In previous posts about next generation sequencing (NGS), we covered the key steps in library preparation (for RNA-seq), top tips for NGS success, and the ins and outs of long-read DNA sequencing. This time we will look more closely at size selection.
What is Size Selection?
Size selection is a relatively cost‐effective part of the NGS workflow that can have a profound impact on experimental results and data quality. It refers to the elimination of suboptimal nucleic acid fragments from the library before applying to the sequencing platform. This ensures that the fragments contained in the library are within the optimum size range for the specific sequencing instrument; for example, this range is 200-500 bp for Illumina platforms but can be up to 700 bp for Roche instruments.
Size selection should not be confused with fragmentation. Fragmentation is the process of shearing DNA or RNA, which is typically performed upstream of NGS library preparation. During fragmentation, the isolated nucleic acids are subjected to either enzymatic activity or some mechanical force that simply breaks the isolated nucleic acids into shorter fragments. Enzymatic methods may permit some control over the length of the sheared fragments by adjusting the reaction time or conditions. Mechanical methods on the other hand, such as sonication or focused acoustics, vary in their ability to produce fragments within a tight size range although some, e.g., ultrasonication, yield predictable and tightly distributed shearing profiles.
Why Do It?
While some of the predictable fragmentation or shearing approaches aim to bypass the need for size selection it is important to bear in mind that intra‐sample differences in shearing profiles will exist that may skew downstream results. Size selection allows you to minimise the impact of such intra‐sample differences in fragment length by selecting fragments within the correct size range for the intended instrument to maximise sequencing capacity and data quality.
Size selection helps you to get the most out of a sequencing run by minimising wasted capacity on very small or very large fragments that are outside the range of what the instrument can read. Furthermore, small fragments of less than 150 bp in length can easily form adapter dimers and primers dimers, resulting in a further waste of sequencing capacity and less usable data.
How to Size Select
Depending on the application, there are generally a few approaches available to select fragments in the desired size range and simultaneously clean up low molecular weight fragments such as dimers, as well as remove unwanted enzymes, nucleotides, and buffer components. Here is an overview of the most commonly used size selection approaches:
1. Gel-Based Size Selection
Although this is a low throughput method, it is a reliable and effective way to perform size selection and it remains a popular choice. Here, fragments in the size range of interest are manually excised from an agarose gel. A molecular weight marker serves as a guide for fragment size. Gel-based selection may be performed on standard agarose gels, which is time-consuming or automated gel-based methods that offer greater reproducibility and save time.
Typically, gel-based size selection is used to eliminate larger fragments from DNA samples i.e. greater than 1,000 bp, or to eliminate miRNA and small RNA fractions from RNA samples. Since the product size for miRNA species is in the range of 18-30 bp and thus very close to adaptor dimers in size, magnetic beads do not provide enough separation for this type of size selection.
2. Magnetic Bead-Based Size Selection
Magnetic bead-based size selection is a cost-effective approach based on inert beads that contain polystyrene cores and are covered in magnetite and a layer of carboxyl molecules. In the presence of polyethylene glycol (PEG) and salt, nucleic acids bind to the beads reversibly. It is possible to control the sizes of the fragments that bind to the beads by adjusting the ratio of PEG, salt, and beads to nucleic acids.
Once nucleic acids have been allowed to bind the beads, a magnetic force is then applied, allowing the separation of the beads and thus the bound fragments from the remainder of the material. Desired fragments can then be collected either through elution from the beads triggered by a change in buffer (larger fragments) or direct collection in the supernatant, typically in the case of smaller fragments. The biggest advantage of magnetic bead-based size selection is that it is automatable and therefore high-throughput. This method is recommended for fragments of 100 bp in length and above.
Choosing the Right Method
Depending on the application in hand and the project goals, one or more size selection methods may be applicable. If you would like to learn more about size selection or obtain guidance on any aspect of the NGS workflow, feel free to reach out to your local Nordic BioSite representative by emailing us at email@example.com.
Head S.R. et al. 2014. Library construction for next-generation sequencing: Overviews and challenges. Biotechniques. 56(2): 61–passim. doi: 10.2144/000114133.