aLibrary preparation quality gives you more information about your samples including concentration, size distribution, profile and quality before you go for sequencing. Quality controls are based on fluorometry, qPCR and microfluidics techniques, and their use depends on the library prep type used.


A good library preparation is essential because, in NGS, very tiny and precise amounts must be loaded in the flow cell; otherwise, it leads to a failure in the sequencing run.

But don’t panic! I know quality control may sound overwhelming, but I will make it simple. So, in this article, I will give you a quick overview of quality control methods in library prep, examples of a good and a bad library, and quality control options depending on the library prep used.


Article Table of Contents

What is quality control in library preparation?

Types of quality control in library preps

Techniques used in library prep quality control

qPCR

Fluorometric techniques

Microfluidic techniques

Examples of good/bad library preps

Example of a good quality library for sequencing

Example of a bad-quality library for sequencing

Quality control based on the applications

Keywords

References


What is quality control in library preparation?

Quality control, or QC, is a step in library preparation that helps you determine if your library meets a specific set of standards needed before you can begin sequencing.

Quality control techniques use qPCR, fluorometric, or microfluidics to determine the library size distribution and concentration before sequencing. Furthermore, quality control measures can provide clues about whether the samples have possible contaminants or are degraded.

A careful QC analysis, apart from telling you about your library profile, also helps maximize your sequencing sources because it provides guidance for downstream applications. Also, QC is required for all sequencing companies to decide if your samples are suitable for sequencing.

What quality control reveals about your library preparation:

  • Library size
  • Library distribution
  • Concentration
  • Possible contamination
  • Possible degradation


how to QC library prep


Types of quality control in library preps

There are two types of quality controls in library prep. They are:

  • Quantitative control
  • Qualitative control

A quantitative control in a library prep tells you the amount of DNA in your samples. Knowing this concentration is important because when it is too low, you may incur an extra cost due to under-clustering during sequencing.

Alternatively, when your library is highly concentrated, over-clustering may occur and affects the flow cell, producing failures in the sequencing run. When this happens, you may have to resend everything.

A qualitative control in a library prep tells you about your library profile size and distribution. Quality control steps are especially important when it comes to larger DNA fragments because larger fragments have a higher chance of being sequenced more compared to smaller DNA fragments.

With larger fragments, more polymerases can be attached compared to small ones because of the long sequence.

Bad qualitative libraries are indicators of possible contaminants and artifacts from your library preparation (e.g., complementary bonded adapters).


Techniques used in library prep quality control

There are three main techniques used for quality control in library prep. Quantitative techniques rely on qPCR and fluorometric methods, while microfluidics is used for qualitative control.

Let’s take a closer look at these three library prep QC techniques.


qPCR

Quantitative PCR (qPCR) is a polymerase chain reaction used to amplify and quantify genomic DNA and cDNA fragments.

With qPCR, you can monitor DNA amplification in real-time. This real-time process ends with the measure of the DNA concentration.

When evaluating your library prep, you can use qPCR because the adapters attached to the DNA fragments already contain primers that can be used in qPCR. At the same time, this specificity allows you to quantify only de adapter-ligated DNA fragments.

qPCR tells you if the library size is correct and also tells you the concentration because it only measures templates with both adapter sequences.

Furthermore qPCR is a very sensitive method to measure DNA compared to other techniques.Check our next tip to differentiate when you have a good or bad library prep.

In PCR-free library kits, the DNA concentration is too small to be quantified using other methods such as fluorometric, so qPCR allows you to amplify and quantify specifically PCR-free libraries.


Fluorometric techniques

A fluorometric technique detects fluorescence from intercalating dyes (e.g., PicoGreenTM) that bind specifically to double-stranded DNA molecules. Fluorometric methods are suitable when the library concentration is between 10 pg/µL to 100 ng/µL (Bruijns et al. 2022).

It is essential to highlight that fluorometric methods are unspecific, meaning dyes may bind to any dsDNA molecules, including residual primer dimers or incomplete library DNA fragments.

Consequently, a fluorometric system does not provide information about the purity of your samples, it only tells you about the concentration of your DNA.


Microfluidic techniques

The microfluidic technique is an electrophoresis system used to check a library qualitatively in terms of library size and distribution.

Library size is important because it allows the sequencers to focus only on the DNA fragments most amenable to producing the longest reads. In this point you don’t want to sequence very short DNA fragments that will create dificculties for downstream analysis (e.g. they are really hard to assemble).

This technique can also be used for quantification but only for narrow-size distributed libraries such as those used in ligation-based RNA library prep kits because the equipment has low accuracy with broad-size library distributions.

This is the preferred technique to evaluate the quality of samples because it tells you in an accurate manner the concentration and quality of the samples.

Bioanalyzer ®, TapeStation ®, and Fragment Analyzer® are microfluidic systems. Some differences between these devices relate to the number of samples they can process simultaneously.

Examples of good/bad library preps

When it comes to quality control of your library, it is important to differentiate a good sample from a bad sample and decide if you should proceed with sequencing. No matter if you go for qPCR, or fluorescence, sequencing centers generally perform a quality control before running the sequences using microfluidics. This is why good or bad library samples are evaluated and compared only with microfluidics.

A good sample is not only going to have a good amount, but it also depends on how the DNA looks (qualitative control).


Example of a good quality library for sequencing

Figure 1 shows the lower and upper standard markers in a microfluidic system. On the X axis, you see the library size in base pairs (bp); on the Y axis, you see the fluorescence units (FU).

A good library prep should have a distribution between these two markers and should not exceed the maximum of these two markers.

Furthermore, your library should have a normal distribution to give the same chance for all types of DNA fragment sizes to be sequenced (figure 1A).

In the case of RNA libraries, the average size should be in the expected insert. For instance, our figure is around 300 bp (figure 1B).

benchmarks for library prep quality control

Figure 1. Example of a good quality library for sequencing. A) For DNA. B) For RNA.


Example of a bad-quality library for sequencing

Figure 2 shows three cases of bad-quality library prep.

  • The first shows an overloaded library; you can see the library exceeds the lower and upper markers (figure 2A).
  • The library is too small in the second figure - its distribution is very low between the markers (figure 2B).
  • In the third figure, you can see the library insert size should be 300 bp but in this case the average is below the 188 bp range (figure 2C).


examples of bad quality library preps

Figure 2.

Example of a bad-quality library for sequencing. A) Overloaded library. B) Low-concentrated library. C) Skewed library size distribution.


Quality control based on the applications

You can’t always perform both quality or quantitative controls for a library. It depends on the prepared library and the NGS application.

Based on the applications, we have a table below of when to use either qualitative or quantitative controls for library preps.

Table 1. QC type selection table

NGS application

Library prep kit

Quantitative control

Qualitative control

qPCR

Qubit

Bioanalyzer

WGS

TruSeqTM PCR free DNA library prep

X

X

WGS

TruSeqTM Nano DNA library prep

X

WGS

Nextera® DNA library prep

X

X

WGS

Nextera® XT DNA library prep

X

RNA

TruSeqTM Stranded total RNA library prep

X

X

RNA

TruSeqTM Stranded mRNA library prep

X

X

RNA

TruSeqTM small RNA library prep

X

Methylation

TruSeqTM DNA methylation library prep

X

X

X

Exome seq

Nextera® Rapid capture exome library prep

X

X

X

Exome seq

Nextera® Rapid capture expanded exome library prep

X

X

X


Keywords

Library prep quality control, library prep, library preparation, qPCR, fluorometrics, microfluidics.




References

Bruijns, B., Hoekema, T., Oomens, L., Tiggelaar, R., & Gardeniers, H. (2022). Performance of Spectrophotometric and Fluorometric DNA Quantification Methods. Analytica, 3(3), 371-384. https://doi.org/10.3390/analytica3030025

Hess, J. F., Kohl, T. A., Kotrová, M., Rönsch, K., Paprotka, T., Mohr, V., Hutzenlaub, T., Brüggemann, M., Zengerle, R., Niemann, S., & Paust, N. (2020). Library preparation for next generation sequencing: A review of automation strategies. Biotechnology Advances, 41, 107537. https://doi.org/10.1016/j.biotechadv.2020.107537

Illumina. (2021). Library quantification and quality control reference guide. Available in: https://support.illumina.com/bulletins/2016/05/lib...

Nietsch, R., Haas, J., Lai, A., Oehler, D., Mester, S., Frese, K. S., Sedaghat-Hamedani, F., Kayvanpour, E., Keller, A., & Meder, B. (2016). The Role of Quality Control in Targeted Next-generation Sequencing Library Preparation. Genomics, Proteomics & Bioinformatics, 14(4), 200-206. https://doi.org/10.1016/j.gpb.2016.04.007

Vashishtha, K., Gaud, C., Andrews, S., & Krueger, C. (2022). Librarian: A quality control tool to analyse sequencing library compositions. F1000Research, 11, 1122. https://doi.org/10.12688/f1000research.125325.1