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Next Generation Sequencing (NGS)

Next Gen Sequencing is an umbrella term describing a collection of high-throughput methodologies and platforms used for the determination of the sequence of base pairs in DNA or RNA.

Next Generation Sequencing (NGS) | Avantor

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What is Next-Generation Sequencing (NGS)?

Next-generation sequencing (NGS) describes several techniques used to determine the sequence of DNA or RNA. Scientists can use that sequencing data to study genetic variations or mutations associated with diseases or biological conditions.

Because NGS allows scientists and researchers to sequence thousands or even millions of DNA molecules simultaneously, it has significantly reduced the cost and the amount of time needed to sequence genes and genomes. That has led to dramatic advances in clinical diagnostics, genetic diseases and personalized medicine.

Sanger Sequencing vs. Next-Generation Sequencing

Sanger sequencing and traditional next-generation sequencing (NGS) are similar in concept, . Both techniques involve using DNA polymerase to add fluorescent nucleotides one at a time onto a growing DNA or RNA template strand. The fluorescent tags allow the identification of each successively incorporated nucleotide.

Where these two sequencing methods differ is in each method’s sequencing volume. The Sanger method sequences DNA or RNA one strand at a time, while next-generation sequencing – initially called “massively-parallel sequencing” – can simultaneously sequence hundreds to thousands of genes at one time.

More recently, new NGS methodologies have emerged. Often referred to as third-generation sequencing, these techniques enable the analysis of much longer DNA and RNA fragments than possible using either Sanger or traditional NGS – delivering significant advantages for a range of applications, including genome assembly and the analysis of large genomic aberrations (e.g. structural variants and repeats). One such technique, nanopore sequencing, enables the analysis of any length of DNA/RNA fragment – from short (20 bp) to ultra long (>4 Mbp). Furthermore, as amplification is not required, nanopore sequencing eliminates amplification bias and allows direct analysis of base modifications (e.g. methylation) alongside the nucleotide sequence While Sanger sequencing is still used frequently in clinical research labs, the use of NGS in these labs is expanding rapidly through the facility to cost-efficiently analyze a greater number of targets and samples at higher sensitivity .

Advantages of NGS

Next-generation sequencing provides multiple benefits when compared to Sanger sequencing, including:

  • Can sequence an entire genome or high numbers of targets at the same time
  • Faster turnaround time 
  • More cost-effective
  • Requires lower sample input
  • Improved accuracy and reliability
  • Higher sensitivity allows detection of variants at lower frequencies
  • Assay a greater range of variation in a single run using nanopore sequencing (i.e. SNVs, SVs, repeats, phasing, methylation)
Typical NGS Workflow
Traditional NGS platformNanopore sequencing
1. Library preparation includes fragmenting DNA or RNA from the rest of the sample, preparing DNA or RNA for sequencing by adding adapters and checking all fragments for size and quality before adding them to the sequencing library1. Attach sequencing adapters to DNA or RNA sample – no fragmentation required, enabling sequencing of any length of molecule, including ultra-long fragments (up to 4 Mb) for enhanced genome assembly and analysis of structural variants, repeats, phasing and transcript isoforms.
2. Clonal amplification generates multiple copies of target areas of interest within a DNA or RNA sample to ensure they provide a robust and detectable signal during the sequencing process.2. Load sample to Flow Cell containing hundreds of nanopores (nano-scale protein holes embedded in an electrically resistant membrane). No DNA/RNA amplification is required, eliminating amplification bias and enabling simultaneous identification of base modifications (e.g. methylation)
3. Sequencing is the process that tells scientists what genetic information each DNA or RNA molecule contains.3. The DNA or RNA molecules pass through the nanopores, disrupting the ionic current and generating distinctive signal that is decoded to provide the specific DNA or RNA sequence. All sequence data is provided in real-time, enabling immediate sample insights and actionable results.
4. Data analysis is the process of applying a wide range of analytical methods to DNA or RNA sequencing information to determine its structure, function, features, or other characteristics.4. Data can be analysed by a wide range of standard tools, including Oxford Nanopore’s EPI2ME platform, which offers a range of optimised best practice analysis pipelines

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