Five tips for high-quality NGS library preparation

[BioDynami]

Next Generation Sequencing (NGS) library preparation is becoming a popular procedure in labs. BioDynami NGS experts have been working on NGS library preparation for many years. Here are some tips that can help you to make high-quality NGS libraries.

1. Quantify DNA with a fluorometer
   To measure DNA concentration for NGS library preparation, fluorometers are more accurate than Spectrophotometers. Spectrophotometers are more likely to be affected by salt, protein, and other contaminants. In some cases, the results from Spectrophotometers are 3 to 5 times higher than those from fluorometers. We use Qubit assays and Picogreen assays in our lab for DNA quantification.
   
   
2. Purify DNA before library preparation
   DNA quality can affect efficiency of library preparation. We suggest purifying DNA with beads after DNA shearing, so the impurities can be removed before library preparation.
   
   
3. Prevent contamination
   Contamination and cross-contamination is a huge problem in some cases. Extra care should be taken when handling multiple samples.
   • Clean your working space. Make it a DNA and RNA free environment before library preparation.
   • Use filtered pipet tips
   • Pipet gently
   • Spin down tubes/plates containing index adaptors and index primers before use. Handle with care.
   • Seal or cap the reaction wells/tubes ASAP.
   
   
4. Minimize PCR cycle numbers
   PCR generates bias. It is a general principle that the number of PCR cycles should be kept minimal. Two ways to monitor the number of cycles:
   • Real time PCR: add SYBR Green dye to the PCR mixture and monitor the amplification. Stop the reaction when necessary.
   • PCR: If you don’t have a real time thermal cycler, you can set a starting cycle number based on the amount of input DNA. Load 5 ul PCR mixtures on 2% agarose gel to confirm the libraries after PCR. Put the PCR plate on ice while running gel. Run additional PCR cycles when needed.
   
   
5. Remove primer dimers and adaptors
   Primer dimers and adaptors are usually removed during beads purification. If primer dimers and adaptors still remain in the libraries, use 0.8X volume of beads to purify regular libraries. Sometimes, a second purification may be needed to remove the residue of primers and adaptors. Gel purification is another option (although it is more time-consuming). Run the libraries on gel and cut the libraries out.

[Normis58]

Thank you for these tips.

[jenniferduran]

Many protocols use bead-based size selection but if you are using gels then use gel cutting tips to avoid cross-contamination, take multiple gel slices just in case something goes wrong downstream and use a Dark Reader to prevent UV damage of your library. Alternatively consider an eGel, LabChip XT or Pippin Prep. Preparation of libraries for Next Generation Sequencing can be challenging for newcomers, often involving multi-step protocols which may stretch over several days. When planning the different steps of library preparation, basic bench rules and molecular biology techniques often get "lost" among all the other considerations.