Five Steps to NGS Success!

blog / Molecular Biology September 26 2018

With the falling cost of NGS services (1) and a wide array of NGS kits and instruments to choose from nowadays, it may seem as though NGS data has never been easier to access. However, as with most other research workflows, obtaining high quality data is never going to be more than a pipe dream without high quality starting material!

In this article, we bring you our top tips for sample quality assessment and handling prior to NGS library preparation to help you on your way to NGS success!

1. Assess Nucleic Acid Quality

Whether you use a manual or automated nucleic acid extraction method, you should always perform QC checks for purity and integrity for every nucleic acid sample prior to downstream processing. Starting material for NGS library construction could be any type of double-stranded (ds)DNA or material that can be converted into dsDNA. Common examples include: genomic DNA (gDNA), PCR products, and RNA.

Sample Purity

Purity can be assessed after nucleic acid extraction using spectrophotometry. The relative abundance of several compounds, proteins and organic solvents can be assessed using the A260/A280 ratio, which should ideally lie between 1.8 and 2.0 for DNA and around 2.0 for RNA. Contamination by salts, polysaccharides as well as several organic compounds can be assessed with the A260/A230 ratio, which should lie between 2.0 and 2.2 for both DNA and RNA.

Sample Integrity

By integrity, we mean intactness. Intact HMW DNA should appear as sharp bands in the upper portion of an agarose gel, while intact whole RNA should appear as two defined ribosomal RNA bands. Depending on the RNA isolation method used, you may also see a low molecular weight band representing the small RNA fraction.

Although good old-fashioned agarose gels are often the preferred way to visualise sample integrity, we recommend supplementing all quality control assessments with one of the many quantitative methods available. For simultaneous quantification and quality control of RNA, we recommend the Agilent BioAnalyzer. For accurate quantification of fragment size distribution of HMW DNA, we recommend either the Fragment Analyzer or FemtoPulse from Advanced Analytical.

To minimise nucleic acid degradation and to get a true molecular snapshot of your sample (e.g., for microbiomics studies), we recommend preserving your starting material (i.e. cells or equivalent) in a commercial stabilisation reagent until you are ready to purify nucleic acids.

In addition, it is recommended never to sequence DNA fragments that have been visualised and excised from an agarose gel, because UV (and ethidium bromide) compromises DNA integrity.

2. Accurately Quantify Nucleic Acids

For newcomers to the NGS world, it may come as a shock to learn that 60-80 % of a sample is typically lost between the nucleic acid extraction and size selection steps during NGS library construction. Therefore, knowing exactly how much starting material you have is critical, so that you can use the right amount in subsequent steps, and optimise isolation protocols to improve yields as necessary.

The currently recommended methods for accurate DNA and RNA quantification for NGS are outlined below. These methods can also provide quantitative details about nucleic acid purity and quantity.

For dsDNA: Qubit* is recommended to quantify dsDNA in the 10 pg/µL to 100 ng/µL concentration range.

RNA and HMW DNA (≥ 40 kb): As mentioned above, Agilent BioAnalyzer is recommended for RNA while Fragment Analyzer or FemtoPulse should be used for HMW DNA.

* Agilent BioAnalyzer and Advanced Analytical Fragment Analyzer are also very useful for non-HMW DNA.

3. Proper Handling

This may seem like common sense, but the importance of proper sample handling throughout the entire workflow cannot be overstated! Here are a few simple tips:

  • Use gloves and keep your work area clean. For RNA work, wipe down your bench with solutions that counteract RNases e.g., commercially available solutions or the homemade remedy of a 0.5 % SDS solution followed by 3 % H2O2.
  • Work with samples on ice as much as possible, and handle samples with care at all times. Avoid vigorous shaking, pipetting and vortexing to minimise nucleic acid fragmentation.
  • Wide-bore pipette tips will help to preserve the integrity of HMW DNA, and will make it easier to pipette viscous solutions, such as those containing HMW DNA.
  • Filter tips are recommended for RNA work to avoid potential contaminants passing from the pipette to the RNA.

4. Nuclease Treatment and Clean Up

Contaminants such as RNA or DNA, proteins or chemicals (e.g., organic solvents, salts and polysaccharides) in DNA or RNA samples can wreak havoc for NGS library preparation and downstream sequencing. An RNAase or DNAse treatment step is therefore highly recommended for DNA and RNA samples, respectively. Bear in mind that RNase and DNase themselves could inhibit NGS library construction, so it is also essential to remove these before downstream processing. You can achieve this with a variety of commercial clean up kits.

For those working with both DNA and RNA for NGS, we highly recommended having separate dedicated workspaces for each to avoid cross contamination.

5. Storage

For long-term storage, keep your purified nucleic acids in Tris-EDTA buffer (final concentration 1x) in aliquots at -80 °C to minimise the degradation that results from successive freeze-thaw cycles. It should be safe to store purified nucleic acid samples at -20°C for several months, but it is advisable to recheck integrity before proceeding to downstream steps. Beware that storing purified nucleic acids in water for prolonged periods of time might lead to hydrolysis!

Share Your Tips with Us!

That was it for our top tips, compiled from our in-house NGS expertise and through interactions with external experts. Please feel free to share your feedback, as well as any tips of your own by dropping a line in the comments section!

References:

1. Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) Available at: www.genome.gov/sequencingcostsdata. Accessed [03-September 2018].