The ABCs of NGS sample and library QC

An adapted version of this newsletter first appeared on the Science Matters Blog

We are probably stating the obvious here, but quality NGS results can only be achieved by assessing sample and library quality at various checkpoints throughout the NGS workflow – from incoming nucleic acid (for example, genomic DNA, cfDNA, total RNA or miRNA) QC to final library validation.

To avoid these pitfalls, one must:

Assess whether the sample material meets the recommended quality (purity and integrity) and quantity parameters  

Base the NGS library preparation step on the recommended size, quality and concentration parameters 

Consider the most effective QC method that complements A and B

Critical QC parameters for starting material

Quantity and concentration: Is there enough material to process? What sample-enzyme ratio should be used? 

Purity: Are there contaminants, such as proteins or chemicals, that could interfere with the assay? Is there unwanted nucleic acid that could lead to an overestimation of concentration?

Size and integrity: Is the nucleic acid degraded?

Critical QC parameters for NGS library

Quantity and concentration: What is the concentration/molarity of the library? 

Size or size distribution: Does the size match the respective acceptance criteria? 

Contamination: Is there contaminating gDNA or adapter–dimers leading to a loss in sequencing coverage?

Fortunately, most labs can answer these questions with innovative quantification and QC methods, such as UV-Vis spectroscopy, Qubit fluorometry or qPCR, and gel and capillary electrophoresis.

Current state of NGS sample and library QC

Automated electrophoresis systems have emerged as a cost-effective fragment separation method, providing higher sample resolution and sensitivity (low detection limit).  

When combined with other techniques in the NGS workflow, such as fluorometry, spectrophotometry or qPCR/digital PCR, these electrophoresis systems provide reliable library quantification and minimize manual handling and analysis time. 

However, different analytes (DNA or RNA) may require more than one instrument and set of consumables for sample and library QC, and researchers may be compelled to trade between analytical sensitivity, convenience and cost-effectiveness. 

This is where automated capillary electrophoresis (CE) instruments such as the QIAxcel Connect – a single instrument for QC of starting material and NGS library assessment are seen to improve the standards of NGS workflows, especially when addressing diseases related to liquid biopsies for cancer. 

Learn more about this automated CE technology in an evaluation study performed by Microba Life Sciences. The study demonstrates that QIAxcel provides higher sensitivity, resolution and precision across multiple library types and concentrations than other electrophoresis systems. Its analysis is versatile, enabling the recognition of varying library fragments, whether they have single or multiple peaks, broad smears or a combination of all. It is beneficial not only for the titration of loading concentration for maximum data yield but also for identifying undesired library fragments. 

Watch this webinar presented by two of our in-house experts to further your understanding of the ABCs of NGS sample and library QC for confident sequencing.  

That's a wrap on our June edition of Benchtop Bulletin!