparation
• Sequencing Techniques
• High-quality Water Purification Systems for a Reliable NGS Workflow
• Related Products

Introduction to NGS

Next-generation sequencing is a high-throughput method that allows for the rapid sequencing of base pairs in DNA or RNA samples. NGS applications include gene expression profiling, chromosome counting, detection of epigenetic changes, and molecular analysis. 

NGS revolutionized genomic research and is now playing a crucial role in the clinical environment. NGS-based tests are invaluable in the assessment of new therapeutic drugs, and are increasingly being used for screening, diagnosis, and molecular characterization of cancers and many other diseases. The development of these molecular pathology tools leads the way to personalized care and preventive medicine.^1,2 Laboratories conducting high-throughput sequencing rely on high-quality reagents for the best results. Water is an essential but often overlooked reagent in the NGS workflow. From system cleaning to sequencing runs, various activities within the NGS workflow require different levels of water quality.

Importance of Quality Water in the NGS Workflow

The various NGS platforms available today rely on different chemistries and sequencing strategies, but most of them follow a similar workflow (Figure 1). Water quality may affect each step of this workflow.

The next-generation sequencing workflow begins with genomic DNA, which is sheared into fragments using acoustic shearing. Pure water is used here to maintain DNA integrity. The sheared DNA is then processed with ultrapure water (Type 1 water) that is nuclease-free for critical steps like end repair, adaptor ligation, and PCR amplification.

After amplifying the NGS library molecules, the final sequencing methods, fluorescence-based NGS and ion-semiconductor NGS, depend on high-quality water to ensure accurate results. Overall, maintaining high water quality throughout the NGS workflow is crucial for reliable sequencing accuracy.

DNA Fragmentation

High-quality sequencing libraries are critical in obtaining reliable NGS data³, and the important first step is DNA fragmentation. If acoustic shearing or sonication is used to fragment DNA, pure, particle-free water, (e.g., from a Milli-Q^® IX pure water purification system), is recommended for the water bath, since impurities could cause variations in the efficiency of energy transfer to the sample. Water should be changed regularly to limit bacteria growth and reduce the risk of sample contamination.

NGS Library Preparation

Water may contain contaminants, albeit in minute amounts, which can directly interfere with DNA and/or with the enzymatic reactions performed during library preparation (e.g., end-repair, ligation, PCR). Enzymatic activity may be affected by traces of organic contaminants (humic acids), and ions or metals (magnesium, iron, etc.) that are potentially present in water. In addition, nucleases may degrade samples and should be avoided. Finally, water contaminants such as particles or bacteria may contaminate instruments or deposit into the lines of automated liquid handlers. Therefore, ultrapure water free of nucleases (e.g., from a Milli-Q^® water purification system fitted with a Biopak^® ultrafiltration polisher) is recommended for these steps.

For library quantification and quality control, gel, chip-based or capillary electrophoresis may be used. In all cases, optimized migration of the sample with respect to its charge depends on water quality, and fluorescence detection may be disrupted by organic contaminants. Therefore, to ensure reliable results, ultrapure water free of ions and organics (18.2 MOhm·cm resistivity; Total Organic Carbon level at 5 ppb or below) should be used to prepare buffer and capillary gels, and for dilutions.

Sequencing Techniques

The sequencing process is highly sensitive to any nuclease activity. Although the impact of nuclease contamination may not be apparent during the initial loading and calibration period, over time, the degradation of signal could prove disastrous for experimental results. For this reason, ultrapure water free of nucleases is recommended for buffer dilutions and system-running buffers.

Fluorescence-based NGS

NGS platforms differ in engineering and in sequencing chemistries, but most of them rely on fluorescence detection. In addition to the aforementioned water contaminants, organic traces must be avoided, as they may affect fluorescence. Indeed, organic contaminants in water could either quench or increase the fluorescent signal, giving erroneous results in both cases.

Ion Semiconductor NGS

This sequencing technology monitors the incorporation of nucleotides by detecting the protons being released.⁴ The detection, instead of being optical, is therefore performed by precise measurements of changes in pH. This technology is sensitive to any pH change that is not due to the extension of the DNA molecule. Instrument manufacturers specifically recommend the use of freshly purified ultrapure water in order to ensure a pH close to 7. This is due to the fact that, upon storage in contact with the atmosphere, ultrapure water absorbs carbon dioxide and becomes slightly acidic (Figure 2), affecting the sequencing process. For this reason, ultrapure water must be freshly purified and protected from contact with the atmosphere when it is used for ion semiconductor NGS.

High-Quality Water Purification Systems for a Reliable NGS Workflow

Water is an essential reagent in NGS, and its quality has a direct impact on the consistency and reliability of sequencing results. A well-designed and maintained water purification system can provide laboratories with high-quality water tailored to meet all their specific needs

Water is an essential reagent in NGS, and its quality directly impacts the consistency and reliability of sequencing results. Contaminants in water, such as nucleases, organic molecules, and ions, can adversely affect enzymatic reactions and lead to erroneous data. Therefore, using high-quality water is crucial for every step of the NGS workflow, from sample preparation to sequencing.

Milli-Q^® water purification systems use advanced technologies to remove impurities, including bacteria, nucleases, and organic compounds, ensuring that the water used in NGS applications is free from contaminants. Milli-Q^® IQ 7 series water systems allow laboratories to produce ultrapure Type 1 water with resistivity of 18.2 MΩ·cm, which is essential for maintaining the integrity of sensitive molecular biology applications.

Acknowledgments

The authors are grateful to Dr. Sean P. Kennedy, Head of Biomics Pole, Pasteur Institute, Paris, France for his technical support and expertise.