Online Purchasing Account You are logged on as Guest. LoginRegister a New AccountShopping cart (Empty)
United States 

NGS Workflow and Fundamentals of Sample Preparation

Rosaria Esposito
Tags: Molecular Biology

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).


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&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.




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).



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!

Never miss a new TechNote!

Receive our TechNotes as soon as they are published.


Follow Us!

 

Did you enjoy the reading?
Share it on your favorite social media

 

Immunoassay E-book

  • What are the Differences Between ELISA Assay Types?
  • 10 Tips for Successful ELISA Assays
  • In-depth Insights on ELISA:
    • %CV in ELISA: How to reduce them and why they’re important
    • Dilutional linearity, parallelism, spike-and-recovery in ELISA: How to QC your results?
    • How to validate sample dilutions and achieve linearity in new sample types

comments powered by Disqus

Related Products

AMPIXTRACT™ General Tissue Section DNA Isolation Kit 

DNA Isolation Kit is designed for isolating DNA from tissue sections.
Print as PDF
 
ENZ-GEN500-0050 50 Reactions 285.00 USD
 
ENZ-GEN500-0100 100 Reactions 455.00 USD
Do you need bulk/larger quantities?
 

AMPIXTRACT™ Paraffin Tissue Section DNA Isolation Kit 

Efficiently isolate DNA from paraffin archives
Print as PDF
 
ENZ-GEN501-0050 50 Reactions 251.00 USD
 
ENZ-GEN501-0100 100 Reactions 436.00 USD
Do you need bulk/larger quantities?
 

AMPIXTRACT™ Urine DNA Isolation Kit 

Isolate DNA from urine
Print as PDF
 
ENZ-GEN502-0050 50 Reactions 189.00 USD
 
ENZ-GEN502-0100 100 Reactions 290.00 USD
Do you need bulk/larger quantities?
 

AMPIXTRACT™ Blood and Cultured Cell DNA Extraction Kit 

Suitable for isolating DNA from blood leukocytes and cultured mammalian cells
Print as PDF
 
ENZ-GEN503-0050 50 Reactions 181.00 USD
 
ENZ-GEN503-0100 100 Reactions 287.00 USD
Do you need bulk/larger quantities?
 

EPIXTRACT® DNA Isolation Kit for Plasma/Serum 

Fastest procedure available to safely isolate DNA from plasma/serum.
Print as PDF
 
ENZ-45018-0050 50 Reactions 176.00 USD
 
ENZ-45018-0100 100 Reactions 254.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ DNA Library Preparation Kit (Illumina) 

Complete set of optimized reagents to carry out a successful DNA library preparation.
Print as PDF
 
ENZ-GEN504-0012 12 Reactions 525.00 USD
 
ENZ-GEN504-0024 24 Reactions 889.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ High-Sensitivity DNA Library Preparation Kit (Illumina) 

A complete set of optimized reagents to prepare a DNA library from very small amount of samples for use in next-generation sequencing applications.
Print as PDF
 
ENZ-GEN505-0012 12 Reactions 525.00 USD
 
ENZ-GEN505-0024 24 Reactions 889.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ High-Sensitivity Bisulfite-Seq Kit (Illumina) 

Designed to bisulfite-convert DNA and prepare an Illumina-based library for bisulfite sequencing, all in one kit
Print as PDF
 
ENZ-GEN508-0012 12 Reactions 642.00 USD
 
ENZ-GEN508-0024 24 Reactions 1,152.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ RNA Bisulfite-Seq Kit (Illumina) 

A complete set of optimized reagents designed to carry out RNA bisulfite conversion, followed by a "post-bisulfite" library preparation process for Illumina platform-based bisulfite sequencing, all in one kit.
Print as PDF
 
ENZ-GEN510-0012 12 Reactions 598.00 USD
 
ENZ-GEN510-0024 24 Reactions 1,152.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ 5-mC RNA Bisulfite-Seq Easy Kit (Illumina) 

A complete set of optimized reagents designed for easily carrying out RNA bisulfite conversion, followed by a "post-bisulfite" library preparation process for Illumina platform-based bisulfite sequencing, all in one kit.
Print as PDF
 
ENZ-GEN511-0012 12 Reactions 598.00 USD
 
ENZ-GEN511-0024 24 Reactions 1,152.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ Post-Bisulfite DNA Library Preparation Kit (Illumina) 

A complete set of optimized reagents to prepare a DNA library, after successful bisulfite conversion, for various Illumina platform-based bisulfite sequencing (bisulfite-seq) assays.
Print as PDF
 
ENZ-GEN512-0012 12 Reactions 571.00 USD
 
ENZ-GEN512-0024 24 Reactions 930.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ ChIP-Seq High-Sensitivity Kit (Illumina) 

A complete set of reagents required for carrying out a successful ChIP-Seq starting from mammalian cells or tissues.
Print as PDF
 
ENZ-GEN509-0012 12 Reactions 660.00 USD
 
ENZ-GEN509-0024 24 Reactions 1,152.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ DNA Size Selection Kit 

A complete set of optimized reagents for quick removal of DNA fragments of <150 bps for library preparation in next generation sequencing applications.
Print as PDF
 
ENZ-GEN506-0048 48 Reactions 155.00 USD
 
ENZ-GEN506-0096 96 Reactions 230.00 USD
Do you need bulk/larger quantities?
 

AMPINEXT™ NGS Barcode (Index) Set-12 

Designed to construct multiplex DNA/RNA libraries used for next-generation sequencing with the Illumina platform
Print as PDF
 
ENZ-GEN507-0144 144 Reactions 155.00 USD
Do you need bulk/larger quantities?
 

Recommend this page

 
Keep in touch

©2020 Enzo Life Sciences, Inc.,