First-hand Tricks for NGS Library Preparation

Next Generation Sequencing (NGS) is a very powerful tool in the biomedical field. The first step of NGS is library preparation. Depending on the purpose of sequencing, the preparation method of the libraries may vary.

Our NGS experts have lots of experience from years of working on the development of library preparation reagents. It is essential that bench scientists should follow the basic procedures of molecular biology or genomics labs, such as bench cleaning, proper pipetting, preparation of master mix when possible, proper reagent storage/usage, etc. However, these are not the focus of this article since these are very basic for laboratories. Here we are going to emphasize on NGS library preparation and provide some useful first-hand tricks that can help you make high-quality libraries.

1. Purify samples before library prep

High quality input DNA/RNA samples is very important for successful NGS library preparation. The library prep involves enzymatic reactions which can be easily affected by contaminants in the input samples. We suggest purifying samples with beads after DNA fragmentation so the impurities can be removed before library preparation.

2. Quantify sample with a fluorometer

There are many ways to quantify DNA/RNA samples, including the old way of measuring OD/260 with spectrophotometers and the more recent method with fluorometers. Spectrophotometers are easier to use at no addition cost (excluding equipment costs), while the fluorescence-based fluorometer method needs reagent prep and sample mixing, and of course there is a cost for the reagent.

Considering the quality of the libraries, we strongly suggest measuring the DNA/RNA concentrations using the fluorometer since it provides more accurate measurements. Spectrophotometers are more likely to be affected by contaminants such as salt, protein, and others. We have found that in some cases, the concentration results from spectrophotometers are incorrectly 3 to 5 times higher than that from fluorometers.

3. Prevent contamination

 

Contamination and cross-contamination is a common problem in the lab. With the high cost of NGS, it is invaluable to take extra care when working on NGS library preparation.

• • • • Clean your work space. Make it a DNA/RNA free environment before library preparation. This is very important for avoiding sample contamination, especially when you have limited samples.
      • Use filtered pipet tips to reduce cross-contamination.
      • Spin down tubes/plates containing index adaptors and index primers before use. Handle with care. This is critical for the prevention of index contamination.
      • Seal or cap the reaction plates/tubes ASAP. This is also critical for preventing index contamination.

4. Minimize PCR cycle numbers

It is well known that PCR generates bias. In some extreme cases, PCR-free library prep is required. Generally, the principle is that the number of PCR cycles should be kept minimal. There are two ways to monitor the cycle numbers easily:

• • • • Real time quantitative PCR: add SYBR Green dye (and ROX if needed) to the PCR mixture and check the amplification curve. Stop the reaction immediately when the concentration is enough.
      • Standard PCR: If you don’t have a real time quantitative thermal cycler, you can mimic the real time PCR by monitoring the reaction manually. You can set the cycle number based on the amount of input DNA. After completion of PCR, load 5 ul of PCR mixtures on a 2% agarose gel to check the library amount. The PCR plate/tube should be kept on ice while running agarose gel. You can decide whether the amount is enough based on the gel image. You can run more cycles with the remaining samples if you need.

 

5. Remove adaptors and primer dimers

Contamination of primers and adaptors not only decreases the yield of sequencing, but also may generate a sequencing problem called “index hopping” when you multiplex samples.  The Primer dimers and adaptors can be removed with beads purification. If primer dimers and adaptors persist in the libraries, you can try with 0.8X volume of beads for regular libraries. In some cases, a second step of beads purification may be needed to remove primers and adaptors completely.

Gel purification is another option, but it is more tedious and time-consuming. Run the libraries on a 2% agarose gel, cut the libraries out and purify the library with a gel purification kit. By using this method, you can choose the size range of your libraries.

6. Store libraries at -80C if possible

Finally, your NGS libraries are done. However, we have found that the completed NGS libraries can degrade over time, even at -20C. Thus, libraries should be stored at -80C, if possible, to prevent the degradation of the completed libraries.

7. Pool samples at the last minute for multiplexing

If you want to multiplex your samples, pool them just before you submit your samples. As we mentioned before, long time storage can cause library degradation and the degradation may not be equal for each library. This last-minute pooling strategy is very useful for making sure that each library in the mixture has the same amount.