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

• Sample extraction. The NGS process starts with the extraction of nucleic acids that will be used for sequencing (i.e. DNA, total RNA, mRNA or chromatin). Depending on the purpose of the experiment, the genetic material can be extracted from a variety of biological samples including blood, cultured cells, biopsies, tissue sections, and urine, as well as microorganisms or plants specimens. The quality and the quantity of the extracted samples is fundamental for a successful NGS. Therefore, care must be taken to choose an appropriate extraction method and establish quality control parameters before proceeding with the next steps.
• Library preparation. At this point, a series of processing steps are necessary to generate a library or, in other words, to convert the extracted samples into the appropriate format for the chosen sequencing technology. DNA (or cDNA) samples are fragmented in short, uniform pieces of dsDNA by physical shearing or enzyme digestion. The 5’ and 3’ ends of the resulting DNA fragments are then ligated to technology-specific adaptor sequences, forming a fragment library. The adaptors may also include a barcode, which is a sequence-based tag unique to that specific sample. This allows for multiple samples to be mixed together and sequenced at the same time, thereby reducing the costs. For most sequencing platforms, a clonal PCR amplification of the library is necessary. Each fragment will thus originate in a distinct cluster. The libraries are now ready for sequencing, and each cluster will act as an individual sequencing reaction.
• Sequencing. Most sequencing technologies are based on the detection of the nucleotides incorporated by the DNA polymerase and are therefore defined “sequencing by synthesis” (SBS) methods. The most common methods are known as “sequencing by reversible terminator” (used by Illumina platforms), “pyrosequencing” (used by Roche platforms), and “ion semi-conductor sequencing” (used by Ion Torrent Platforms). For more details on the topic, check out the TechNote “What is Next Generation Sequencing (NGS)?”. The sequencing generates a huge amount of complex raw data, generally handled by bioinformatics specialists. Very briefly, in a first phase the specific nucleotides present at each position in a single sequencing read are identified (base calling); the reads are then aligned on the reference genome (read alignment); finally the DNA variants are identified and annotated, allowing for the interpretation of the results (variant calling and variant annotation).

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&trade, and EPIXTRACT&trade 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:

• Fast: Depending on the sample type, the entire procedure can be completed within 2 hours.
• Effective: High efficiency of nucleic acid isolation from various sample types with low amounts of start material (as low as 1 ng).
• Reliable: Our specially designed columns allow for nucleic acids to be conveniently recovered.
• Safe: Use of non-toxic reagents and no phenol chloroform.

Product Name	Sample Input	Elution Volume	Protocol Time
AMPIXTRACT™ General Tissue Section DNA Isolation Kit	1 ng (optimal range 10 ng – 1 μg)	8 - 20 μL	2 hours
AMPIXTRACT™ Paraffin Tissue Section DNA Isolation Kit	1 ng (optimal range 10 ng – 1 μg)	8 - 20 μL	2 hours
AMPIXTRACT™ Urine DNA Isolation Kit	1 ng – 2 μg (optimal range 10 ng – 1 μg)	8 - 20 μL	20 minutes
AMPIXTRACT™ Blood and Cultured Cell DNA Extraction Kit	1 ng – 4 μg (optimal range 10 ng – 1 μg)	10 - 20 μL	20 minutes
EPIXTRACT® DNA Isolation Kit for Plasma/Serum	0.1 ng (optimal range 1 ng – 1 μg)	8 - 20 μL	15 minutes

Table 1: AMPIXTRACT™ and EPIXTRACT™ 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).

Product Name	DNA		Bisulfite-Seq			Post Bisulfite-Seq	ChIP-Seq
	AMPINEXT™ DNA Library Preparation Kit	AMPINEXT™ High-Sensitivity DNA Library Preparation Kit	AMPINEXT™ High-Sensitivity Bisulfite-Seq Kit	AMPINEXT™ RNA Bisulfite-Seq Kit	AMPINEXT™ 5-mC RNA Bisulfite-Seq Easy Kit	AMPINEXT™ Post-Bisulfite DNA Library Preparation Kit	AMPINEXT™ ChIP-Seq High-Sensitivity Kit
Input Type	DNA	DNA	DNA	RNA	RNA	DNA	Cells	Tissues
Input Range	5 ng – 1 μg	0.2 ng – 100 ng	0.2 ng – 200 ng	5 ng – 1 μg	5 ng – 500 ng	0.5 ng – 1 μg	105 – 106 Cells	5 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 hours	6 hours		5 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!

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