Don’t neglect your water

Next generation sequencing is a complex endeavour with advanced equipment, well developed methodologies and incredibly detailed analysis. But, say Estelle Riché and Sean P. Kennedy, don’t forget the basics – neglect water quality and your sequence will suffer  

High quality reagents are indispensable for laboratories conducting next generation sequencing (NGS). To ensure quality and reproducibility, NGS laboratories go to great lengths to standardise kits, protocols, reagents and procedures. Even so, an essential but often overlooked reagent in the NGS workflow is water, which should also be part of this rigorous quality control effort.

Water quality has a direct impact on the consistency and reliability of sequencing results. From system cleaning to sequencing runs, various activities within the NGS workflow require different levels of water quality. An understanding of the various water contaminants and their potential impact on sequencing results can help laboratories make the best choice among the wide range of water sources available to meet their specific needs.

The highest water grade commonly used in laboratories – ‘ultrapure’ – should always be used for very sensitive applications, including the preparation of reagents and buffers used in sequencing. ‘Pure’ water can be used for general laboratory applications and for rinsing.

[caption id="attachment_48953" align="aligncenter" width="620"] Basic principle of laboratory water purification.[/caption]

Water may contain many contaminants, including ions (e.g., magnesium, iron), organic matter (e.g. humic acids), bacteria and by-products (e.g., nucleases), particles and gases. Even if many of these contaminants are present only in low amounts, they can still directly interfere with DNA and/or with the enzymatic reactions performed during the sequencing workflow. In addition, contaminants such as particles or bacteria can contaminate instruments or deposit into the lines of liquid handlers. It is therefore critical to carefully select the water quality used for each step of the NGS process.

[caption id="attachment_48954" align="aligncenter" width="620"] NGS workflow and specific water quality requirements[/caption]

Nucleic acid library preparation The preparation of high quality sequencing libraries from nucleic acids is critical to obtain reliable NGS data. If an automated sonicator is used to fragment DNA, pure water is recommended for the water bath since impurities could impact the efficiency of transfer of precise energy levels to the sample. For library quantification and quality control, gel, chip-based or capillary electrophoresis may be used. In all cases, migration of the sample with respect to its charge depends on water quality. Further, in the case of capillary electrophoresis, instruments rely on fluorescence detection, which may be disrupted by organic contaminants. Therefore, using ultrapure water for buffer and capillary gel preparation, and for dilutions, will ensure reliable results. In addition, many enzymatic reactions are performed when preparing samples for NGS (e.g. PCR, ligation, end-repair), and all are dependent on water quality.

Fluorescence-based NGS The sequencing process is highly sensitive to any nuclease activity. Although the impact of DNase contamination may not be apparent during the initial loading and calibration period, over the course of one week the degradation of signal could prove disastrous for experimental results. For this reason, only ultrapure water free of nucleases should be used for all buffer dilutions and system running buffers.

Nucleases cleave the phosphodiester bonds of nucleic acids. These robust enzymes are present everywhere in the laboratory environment and they are notoriously difficult to eliminate. Autoclaving is not sufficient to remove nucleases from laboratory water: although they may become denatured during the process, nucleases can regain their native structure and function. In many cases, the water is first chemically treated with diethylpyrocarbonate (DEPC) and then autoclaved to ensure the eradication of nuclease activity. The challenge with this approach is that the alcohol and CO­­₂ generated by the chemical treatment can interact with enzymatic reactions and thus affect experimental results. In addition, DEPC is a hazardous chemical, and the overall DEPC treatment procedure is lengthy.

An alternative method to DEPC treatment of water is ultrafiltration. This process utilises hollow fibers to separate contaminants based on their size. It ultimately removes nucleases from water instead of merely inactivating them. Ultrapure water treated by ultrafiltration is nuclease free. The efficiency of RNase removal by ultrafiltration is equivalent to the inactivation of RNase activity by DEPC treatment, but without the drawbacks linked to the generation of alcohol and CO­­₂.

[caption id="attachment_48955" align="aligncenter" width="620"] Efficiency of RNase removal by ultrafiltration. Ultrapure water spiked with RNase A was either 1: treated with ultrafiltration, 2: not treated. Ribosomal RNA was added to each solution, and gel electrophoresis was performed.[/caption]

Ion semiconductor NGS This technology is sensitive to any pH change that is not due to the extension of the DNA molecule, including contamination, temperature changes, and/or atmospheric CO­­₂ being dissolved into the running solutions. Often the user manuals of ion semiconductor NGS systems state that solutions need to be prepared daily with ultrapure water (resistivity: 18 MOhm.cm) directly from a water purification system, and that water shall not be stored.

Ultrapure water is an excellent solvent; as such it tends to become contaminated by the environment and/or the containers it is in contact with. Ultrapure water stored in a container, for example, will very quickly absorb atmospheric CO­­₂ to form carbonic acid, and then bicarbonate and carbonate; as a result, the pH will become acidic. After just one hour, the resistivity of ultrapure water can drop from 18 to 4 MOhm.cm. This change in pH would have a detrimental effect on the sequencing process. Freshly purified water from a water purification system is therefore necessary for ion semiconductor sequencing platforms. Once collected, the water is topped with high purity nitrogen gas in order to avoid the dissolution of atmospheric CO­­₂.

Water purification Water purification is generally conducted in two steps: pre-treatment and polishing. Pre-treatment removes the largest amount of contamination from the water – usually 95 to 99% of all contaminants. Polishing removes trace contaminants that are still present in the water. A single technology does not exist that can reliably remove all contaminants from water. Therefore, a combination of technologies is used in each of the purification steps to obtain the purest water possible.

There are two common ways of monitoring the quality of laboratory water; resistivity and total oxidisable carbon (TOC).

• Resistivity is a reflection of ionic purity. Tap water typically contains a level of parts per million (ppm; equivalent to mg/L) of ions. These levels drop to sub-parts per billion (ppb; equivalent to µg/L) after the pre-treatment step, and then are reduced further after the polishing step to parts per trillion (ppt; equivalent to ng/L) or below. • The TOC reflects the extent of organic contamination in water. Tap water typically contains ppm levels of organic contaminants. These levels drop below 50 ppb after pre- treatment, and to less than 5 ppb after the polishing step. An additional filter may be attached at the outlet of the water purification system, such as a 0.22 µm screen filter (to retain particles and bacteria) or an ultrafilter (to retain nucleases and other bacteria by-products) for sequencing work.

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 their specific needs. Additionally, some purification systems include specific software tools for users in GxP environments who must comply with guidelines such as U.S. FDA 21 CFR Part 11 or similar requirements set by other global regulatory organisations.

The authors: 

Estelle Riché, PhD is Senior Scientist at Millipore SAS in France. Sean P. Kennedy, PhD is Head of Biomics Pole at the Pasteur Institute in Paris, France.

Contact:

estelle.riche@merckgroup.com