Evaluating Quality of DNA for Next-Gen Sequencing
Next-Generation sequencing (NGS) has revolutionized genomic research, allowing for entire genomes to be sequenced in a single day. This form of sequencing provides an ultra-high throughput, scalable, and rapid method for scientists to answer genetic questions from a wide spectrum of applications and biological systems. Today, NGS is an essential tool for any biologist.
With the explosion of NGS, there are greater requirements for high quality and quantity DNA going into sequencing pipelines. Here, we detail the ideal requirements for NGS.
What is NGS?
Next-Gen Sequencing (massively parallel, deep sequencing or second-generation sequencing) is used to describe several modern sequencing technologies that allow for millions of DNA fragments to be sequenced in parallel. Once all these short reads have been generated, bioinformatic analyses pieces together these fragments together. The process sequences the entire genome multiple times, delivering high depth and accurate data.
How to Validate DNA for NGS
The quality and quantity of DNA going into the NGS pipeline is of utmost importance. Sequencing low-quality nucleic acid will result in impaired performance and even failed runs. Because of this, it is crucial to have an optimized DNA extraction pipeline in place that can scale easily.
For the most consistent and robust sequencing pipeline, we recommend validating DNA for NGS with the following steps:
A nucleic acid profile needs to be generated to evaluate what is present within the sample. This can be performed with an agarose gel or a Bioanalyzer. The sample should have the following:
High Molecular Weight Genomic DNA
The genomic DNA should be intact and un-sheared, without any large smears across the lane. Some NGS workflows will fragment DNA in the sequencing process further. Should this happen, it can result in lower results or they could be selected out of the sequencing run completely.
No RNA Contamination
To sequence genomic DNA, there should not be any RNA present within the sample. While the RNA may not impede the sequencing process, it can artificially inflate the amount of DNA present within the sample (especially with spectrophotometry).
The purity of the sample needs to be evaluated via spectrophotometry (e.g., NanoDrop™ 2000). By analyzing the absorbance at various wavelengths, you can determine if there are any contaminants present within the sample based on absorbance values. Ideally, a DNA sample for NGS should have the following measurements:
260/280 Absorbance Ratio: ~ 1.8
This ratio provides a general assessment of the amount of DNA to RNA present within a sample. A ratio of ~1.8 typically corresponds to sample with high amounts of DNA, while a ratio of ~2.0 corresponds to a sample with high amounts of RNA. Values lower than 1.8 can indicate protein contamination.
260/230 Absorbance Ratio: > 2.0
Salts, EDTA, phenol, carbohydrates, and other contaminants all absorb around 230 nm. A high 260/230 ratio (above 2.0) indicates that there are very few of these contaminants present within the DNA sample. With 260/230 ratios < 1.5, there are a large number of contaminants present within the sample which can negatively affect many kinds of enzymatic reactions in the NGS workflow.
Fluorometric methods of quantification are the gold standard for NGS pipelines (such as Qubit™ or Pico/RiboGreen™) since they only allow for the DNA to be quantified. However, the yield can also be ascertained through spectrophotometry, so long as there is no RNA contamination present within the sample.
Regardless of the method, the DNA should be high-quantity and high concentration.
Abiding by these requirements before preparing samples for NGS will greatly help to reduce any issues in the sequencing pipeline. If your samples do not meet these purity requirements, be sure to use a DNA clean up kit to get high-quality nucleic acid.