Next Generation Sequencing (NGS) sample preparation is the first step in library preparation. Samples consist of DNA or RNA extracted from tissues. Then quality controls are carried out before proceeding with library preparation.
The preparation of the input sample is fundamental for ending with a high-quality library prep. A good sample preparation is critical in successful NGS because many downstream processes, such as library preparation, sequencing, data analysis, and validation, could be compromised when sample preparation is not properly performed.
In this article, I will focus on input sample preparation and things to consider when extracting DNA/RNA molecules to help you succeed in NGS. The other two steps are explained in separate articles.
In this article
Extracting DNA or RNA molecules from tissues is key for the creation of libraries. Therefore, DNA or RNA extraction quality is vital for achieving good results in NGS.
Low-quality DNA/RNA extractions could be contaminated with proteins or organic compounds such as polyphenols. And it may make library preparation problematic in several forms.
For instance, for those libraries where PCR is used, extraction contamination makes amplification difficult and may skew the results. In other cases, contaminants may reduce the efficiency in the sequencing of target molecules.
Preparing a sample for NGS can be challenging because many things must be considered, such as the sequencing application, source of the tissue, and available kits or reagents in your lab to perform the extraction.
We have a step-by-step overview on sample preparation that I used during my work in NGS.
To prepare samples for NGS, you must first define the application in NGS. You can start from genomic DNA for whole genome sequencing (WGS), Exome-Seq, or Methyl-Seq.
For RNA-Seq, you should start from RNA extraction, but keep in mind that RNA must be transformed into DNA, producing complementary DNA or cDNA. The conversion to cDNA is important because manipulating RNA molecules directly in short read sequencing is challenging due to their instability.
Once you have chosen the NGS application, then it’s time to focus on the tissue from which you will get the RNA or DNA extraction.
Some tissues are more complex than others due to their fragility during the extraction or high content of polyphenols or polysaccharides that make it difficult to achieve a clean extraction.
Furthermore, FFPE or fixed tissues naturally produce low yields, so the manipulation of FFPE samples requires special kits already available for this.
Reports give you an idea of how easy or difficult your tissue is.
You will also need to consider when, where, and how to collect your tissues.
For example, if you go into the field to collect samples, you may want to use preservation solutions that are high salt concentrated buffers preventing tissue damage (and therefore the content of nucleic acids internally) while being transported to the laboratory.
Usually, 1:5 tissue: volume is recommended. It means, if you put your tissue in a tube and your tissue is about 1 ml, you would need to add 5 ml of the preservation solution. This procedure prevents the tissue from damage before the nucleic acid extraction.
On the other hand, if you already have tissues available in your lab (e.g., from experimental plants or stored organs), you can use liquid nitrogen to preserve the tissues before the extraction.
After collecting the samples, you could keep the samples in the freezer at -80°C until you are ready for extraction.
Next, I recommend looking for a DNA/RNA extraction protocol already reported from tissues or species related to your target organism.
Similarly, you may ask in your lab what kits or reagents are available to perform DNA/RNA extractions. The extraction protocol is critical to obtain high-quality RNA.
From there, I strongly recommend understanding what happens in each extraction step. Usually, when kits are used, the biology behind them is skipped, which is critical to improving the protocol. So, I recommend studying the protocol enough, identifying the purpose of each step, and then evaluating the protocol with a small portion of your tissues.
Depending on the quality of your results, you can come back to revise the protocol and determine which steps need to be enhanced.
Remember, when working with RNA, there is a necessary intermediate step of transforming your RNA into cDNA. Therefore, protocols, including RT-PCR, are advisable.
This process can be done by yourself or be requested by a sequencing center.
I also recommend reading our GoldBio article Overview About How To Do RT-qPCR.
It is crucial to store the samples correctly because you need to check the quality of your extractions, or you are ready to submit them to the sequencing center.
The reason proper storage is so critical is because temperature changes may degrade your DNA/RNA molecules and make them prone to the enzymatic activity of RNases or DNases, and samples can be lost.
Storing your samples at -20°C for a short period is ok. For longer sample storage (more than three months), I recommend storing your samples at -80°C.
Checking the quality of your extraction is paramount for NGS success.
Different techniques are used to check the quality of DNA/RNA extraction, such as spectrophotometric, fluorescence, and microfluidics.
Spectrophotometric: The most common and relatively cheap technique to determine the concentration and quality of your DNA or RNA extraction is using the NanoDropTM system.
This equipment uses spectrophotometry to measure the quantity and quality of nucleic acids in a sample. It is based on the ratios 260/280 and 260/230.
The maximal absorption for DNA is at 260nm. The quantity is expressed in µg/µL. The ratios are also used as indicators of contamination. If 260/280 is lower than 1.8, the DNA sample is considered to have high purity. If the absorbance at 260/230 is lower than 1.8, it means there is possible contamination with organic compounds like polyphenols.
Fluorescence: For fluorescence, the most common device is QubitTM. This system is based on the fluorescence of dyes like PicoGreenTM, which binds to double-stranded DNA molecules. Here, the amount of DNA is proportional to the fluorescence intensity. Qubit is used to determine the concentration of DNA, however it does not show the contamination.
Microfluidics: A system based on microfluidics provide information about the quality and quantity of DNA and RNA molecules. It tells you if the samples are degraded or not and the amount of nucleic acids you have. It displays a figure similar to a gel electrophoresis where bands are shown. The quality of a sample is expressed in terms of RNA integrity number (RIN) or DNA integrity number (DIN). This number goes from 0 to 10, where generally, high-quality samples are between 7 and 10. The quantity is expressed in µg/µL.
The easiest way to ship samples properly is to ask the sequencing center in advance about how to prepare them.
Tell the sequencing center bout your protocol and the final solution to precipitate your samples. They may recommend what to do.
Things are easier when your university has a sequencing center. It is just a matter of taking your samples out of the fridge, putting them on ice, walking to the center, and providing your samples to the technician.
Some issues can arise when you submit your samples out of your lab to another city or facility. Careful shipping is critical because bad shipping can ruin your work. So, my recommendation when shipping RNA is to use dry ice.
In cases where you have backup samples and dry ice is not possible, you can submit your RNA samples in 100% ethanol.
You can dissolve the samples in TE buffer or 10mM Tris pH 8.0 buffers with 4°C blue ice for DNA shipping. Generally, dry ice is also recommended.
Again, the quickest way to determine the best shipping approach is to ask your sequencing center.
At GoldBio, we provide you with the tools and resources to succeed in NGS. Below there is a list of articles and helpful videos about NGS.
- Library Preparation Methods for Sequencing
- Quality Control for Library PrepsQuality Control for Library Preps
- Overview About How To Do RT-qPCR
- Guide to Plant DNA Extraction
- Plant RNA Extraction Overview: Methods, Tips, Steps and More
- Plant DNA Extraction Protocol
- Plant RNA Extraction Protocol
- DNA Extraction from Mouse Tail
Healey, A., Furtado, A., Cooper, T., & Henry, R. J. (2014). Protocol: A simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods, 10(1), 21. https://doi.org/10.1186/1746-4811-10-21
Nath, O., Fletcher, S. J., Hayward, A., Shaw, L. M., Agarwal, R., Furtado, A., Henry, R. J., & Mitter, N. (2022). A Comprehensive High-Quality DNA and RNA Extraction Protocol for a Range of Cultivars and Tissue Types of the Woody Crop Avocado. Plants, 11(3), 242. https://doi.org/10.3390/plants11030242
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Psifidi, A., Dovas, C. I., Bramis, G., Lazou, T., Russel, C. L., Arsenos, G., & Banos, G. (2015). Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples. PLOS ONE, 10(1), e0115960. https://doi.org/10.1371/journal.pone.0115960
Rana, M. M., Aycan, M., Takamatsu, T., Kaneko, K., Mitsui, T., & Itoh, K. (2019). Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue. Methods and Protocols, 2(2), 54. https://doi.org/10.3390/mps2020054
Scholes, A. N., & Lewis, J. A. (2020). Comparison of RNA isolation methods on RNA-Seq: Implications for differential expression and meta-analyses. BMC Genomics, 21(1), 249. https://doi.org/10.1186/s12864-020-6673-2
Wong, R. K. Y., MacMahon, M., Woodside, J. V., & Simpson, D. A. (2019). A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma. BMC Genomics, 20(1), 446. https://doi.org/10.1186/s12864-019-5826-7
Yockteng, R., Almeida, A. M. R., Yee, S., Andre, T., Hill, C., & Specht, C. D. (2013). A method for extracting high‐quality RNA from diverse plants for next‐generation sequencing and gene expression analyses. Applications in Plant Sciences, 1(12), 1300070. https://doi.org/10.3732/apps.1300070