May 02, 2018

Next-generation sequencing: 3 main challenges (& solutions!) in NGS sample preparation

By Cytiva

Sample prep can have a big impact on next-generation sequencing (NGS) outcomes. Here are some simple things you can do to get your NGS off to a good start.

How to address common challenges in next-generation sequencing sample preparation

Getting reliable data in next-generation sequencing (NGS) is all about the DNA (or RNA) you put in. How can you make sure your input DNA gives you the quality sequencing results you need?

DNA for sequencing might come from a variety of sources, including fresh tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cultured cells, and liquid biopsies. Each source comes with its own challenges for maximizing the three key aspects:

  • Quantity
  • Integrity
  • Purity

Challenge #1: Yield

Different workflows and kits vary significantly in the amount of starting material required. Your workflow might require you to use a specific type of kit, and therefore starting DNA, or vice versa.

It’s important to understand which workflows and kits suit your application and the typical amounts of starting material they need. If the two don’t match up, can you try another approach?

If your sample is insufficient, what can we learn from those studying at the single-cell level? Commercially available whole genome amplification (WGA) kits provide the opportunity to expand your starting material from nanograms to micrograms in a matter of hours. This technique provides improved coverage compared to PCR-based amplification and is associated with fewer amplification errors.

Your fragmentation method can also affect your final DNA yield. Physical fragmentation can result in unexpectedly small DNA fragments which can be lost, reducing the amount of DNA available for sequencing. If you have the option, enzymatic fragmentation can provide better predictability and control over fragmentation.

Challenge #2: Integrity

Having enough DNA won’t make for accurate sequencing if your DNA is degraded. Degradation can affect all kinds of samples, but long-term storage and exposure to fixatives, as you might find in FFPE samples, can exacerbate the damage.

A DNA integrity number (DIN) measurement can indicate the level of DNA damage. Although not a perfect predictor of usability, or parameters such as library complexity, DIN measurement is an easy method to check DNA integrity.

Extracting a little more DNA can compensate for low quality to some extent. However, there’s not much you can do about previous storage conditions, unless you can choose newer samples or those that haven’t gone through such harsh processing.

If you can’t acquire better samples, DNA repair might improve your outcomes. Several commercial kits can, for example, modify blocked 3’ ends or fix DNA nicks. These simple repairs help make more fragments suitable for sequencing.

Challenge #3: Impurities

Okay, so you have enough DNA, and it’s in pretty good condition. What else do you have in that tube?

Producing reliable results in sequencing requires samples free of proteins, organic solvents and surfactants. You might also have tissue-specific contaminants to consider.

Researchers often measure DNA purity by looking at the 260:280 nm absorbance ratio. A high-purity sample should have a 260:280 ratio of 1.8 to 2.0. Nucleic acids have an absorbance maximum at 260 nm and finding a ratio below 1.8 can indicate contamination.

As a secondary check, measure the 260:230 ratio, which will detect the presence of commonly used solvents and surfactants, such as phenol and EDTA. Values between 2.0 and 2.2 indicate high purity.

  • Remove hemoglobin by preferential lysis of red blood cells early in your workflow.
  • Remove heparin by washing.
  • Do a phenol–chloroform extraction to reduce protein contamination.
  • Use a phenol-free extraction kit to remove phenol contamination.

In brief…

Possible solutions
Low yield
  • Check whether other workflows/kits are suitable
  • Use whole genome amplification (using MDA kit)
  • Use enzyme-based fragmentation
Low integrity
  • Measure DNA integrity for all samples
  • Use more DNA
  • Use fresh or newer (FFPE) samples
  • Carry out a DNA repair step
Low purity
  • Measure 260:280 ratio (should be 1.8 to 2.0)
  • Check strategies for tissue-specific contaminants
  • Remove proteins by phenol–chloroform extraction
  • Use phenol-free extraction

At Cytiva, we provide a range of kits, tools, and resources to help you improve your NGS outcomes.

Find out more about our NGS products or contact us for more information.

Learn how to support your sequencing workflows here