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.
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:
- Isolation of input DNA or RNA: note that the quality of the input material affects the data output quality
- 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
- Sequencing: available systems are iSeq 100®, MiniSeq®, MiSeq®, NextSeq® and NovaSeq 6000®
- 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.