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How To Prepare Your Samples For NGS

How To Prepare Your Samples For NGS

With more and more advancements in the fields of genetics, biochemistry, and biotechnology, efficient and more reliable techniques of handling genomic information of living forms are surfacing up. One of them is Next Generation DNA Sequencing. After the successful implementation of other DNA sequencing and manipulation methods like PCR and Sanger Sequencing, NGS is taking genetics to a new stage.

What is Next Generation DNA Sequencing?

NGS is a simple method of DNA Sequencing. The factors that make it different than the other previous similar techniques are

  1. It is faster.
  2. It is more reliable.
  3. The technique is relatively affordable.
  4. It is easier to handle.

Preparing DNA for any type of sequencing is a complex set of steps, but if the chosen technique is NGS, the handling is less complicated and time-consuming. When it comes to NGS, the preparation of samples depends on two substantial parameters:

  1. The characteristics and type of samples.
  2. The nature of the NGS platform.

Additionally, how the user prepares the sequencing library also affects the results of the NGS sequencing.

If you want to know how one can go about preparing the DNA samples for NGS, continue reading. Below are the fundamentals that cover everything there is about the modern way of sequencing DNA – NGS.

Fundamentals of NGS Sampling

DNA Extraction Protocol

The first and foremost step in the extraction protocol of any DNA sequencing technique is the isolation of the targetted genetic material from the cells and tissues of the subject organism. It could be either DNA or RNA. The presence of other molecules in the experiment mix like proteins and RNA bodies interfere with the results of the sequencing and can sidetrack the target goal of the methodology. Hence, their removal through safe ways is a necessity to ensure 100% accuracy. Furthermore, the deciding factors of the extraction process of NGS sequencing are the storage conditions and the characteristics of the target tissue.

A typical extraction involves the breaking down of the extracellular matrix. What follows is the penetration into the cell membrane with the help of chemical agents like surfactants, solvents, and enzymes. Afterwards, the DNA from the cell membrane is isolated. The two popular methods of extraction that are widely used for the preparation of NGS samples are:

  1. Phenol-based extraction.
  2. Chloroform-based extraction.

Amplification

After extraction, the choice of amplifying the resultant isolated DNA depends on the sample size and application of the user. For example, if you are carrying out whole genome sequencing with a sample size of 2µg, there will be no need for amplification. However, if the sample size of the starting material ranges from nanograms to picograms, amplification becomes a requirement to ensure enough coverage of the sequencing cells.

The two amplification methods that are most common in NGS sequencing are polymerase chain reaction (PCR) and isothermal amplification. PCR takes advantage of generic primers that increase the sample size of the starting material in a uniform and convenient manner. However, this method is known to be more error-prone than others.

More modern amplification methods are MDA and MALBAC. Both of these techniques offer excellent accuracy and extremely low rates of false-negatives and false-positive in the final results of the DNA sequencing.

Library Preparation

In NGS sequencing, the platforms study the DNA in the form of bite-sized fragments through a process called fragmentation. With this process, a library of equal and optimal sized DNA fragments is achieved that is essential for accurate results. There are two ways one can carry out fragmentation in the process of sample preparation for NGS

  1. Mechanical fragmentation (shearing)
  2. Enzymatic fragmentation.

Both of these methods are reliable and result in the quality and correct fragmentation of DNA samples. The only difference is that with enzymatic fragmentation, creating a de novo assembly is more challenging as compared to mechanical fragmentation.

Size selection and Purification

Size selection before finalizing your samples is necessary to speed up the workflow. In other terms, you can also call this step the “clean-up” step. Removing fragments that can affect or alter the quality of the data will ensure that your hard work pays off by the end of the experiment. For example, if the requirements for the NGS sequencing are of narrow size fragments, eliminating samples that are too large or too small can help in producing useful results and accurate sequencing.

Quality Control

The concluding step in the preparation of the NGS sampling is to ensure that the quality and quantity of the DNA samples are precise. Both of these factors play a substantial role in the confidence level of your sequencing data. You can analyze the quantity of your input DNA samples through qPCR based methods and fluorescent-based methods (Cytiva, 2018).

Tips to remember during NGS Sampling

According to the experts in the field of life sciences, the following are the tip you need to follow if you acquire 100% accurate results.

#1 The quality of the input is the factor that will decide how accurate is the data of your DNA sequencing. No matter how well you are experimenting, if the sample size is not sufficient, the preciseness of your data will suffer.

#2 The second most crucial parameter of NGS sampling is the quality of the Nucleic Acid. Therefore, you have to choose the most complimentary purification method that goes with the nature of the library platform. You have to implement a technique that not only generates a large amount of DNA or RNA but also produces material that can be used without any complexities.

#3 Make sure that the mass size and size distribution of both the DNA sample and final library platform is accurate. If these metrics are not handled well, the results of the DNA sequencing will not be as fruitful as it should be.

#4 Selection of the right library preparation kit and panel is essential to experiment with an impressive workflow speed and unerring results. The factors that decide which preparatory kit will work the best for you are

  1. Application.
  2. Characteristics of the sample.
  3. The sample size of the DNA input.
  4. The library platform.
  5. The capacity of the library (Biocompare, 2019).

References

Biocompare. (2019, Feb 11). NGS Sample Preparation: Tips from Five Experts. Biocompare. https://www.biocompare.com/Media/37/Document/BC_NGS_Sample_Preparation_Tips.pdf

Cytiva. (2018, April 3). Fundamentals of NGS sample preparation. Cytiva. https://www.cytivalifesciences.com/en/us/news-center/next-generation-sequencing-sample-preparation-guide-10001

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