What do you call a collection of millions of DNA fragments sharing the same short sequences on the 5’ and 3’ ends? The answer is a next generation sequencing, or NGS, library. Today, we are going to focus on the four basic steps of NGS library preparation that can be broadly applied across different preparation methods.
A key step in the NGS workflow is preparing the input for sequencing, known as creating a library. An NGS library is a collection of similarly sized DNA fragments with known adapter sequences added to the 5’ and 3’ ends. A library corresponds to a single sample and multiple libraries, each with their own unique adapter sequences, can be pooled and sequenced in the same sequencing run.
NGS library preparation has four general steps: 1. DNA Fragmentation or Target Selection, 2. Addition of adapter sequences, 3. Size selection, and 4. Final library quantification and QC.
The first step, DNA fragmentation or Target Selection. In order to get the starting DNA into smaller pieces, isolated DNA may be fragmented using physical or enzymatic methods. These libraries are referred to as fragment libraries. Alternatively, if the sequence of specific DNA targets is known, PCR amplification of those targets may be used to produce DNA amplicons within the desired size range. These libraries are referred to as amplicon libraries.
Next, specific DNA adapter sequences are annealed to the 5’ and 3’ ends of the fragmented or amplicon DNA. The double-stranded DNA adapters are approximately 20 to 40bp fragments that contain known sequences. Generally, there are two different adapter sequences that can anneal to the DNA fragments in either the 5’ or 3’ orientation. One adapter sequence contains the primer annealing site for the sequencing primer, while the second adapter sequence is generally used to anchor the DNA fragment to a surface for sequencing; for example beads or a solid surface containing a complimentary DNA sequence.
Now we have our DNA fragments with known adapter sequences on either end. The next step is to select the library fragment sizes we need for our sequencing run. There are two common size selection methods, the first is a gel electrophoresis based-method, while the second is bead-based size selection method. For the gel-based method, the adapted library fragments are run on a gel to separate the fragments by size and the band corresponding to the size of interest is collected. Using the bead-based method, magnetic beads are used with varying concentrations of buffers to isolate the DNA fragment sizes of interest. Final library fragment size is important for efficient, high quality DNA sequencing downstream.
Bonus, when preparing amplicon libraries, size selection is usually not necessary, as long as the PCR products were already designed to be within the desired size range. We are almost done! The last and very important step is library quantification and QC. Accurate library quantification is important for successful template preparation and sequencing. There are a few library quantification methods commonly used. The first is analysis by the Bioanalyzer system. This method gives you both library concentration and fragment size information. The second is qPCR. This method provides the most accurate library quantification information, as it only measures amplifiable library fragments, but lacks library size information. Which method you prefer is entirely up to you (and probably what’s available in your lab).
And that’s NGS library preparation in a nutshell. Of course there are variations on this theme depending on your application, for example, gene expression or DNA methylation analysis, but the fundamentals stay the same. Since your library quality dictates the success of all downstream processes from template preparation to sequencing, understanding the library preparation is important to help ensure you get the highest quality sequencing data.
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