In this blog we cover the fundamentals of preparing your samples for NGS, and considerations for each step. These steps include DNA extraction, amplification, library preparation, selection or purification, and quality control.
Next-generation sequencing (NGS) has enabled us to extract genetic information from samples faster, more reliably, and at lower cost than ever before. Getting your DNA ready for sequencing requires the preparation of a sequencing library as well as a few other steps that depend on the type of sample and the NGS platform.
In this blog we cover the fundamentals of preparing your samples for NGS, as well as considerations for each step: DNA extraction, amplification, library preparation, selection or purification, and quality control.
Next-generation sequencing: DNA extraction protocol
The first step in every sample prep protocol is extracting the genetic material– DNA or RNA– from cells and tissues. Other molecules, such as RNA and proteins, interfere with the sequencing process and must be removed before doing anything else. The specific tissue type and storage conditions determine the details of this extraction process.
Extraction entails breaking down the extracellular matrix and opening the cell membranes using enzymes, solvents, or surfactants. The DNA in the resulting mixture must then be isolated.
The traditional gold standard in DNA isolation is phenol-based extraction. Phenol is a hydrophobic solvent that denatures and dissolves proteins, removing them from the DNA-containing aqueous phase. However, it can be tricky to work with, and users need to be careful not to contaminate the aqueous phase with phenol.
Spin columns that specifically bind DNA provide an alternative and are an easy-to-use, but more expensive, method to wash away the debris. Chloroform-based extraction, another alternative, enables you to isolate high-quality DNA without phenol, and commercial kits can include a resin that minimizes the risk of contamination.
Next-generation sequencing: amplification methods
Amplification after extraction is optional, depending on your application and sample size. For example, whole genome sequencing (WGA) with 2 µg of starting material does not necessarily require further amplification. But, with nanograms—or even picograms—of starting material, amplification becomes essential to obtain sufficient coverage for reliable sequence calls.
Isothermal amplification and polymerase chain reaction (PCR) are two common methods to increase the amount of input DNA. PCR uses generic primers to amplify the starting material in a highly uniform manner, but tends to be more error-prone than multiple displacement amplification (MDA).
MDA is an isothermal method, often based on Phi29 polymerase, and excels in accuracy with low rates of false-positives and false-negatives. MDA’s main drawback is overrepresentation of some regions of the genome.
More recently developed hybrid methods, such as MALBAC, aim to correct this issue with MDA, but these methods also rely on PCR, and have some of the same associated drawbacks.
The different advantages and disadvantages of these methods mean that each is better suited to detect some features over others. For example, MDA outperforms the other two methods in detecting single-nucleotide variants (SNVs), whereas PCR and MALBAC are better for studying copy number variation (CNV), as described in this Nature review article.
DNA library preparation for next-generation sequencing
Most NGS platforms analyze DNA in uniform, bite-size pieces, created by DNA fragmentation. This process generates a ‘library’ of fragments with a narrow length distribution that is optimal for the sequencing platform.
DNA fragmentation
Both mechanical fragmentation (shearing) and enzymatic methods are suitable for NGS. Mechanical methods enable random shearing to produce a variety of overlapping fragments for any given region of the genome. This is ideal for de novo assembly.
Enzymatic methods are relatively fast and require less investment upfront but have some ‘bias’, cleaving some sites preferentially, making de novo assembly more challenging without the variety of overlapping fragments.
DNA end-repair
The fragments generated have single-stranded, ‘sticky’ ends. The next step, end-repair, fills in these sticky ends to create blunt ends, ready for adaptor ligation.
Adaptors
Adaptors are then bound to both the 5’ and the 3’ ends of the library fragments. They are specific to the sequencing platform, but ultimately all serve to enable in-platform clonal amplification, i.e. Illumina’s bridge amplification or BGI’s rolling circle amplification.
The adaptors are designed to bind to the sequencer-specific substrate, such as a patterned flow cell, contain sequences to enable amplification, and can have barcodes for fragment identification.
Targeted sequencing
These library preparation steps are generally applicable to whole genome sequencing. If you’re looking to perform targeted sequencing, library preparation differs.
In amplicon-based target enrichment, the fragmentation and end-repair steps tend to be unnecessary. Pulling the targeted regions out as amplicon fragments with blunt ends enables you to go directly to adaptor ligation.
Hybridization-based enrichment does require fragmentation. The hybridization probes pull out the regions of interest from the library of overlapping fragments, ready for end-repair.
DNA sequencing: size selection and purification
To speed up your workflow, it might be necessary to ‘clean up’ your library before sequencing by removing fragments that won’t produce relevant data. For NGS workflows that have narrow size requirements, discarding fragments that are either too large or too small to produce useful results can improve sequencing efficiency.
There are different protocols for size selection, which might involve gel electrophoresis or magnetic bead-based selection. Magnetic beads also provide a quick and easy method for final clean-up.
DNA quality control
A final step before proceeding to sequencing is to confirm the quality and quantity of your DNA. Both parameters contribute to the confidence in your sequencing data. You can measure the quantity of your DNA using fluorescence- or qPCR-based methods.
For qualitative validation, many protocols use the Agilent TapeStation™ or Bioanalyzer™. Have a look at our blog on the challenges in NGS sample preparation for possible solutions for quality or quantity issues.
These are the basic steps that researchers use to prepare DNA for sequencing. You can find more information about specific NGS workflows and applications in our other NGS blogs.
At Cytiva, we provide a range of kits, tools, and resources to help you improve your NGS outcomes. To find out more about our NGS product range or for support with your NGS sample preparation, contact us.