The Sanger method of DNA sequencing is a dideoxy-chain terminating sequencing method that has been recognized as the gold standard of DNA sequencing for nearly 40 years. Sanger DNA sequencing matured from a manual method to an automated one through the 1980s as PCR amplification was in its early beginnings, di-deoxynucleotides (ddNTPS) were modified without a 3’ hydroxyl group (“-OH) labeled plus fluorescent dye-terminator reagents for single-reaction sequencing rather than the usual four separate PCR reactions and the introduction of sequencers with automated capillaries. Automated Sanger DNA sequencing enabled researchers to input 10 to 50 ng of input DNA to produce single reads of 700 -1000 bases of very accurate sequence data. This method was used to carry out the Human Genome Project from 1990 to 2004; a science research initiative that led to deciphering the whole genome sequence after several million dollars and ushering the first generation technology. The next initiatives began to focus on miniaturization of volumes required for upscaling sequencing reactions and undergoing massive parallelization of sequencing reactions simultaneously to produce “higher-throughput” at a cheaper cost on time and money. This sparked the genesis of the second generation sequencing technologies that are referred to as “Next-Generation”.

Next-generation sequencing (NGS) in broad terms refers to the massively parallel, high-throughput sequencing technologies for DNA, RNA and methylation sequencing that have evolved from traditional fragment-based Sanger DNA sequencing technologies. It is “massively parallel” due to an approach termed “shotgun” due to its inherent random scattershot approach which reads 100s of millions of reads that number in millions to billions across days to weeks. NGS methods have been continually developing as a field providing researchers with larger-scales of genomic sequences available to analyze than ever previously known. NGS technologies are able to sequence genomes with millions to billions of base pairs across days to weeks versus the first generation technology which took 13 years to sequence an estimated 3.3 billion base pair human genome.

Second generation NGS technologies or (short-read sequencing) are characterized by several advantages when compared to the first generation Sanger technology. First, end-users are able to shotgun sequence random fragment genomic DNA or cDNA (reverse transcribed) without the need for cloning via a foreign host cell through the preparation of DNA libraries. DNA libraries are a collection of DNA fragments that are stored and propagated in a population of micro-organism for the process of cloning. Secondly, the use of a linker and/or adapter sequences ligated to the fragmented DNA or cDNA and a clonal amplification step that is performed on a solid surface or on beads within miniature emulsion droplets allows for thousands to billions of sequencing reactions to be able to occur in parallel versus hundreds from an automated Sanger method. Lastly, unlike the traditional Sanger method, nucleotide incorporation is detected without the need for capillary gel electrophoresis through capturing luminescence or changes in electrical charge directly during the sequencing reaction. NGS workflows from second generation technologies have three components – library preparation, template preparation and sequencing. The differences between different NGS platforms lie mainly in the technical details of the sequencing reaction.

Pyrosequencing

Pyrosequencing is a sequencing approach that use non-electrophoretic, bioluminescence method that measures the release of inorganic pyrophosphate by proportionally converting it into visible light through a cascade of enzymatic reactions. The enzyme detects the presence of diphosphate (PPi) by combining free PPi with adenosine-5’-phosphosulfate in the presence of ATP sulfyrase enzyme to produce ATP. This ATP serves as fuel for the enzyme luciferase to covert luciferin into oxyluciferin which in the process produces visible light which can be captured by a CCD-based imaging assembly in the sequencing instrument.

Sequencing by ligation

Sequencing by ligation is a DNA sequencing method that uses the enzyme DNA ligase instead of DNA polymerase to identify the nucleotide present in a DNA strand. After template preparation via emulsion PCR, a ligase will discriminate between perfectly matched fluorescently-labeled probes and the non-perfectly matched ones. During ligation, a probe is attached to a primed strand that existed on an emulsified, amplified bead and then imaged. The ligation occurs on the opposite DNA orientation (3’ to 5’) than what a polymerase will do (5’ to 3’). After the imaging, the fluorophore is cleaved and the cleaved end is prepared for another round of ligation synthesis.

Sequencing by reversible terminator chemistry

Sequencing by reversible terminator chemistry is a sequencing approach that relies on solid-phase amplification of DNA templates immobilized to a solid surface (usually a flow cells) separated across it to generate clusters on the order of millions. In this method, this is achieved when the DNA library that is constructed is adaptor-flanked to immobilize it to the flow cell and then template amplification is performed via Bridge PCR. Bridge PCR refers to the fact that during the annealing step, the extension product from one primer forms a bridge to the other bound primer and its complementary strand is produced. This sequencing techniques uses reversible terminators bound to dNTPs in a cyclical method that comprises of nucleotide incorporation, fluorescence imaging and cleavage. A fluorescently-labeled terminator is imaged as a dNTP is added and then cleaved to allow incorporation of the next base. An imaging step follows each base incorporation step – so after a single base is added, the entire flow cell is imaged, using CCD camera setup to discriminate different spectral characteristics to discriminate each of the four color dyes for the respective base. During the cleavage step, the blocked group is chemically removed which sets the strand up for the next nucleotide to incorporate along the strand by DNA polymerase. These steps usually repeat for a set number of cycles defined by the end-user’s settings.

Enzo’s AMPINEXT™ DNA Library Preparation Kit (Illumina) is a complete set of optimized reagents suitable for DNA library preparation using Illumina’s sequencing platforms. Using this kit, our end-users have all the required components for each step of DNA library preparation, which are sufficient for end repair, dA tailing, ligation, clean up, size selection and library amplification (Figure 1). The procedure has been optimized to take an end-user from fragmented DNA to size selection in less than 2 and a half hours. Furthermore, this kit is able to be used with both nonbarcoded (singleplexed) and barcoded (multiplexed) DNA library preparation. This kit has reported a broad range of input DNA from 5 ng to 1 ug. For a PCR-free library prep, it can be performed with 500 ng or greater of DNA.

Figure 1: General workflow for the AMPINEXT™ DNA library prep kit.

The AMPIXTRACT™ General Tissue Section DNA Isolation Kit provides the essential components to be able to efficiently isolate DNA in any targeted microscopic tissue area on a slide. We’ve provided our end-users with a unique protocol that involves the removal of a tissue section, followed by treatment in our proprietary DNA digestion buffer and recovered through an Enzo designed Fast-Spin column to elute DNA of interest (Figure 2). This kit allows isolation of DNA sizes from 50bp to 20kb and produces highly efficient isolations from samples with as little as 1ng of DNA. Compared to other extraction methods for NGS samples, our method provides our end-users with a solution as quickly as 2 hours reducing the overall time investment for this portion of their NGS workflow. This can be used for all molecular biology applications such as PCR, restriction digestions, and cloning, in addition to sequencing.

Figure 2: Visual schematic of the procedure for AMPIXTRACT™ General Tissue DNA Isolation Kit.

Enzo is a recognized pioneer and innovator of life sciences tools, backed by patented DNA and RNA labeling chemistries for genomics research and development. We now provide our end-users with comprehensive NGS solutions for sample extraction and library preparation. Please check out our Next-Generation Sequencing and Genomics portfolios for more information and our Successful Research Tips. Additionally, contact our Technical Support Team for further assistance.

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