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Next-Generation Sequencing

This page will discuss the uses of Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS) is a combination of high-throughput technologies that can determine a sequence of DNA or RNA. In doing so, it can be used to benefit progress in the fight against cancer where appropriate, as targeted therapies can be used to fight certain types of tumours. This method of sequencing can also be used to identify unique cancer mutations and detect individuals whose genetics result in a predisposition to getting cancer.

NGS is capable of sequencing millions of fragments at the same time, more accurately whilst ensuring that expenses are still cheaper than traditional sequencing methodologies.

The process is separated into four main stages demonstrated below.

1 - Sample Preparation – Nucleic acids, both DNA and RNA are extracted from the target samples for analysis, typically from bone marrow, blood or mouth/nose swabs. These samples then undergo quality checking to ensure they will be appropriate for sequencing, which is done in a variety of ways such as spectrophotometry, fluorometry or gel electrophoresis. Some library prep kits may include reverse transcriptase that synthesises cDNA from RNA which must be carried out at this stage.

2 - Library Preparation – This is to ensure the samples are in a sequencer compatible form and commonly encompasses fragmenting DNA or cDNA. This fragmentation is often carried out by enzymatic treatment or sonication and once complete, the addition of adapters is applied to both ends of the sequence. These adaptors are smaller DNA fragments that have been ligated allowing for the sequencer to carry out ‘multiplex sequencing’ on individual sequences from multiple samples, thus enabling many libraries to be pooled and sequenced.

From this, size selection is performed with a variety of methods using magnetic beads such as purification or normalisation. Gel electrophoresis is also an option to remove fragments that are too long or short from the sequencer or chosen procedure.

Amplification is then carried out to increase the size of the library via PCR and is then followed by a clean-up step to remove undesired fragments thus improving the efficacy once in the sequencer. qPCR is then an optional step if the protocol indicates such, this acts as a form of QC checks as well as ensuring the correct dilutions have been carried out.

3 - Sequencing – Depending on the selected protocol, clonal amplification is carried out to detect a strong signal once on the sequencer. This process can be carried out either prior to sequencer loading in the form of emulsion PCR or once the sequences are loaded in the form of bridge PCR.

4 - Data Analysis – Once complete, the sequencer will direct the data generated to the desired software in preparation for the most appropriate analysis based on the objective of the organisation, business or individuals.

Nect-Generation Sequencing Diagram

The prospects of NGS are tremendously positive, with a predicted 60 million individuals expected to have their genome sequenced in the next 3 years as well as its imminent role in artificial intelligence (AI) and machine learning (ML). AI and ML results in the building of statistical models that utilise billions and trillions of data points, thus being able to predict the impact of a treatment using clinical factors as input features. These forms of deep learning algorithms are exceptionally innovative, yet they still demonstrate success in a variety of sequencing applications and could be critical in advancing NGS from bench to bedside.

For further information about the future of NGS, below are some thought-provoking journals and papers which provide a deeper investigation into these novel techniques that utilise AI and ML.

An Overview of AI:

Cloud compatibility and AI in NGS:

AI in Precision Oncology:

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