Next Generation Sequencing (NGS) is a powerful and accessible high-throughput tool to gain insight into potentially any species at the genomic, transcriptomic or epigenetic level. Since the introduction of the groundbreaking Sanger method in 1977, fast advancements in sequencing technologies have permitted a substantial enhancement in the throughput capabilities which has been accompanied by a drop in cost. The possible applications in research (e.g. metagenomics, evolutionary studies, population genetics), medicine (e.g. studies on the genetic diseases, diagnostics, prognostics), and biotech fields (e.g. food safety, agriculture and farming improvement) are extremely broad. Based on the specific needs, it is possible to choose among multiple sequencing platforms, each one of them using a typical technology, thus resulting in different sequencing capabilities and read lengths. However, regardless of the application or even the selected sequencing technology, the NGS workflow is characterized by three key phases: sample extraction, library preparation, and sequencing/analysis (Figure 1).


The NGS Workflow

Figure 1: NGS Workflow.

The Importance of Quality Controls in Sample Preparation

The preparation of libraries is certainly the most critical step in the NGS workflow, since the final result is strictly dependent on their quality. Therefore, high-quality, purified, nucleic acids should be used as starting material as they directly affect the efficiency of the subsequent enzymatic reactions leading to the library construction. In fact, the presence of contaminants, the concentration of the samples or the integrity of the input material, are all factors that have an impact on the quality of the library and need to be evaluated.

Contaminants often act as inhibitors of enzymatic reactions by blocking or degrading the template sample, competing with the ions in solution, or even denaturing the enzymes. Some contaminants are inherent to the sample type, such as polysaccharide complexes or tannic acids in plants, hemoglobin in blood, or urea in urine samples. The extraction procedure itself can also introduce some impurities. Typical examples are the reagents commonly used in “manual” extraction protocols, such as chaotropic salts (e.g. guanidinium chloride), alcohols (e.g. ethanol, isopropanol), salts, or phenol: chloroform. For this reason, filter-based column purification systems are generally preferred, as they reduce the risk of carryovers from the extraction reagents or the biological samples themselves.

Regardless of the starting material and the chosen purification method, purity assessment is crucial before proceeding with library preparation. A very common method is the measurement by UV spectrophotometry of the absorbance ratios at 260 vs 280nm and at 260 vs 230nm. The A260/280 ratio, used to assess protein contamination, should be ~1.8 for DNA and ~2.0 for RNA preparations. The A260/230 ratio is related to the possible presence of contaminants absorbing at 230nm, such as organic compounds (i.e. phenol) or chaotropic salts. This value should be in the range of 2.0–2.2. In case of significant deviation from these ratios, it might be necessary to re-purify the samples.

Other typical approaches to QC the starting material are the fluorometric (e.g. Qubit assay) and the automated capillary electrophoresis (e.g. Bioanalyzer) methods. The fluorometric system is particularly useful for an accurate determination of the concentration, as it quantifies only double-stranded DNA, thus avoiding the risk of over estimating the amount of starting material. The capillary electrophoresis provides robust information concerning the integrity (i.e. size) of the nucleic acids. Once the QC parameters have been verified, it is possible to proceed safely with the library preparation.


Enzo’s Solutions

Enzo Life Sciences, a pioneer in labeling and detection technologies, offer comprehensive mix of kits for nucleic acid extraction and preparing library samples for NGS analysis. Our AMPIXTRACT™, and EPIXTRACT™ nucleic acid extraction kits provide the essential components for rapid isolation of pure genomic DNA from different sample types (see Table 1). Our nucleic acid extraction solutions are:

Product NameSample InputElution VolumeProtocol Time
AMPIXTRACT® General Tissue Section DNA Isolation Kit1 ng (optimal range 10 ng – 1 μg)8 – 20 μL2 hours
AMPIXTRACT™ Paraffin Tissue Section DNA Isolation Kit1 ng (optimal range 10 ng – 1 μg)8 – 20 μL20 minutes
AMPIXTRACT® Urine DNA Isolation Kit1 ng – 2 μg (optimal range 10 ng – 1 μg)8 – 20 μL
AMPIXTRACT® Blood and Cultured Cell DNA Extraction Kit1 ng – 4 μg (optimal range 10 ng – 1 μg)10 – 20 μL20 minutes
AMPIXTRACT® DNA Isolation Kit for Plasma/Serum0.1 ng (optimal range 1 ng – 1 μg)8 – 20 μL15 minutes
Table 1: AMPIXTRACT™ and EPIXTRACT™ nucleic acid extraction kits.
Figure 2: Schematic procedure for using the AMPIXTRACT™ and EPIXTRAC™ nucleic acid extraction kits.

Your purified nucleic acid samples are now ready for Enzo’s AMPINEXT™ DNA Library Preparation Kits, a complete set of optimized reagents suitable for DNA library preparation using Illumina’s sequencing platforms (Table 2).

DNADNABisulfite-SeqBisulfite-SeqBisulfite-SeqPost Bisulfite-SeqChIP-SeqChIP-Seq
Product NameAMPINEXT™ DNA Library Preparation KitAMPINEXT™ High-Sensitivity DNA Library Preparation KitAMPINEXT™ High-Sensitivity Bisulfite-Seq KitAMPINEXT™ RNA Bisulfite-Seq KitAMPINEXT™ 5-mC RNA Bisulfite-Seq Easy KitAMPINEXT™ Post-Bisulfite DNA Library Preparation KitAMPINEXT™ ChIP-Seq High-Sensitivity KitAMPINEXT™ ChIP-Seq High-Sensitivity Kit
Input TypeDNADNADNARNARNADNACellsTissues
Input Range5 ng – 1 μg0.2 ng – 100 ng0.2 ng – 200 ng5 ng – 1 μg5 ng – 500 ng0.5 ng – 1 μg105 – 106 Cells5 mg – 50 mg
(Optimal Range)(100 ng – 200 ng)(10 ng – 50 ng)(10 ng – 50 ng)(200 ng – 500 ng)(100 ng – 200 ng)(100 ng – 200 ng)(4×105 – 5×105 Cells)20 mg – 30 mg)
Protocol Time< 2.5 hours< 1.5 hours< 6.5 hours6 hours6 hours5 hours< 7 hours< 7 hours
Table 2: AMPINEXT™ library preparation kits.

Do you have more questions on NGS workflow? Do you need advice to set up your experiment? Want to learn more about our NGS portfolio? Check out our Next-Generation Sequencing and Genomics portfolios and reach out to our Technical Support Team. We will be happy to assist!

Leave a comment