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What is Next Generation Sequencing?

Next Generation Sequencing is a technology that enables massive parallel decoding of DNA or RNA fragments, based on the sequencing-by-synthesis or single molecule sequencing principles. It generates huge amounts of sequencing data output that can only be analyzed using dedicated bioinformatics tools. Examples of major players in the market are Illumina® Inc., Pacific Biosciences® (PacBio), Oxford Nanopore Technologies® and Thermo Fisher Scientific®. Next to hardware, many application specific library preparation providers enable NGS users to work with their perfect custom solution.

Current status of Next Generation Sequencing

Next Generation Sequencing is booming. Latest innovations enable high throughput multiplexing of samples, up to 20 billion reads per run, and the sequencing of genomic DNA fragments, up to 40Kb. This translates itself toward science and opens doors with respect to translational genomics and molecular diagnostics. CleanNA will tell you more about it, like about our CleanNGS.

Impact of NGS

NGS platforms can be broadly applied for monitoring, detecting, identifying, discovering living organisms or for studying cellular processes.

For example, NGS can be applied in clinical diagnostics by targeting free-circulating DNA in order to monitor tumor development during the treatment of patients. Furthermore, NGS can be a powerful tool to detect and or identify pathogenic microbes or to map microbial populations. In the agricultural sector NGS can be used for genetic selection for crop improvement. With respect to food safety, NGS enables screening for food borne pathogens. Overall, NGS offers fundamental and applied sciences a tool for holistic approaches by mapping entire genomes or gene expression profiles.

NGS applications

Examples of molecular applications of NGS are Whole genome sequencing, Exome sequencing, Amplicon panel sequencing, Hyb Capture panel sequencing, RNA sequencing and Methyl-seq.

Sequencing-by-synthesis based NGS platforms

Illumina® Inc. and Thermo Fisher Scientific® platforms. The power of short fragment sequencing is it relatively high accuracy and the enormous amount of data that can be generated by massive parallel sequencing. The downside of these platforms is that the produce relatively short read lengths.

In general, an Illumina sequencing workflow looks like this:

  1. Isolation of input DNA or RNA: note that the quality of the input material affects the data output quality
  2. Library preparation: depending on the specific needs, a wholistic or targeted library preparation will be performed. Important aspects to be considered are amongst others, fragmentation randomness, adaptor ligation efficiency, bias introduction by library amplification, size selection quality and library quantification methods
  3. Sequencing: available systems are iSeq 100®, MiniSeq®, MiSeq®, NextSeq® and NovaSeq 6000®
  4. Data analysis: data analysis apps available

Long fragment sequencing or single molecule sequencing

Pacific Biosciences® and Oxford NanoPore technologies® platforms enable single-molecule sequencing and generate low fragment lengths. These are ideally suited for de novo whole genome sequencing purposes. For these applications the input material should be treated mildly, reducing the risk of breakdown of high molecular weight DNA. Therefore, a well-considered choice of an upfront DNA isolation method is key. In addition, our CleanNGS kit can be used for purification, while maintaining a high molecular weight.