How Next-Generation Sequencing Works & How Our Services Save Time & Money

into digital information (0, 1) on a semiconductor chip, similar to the one you might find in your digital camera. Some systems essentially act as the world’s smallest solid-state pH meter to determine DNA sequences.

The DNA is fragmented, attached to beads, and deposited in millions of wells across the surface of the chip. The wells are then sequentially flooded with one nucleotide after another.

If a nucleotide is incorporated into the strand of bead-bound DNA, a hydrogen ion is given off, a chemical change is measured by an ion sensor beneath the well, and a base is called.

This process takes place in millions of wells simultaneously, enabling sequencing in only a few hours.

DNA ligase is used to determine the underlying sequence of the target DNA molecule. A fluorescence-labeled di-based probe hybridizes to its complementary sequence adjacent to the primed template.

The dye-labeled probe is then joined to the primer following the addition of DNA ligase. Non-ligated probes are washed away, and the ligated probe is identified using fluorescent imaging.

Each base is effectively probed by two independent ligation reactions using two different primers.

SMRT technology, in which DNA polymerase attaches itself to a strand of DNA to be replicated, examines the individual base at the point it is attached, and then determines which of four building blocks, or nucleotides, is required to replicate that individual base.

After determining which nucleotide is required, the polymerase incorporates that nucleotide into the growing strand that is being produced.

After incorporation, the enzyme advances to the next base to be replicated and the process is then repeated.

Sequencing templates are immobilized on a proprietary flow cell surface that is designed to present the DNA in a manner that facilitates access to enzymes, while ensuring high stability of surface-bound template and low non-specific binding of fluorescently labeled nucleotides.

Solid-phase amplification creates up to 1,000 identical copies of each single template molecule in close proximity, generating a cluster, and because this process does not involve the positioning of beads into wells or mechanical spotting, much higher densities are achieved.

SBS technology uses four fluorescence-labeled nucleotides to sequence the tens of millions of clusters on the flow cell surface in parallel, using a proprietary reversible terminator-based method.

This enables the detection of single bases as they are incorporated into growing DNA strands. Since all four reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias.

The result is base-by-base sequencing that enables highly accurate data for a broad range of applications.

Nanopore technology is an exciting new method currently being developed.

The technology records characteristic changes in electric current as nucleic acids pass through a synthetic or protein nanopore and will theoretically allow sequencing of a complete chromosome in one step, without the need to generate a new DNA strand.

NGS can be compared to a number of existing sequencing technologies, such as microarrays, quantitative PCR (qPCR), and Sanger sequencing. However, NGS and Sanger sequencing are often compared the most. While both are similar in principle, the main distinction between them is each method’s sequencing volume.

Both techniques use DNA polymerase to add fluorescent nucleotides one by one onto a growing DNA template strand in order to identify incorporated nucleotide with a fluorescent tag. However, Sanger sequencing, considered by some to be the golden standard of DNA sequencing technology, can only sequence a single DNA fragment at a time. On the other hand, NGS can can sequence millions of segments per run.

Today, NGS is often paired with Sanger sequencing. It is often used to verify NGS results because of its high levels of accuracy. Both Sanger and next-generation sequencing methods are used in genetic analysis in many of today’s labs, as the two methods can actually support one another, producing more accurate and reliable results.

While NGS is considered an established analysis technology for research applications across the life sciences, analysis workflows still require substantial bioinformatics expertise.

Bioinformatics, in the context of genomics and molecular pathology, uses computational, mathematical, and statistical tools to collect, organize, and analyze large and complex genetic sequencing data and related biological data.

Some common challenges in setting up large-scale analysis workflows include the appropriate selection of analytical software tools, the speed-up of the overall procedure using HPC parallelization and acceleration technology, the development of automation strategies, data storage solutions, and the development of methods for full exploitation of the analysis results across multiple experimental conditions.

More recently, NGS has begun to expand into clinical environments, where it assists diagnostics, enabling personalized therapeutic approaches. One of the most common NGS assays for cancer patients is targeted panel sequencing, which typically interrogates hundreds of targeted genes.

Sequencers produce vast amounts of quantitative and complex sequencing data that many clinical laboratories rely on to identify genetic alterations of clinical relevance. Creating a resource-intensive data processing pipeline to analyze all the NGS data generated has become necessary in today’s clinical environments.

The Bioinformatics CRO, in parntership with Excedr, offer end-to-end bioinformatics services for labs of all sizes. If you’re looking to lease a next-generation sequencer and foresee the need for a bioinformatics team, look no further. From experimental design through to final analysis, The Bioinformatics CRO can help.