DNA fragmentation, or shearing, is essential in the sample preparation for Next Generation Sequencing (NGS). This is the first step in preparing your DNA samples.
But why do you need to break up the DNA? What are the existing methods for breaking up DNA? What should you consider when it comes to DNA fragmentation? And how does DNA fragmentation influence NGS?
Currently, there are no sequencing platforms that sequence whole DNA samples. For instance, complex genomes like the human genome or plant genomes cannot be sequenced with a single DNA polymerase.
Therefore, the input DNA must be fragmented into tiny pieces before sequencing.
After fragmentation, these small DNA pieces are later ligated to adapters (oligonucleotides that bind the DNA molecule to the flow cell, shown on figure 1) to generate a library.
Figure 1. Illustration of a DNA fragment for next generation sequencing, which is attached to an adaptor. The adaptor sequence can then bind to a complementary DNA molecule on the flow cell.
Finally, libraries composed of unknown DNA molecules are sequenced using any of the NGS technologies such as Illumina®, HeliScope™, SOLIDT™, or 454 platforms.
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A DNA polymerase can only add to the template out to a certain number of bases before running into errors and mismatches (impaired matches between nucleotide bases).
Here is when we talk about high fidelity sequencing in NGS. To get a sequence that is the same as the template, researchers designed DNA polymerases that create short sequences in order to obtain high fidelity reads. Consequently, the input DNA should be fragmented to facilitate the DNA polymerase’s work.
Furthermore, there is no DNA polymerase able to sequence a complete complex genome like eukaryotes.
Next-Generation Sequencing gives you millions of reads of DNA fragments. These reads are very short sequences of DNA.
A read is defined as the sequence of a given DNA fragment. Illumina® and Long read lengths are currently under 600 bases.
The three current methods for fragmenting DNA are physical fragmentation, chemical fragmentation, and enzymatic fragmentation. As the names imply, physical fragmentation involves physical methods, chemical fragmentation involves the use of chemicals to break up DNA, and enzymatic fragmentation uses enzymes.
Let’s dive into each of the three methods in greater detail.
One of the most common physical methods of DNA fragmentation is sonication.
Sonication uses ultrasound waves to break apart chemical bonds of DNA. The sonication mechanism consists of applying acoustic waves to generate cavitation bubbles.
In the cavitation phenomenon, sound vibration creates bubbles that collapse after each acoustic stimulation. It generates high resonance vibration and thereby leads to the breakage of DNA strands. Sound waves cleave hydrogen bonds in the DNA molecule and cause single- and double-strand ruptures.
Depending on the sound energy, DNA can be broken into different sizes. For instance, some acoustic devices can break DNA into 100-5kb, and others can break the DNA into 6-20 kb.
The fragmentation need depends on the type of library you will sequence. For example, Mate-Pair libraries require an input DNA size lower than 20 kb, while RNA-seq libraries can accept a bigger input size, about 100-150 kb.
The DNA sizes can be controlled by modifying the length of the sonication treatment. For example, longer sonication times produced shorter DNA fragments, while a short sonication produced longer DNA fragments.
In general, a large proportion of DNA fragments are within the desired ranges. However, there is a need to optimize the sonication protocol.
Some sophisticated equipment already available to perform sonication are Diagenode’s Megaruptor®, Bioruptor®, and Bioruptor® Pico. These instruments are designed to automate DNA shearing.
Due to the ease of obtaining random and size-appropriate DNA inserts, sonication is the most widely adopted method for shearing DNA.
Chemical DNA fragmentation uses a combination of a divalent metal cations such as magnesium or zinc and heat to break nucleic acids.
Although DNA can be broken using chemical shearing, this method is mostly used for breaking RNA.
For instance, if you want to perform RNA-Seq (NGS technique) to sequence a transcriptome (the pool of all RNAs present in a sample), like DNA, the RNA should also be cut into small pieces before sequencing.
Also, depending on the incubation time (heat + metal cation), RNA fragments can be smaller or longer. The longer the incubation time, the shorter will be the RNA fragments.
Enzymatic DNA fragmentation methods use enzymes to break intact DNA and long sequences into ideal fragments for sequencing. The enzyme-based methods of DNA fragmentation for sequencing include transposase, restriction enzymes, and nicking enzymes.
For instance, in the Nextera library prep, a transposase enzyme is used to cut DNA and insert the adapters simultaneously into the double-strand DNA (dsDNA). However, this enzyme can be prone to creating insertions and deletions in the cut DNA molecule.
Enzymes can break both strands of dsDNA (e.g., restriction endonucleases) or generate nicks (discontinuity in a dsDNA without a phosphodiester bond) on each strand of dsDNA. This method is highly flexible and can be used to generate fragments from low bp to many kb in length.
The main concern with this method has been sequence bias, as many DNA cut enzymes have recognition sequences or sequence preferences.
Table 1. Advantages and disadvantages for the DNA fragmentation methods.
Before proceeding with your fragmentation method selection, you must define the study goal. That will give you the guideline to choose the sequencing platform, DNA insert size, and, therefore, the fragmentation method.
For instance, depending on the sequencing platform, a range size of the DNA fragments must be generated. For example, Illumina and Ion Torrent platforms can accept several hundred base pairs for short-read sequencing.
Some NGS applications require a minimal amount of DNA template. In those cases where you have low input DNA, the library prep may include a PCR step, otherwise, the library prep can be PCR-free.
There are four main types of library preps for whole-genome sequencing using NGS.
- TruSeq PCR free library prep kit: Ideal if you have 1-2 µg of DNA. This is PCR-free.
- TruSeq Nano DNA library prep kit: Ideal if you have 100-200 ng of DNA. It has a PCR step.
- Nextera DNA library prep kit: Ideal for complex genomes like the human genome. This library prep method uses the transposome complex to cut and ligate (this combined process is called ‘tagmentation’).
- Nextera DNA XT library prep kit: Ideal for small genomes like bacteria and plasmids and amplicons (PCR products). Use the transposome complex to cut and ligate (this combined process is called fragmentation).
Equipment and setup considerations (sonication and enzymatic processes)
Sonication requires special tubes compatible with the sonicator. Furthermore, the sonicator device must be available for fragmentation.
Some sophisticated sonication instruments can be expensive; however, if used for dedicated sequencing projects, they help automate and save time.
It is essential to control the temperature of the sonicators, as temperature also influences DNA shearing. For the chemical method, you should get access to a temperature-controlled water bath.
Finally, if using the enzymatic process, standard lab equipment can be used for DNA fragmentation. However, be aware of biases in the sequences.
If you want to get better results, simple adjustments may be necessary. Temperature is a key factor in physical methods, while cation concentration is important in chemical methods. Finally, for the enzyme-based method, the type and concentration of the enzymes are critical for improvements.
Although each fragmentation method has its advantages and disadvantages, a narrowly distributed peak of DNA fragments is ideal for the NGS library if you plan to perform whole-genome sequencing (WGS) (Panteleeva et. al., 2016).
Furthermore, a study comparing physical and enzymatic methods showed the overall performance was equal for both methods with minor differences in NGS sequencing.
Authors suggest that fragmentation methods can be chosen solely according to lab facilities, feasibility, and experimental design (Kechin et. al., 2021).
We have moved from an era of low-gene testing to high-throughput testing. This has opened new scientific horizons where the continued expansion of sequencing technologies will largely depend on overcoming the limitations associated with sample preparation. This will allow us to generate sequences with few deviations representing a highly faithful copy of the original sample.
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Clinisciences. (2021b). DNA library preparation kits for Illumina® - Physical shearing. Clinisciences.
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