a brief introduction to next generation sequencing

Next Generation Sequencing (NGS) is a modern sequencing technology also known as high throughput sequencing. It allows RNA/DNA sequencing to be carried out much more quickly than Sanger sequencing.

NGS sequencing includes Illumina, Roche 454, Ion Torrent, and Solid sequencing.

Illumina Sequencing

It’s a single stroke sequencing of vast numbers of short threads. Illumina sequencing utilizes approximately100-150bp of reads. On the other hand, you should tie longer fragments up to generic adaptors, then hardened to a slide using adaptors. Moreover, amplifying the reads using Polymerase chain reaction (PCR) individually creates a spot with numerous copies of the same read. Separation of each read into single strand is then done before sequencing.

454 Sequencing

Like Illumina, 454 sequencing uses longer reads. It does this by reading optical signals by sequencing multiple reads at once as you add their bases. Secondly, you should split RNA/DNA into 1kb of shorter reads. Furthermore, Polymerase chain reaction (PCR) amplifies the fragments using specific primers and placed in a single well of a slide. Finally, flooding of the slide with one of the four nucleoside triphosphate (NTP) is then done for sequencing.

ION Torrent/Proton Sequencing

ION Torrent/Proton sequencing doesn’t make use of optical signals like Illumina and 454 sequencing. They capitalize on the fact that the addition of dNTP to DNA polymer releases H+ ion. However, like other NGS you should split input RNA/DNA by ~200bp.

Applications of Microsampling Technology in NGS

Microsampling devices like the hemaPEN^® and the Mitra^® device based on VAMS^® technology can allow for the production of accurate results from molecular research labs. Blood collection via remote microsampling is known for being minimally invasive, less costly than other blood collection methods, and very efficient in both clinical and remote settings.

Recently, the Mitra devices based on VAMS were used to validate a biological, analytical method by Metabolon on their Global Discovery Panel for metabolomics studies. Analytical validation ensures that the blood microsamples maintain sensitivity, precision, and stability throughout the sample journey, from collection by the study subject to processing and analysis in the lab. Biological validation confirms that known signatures in other blood sample types will be detectable in a reproducible way.

Mitra microsampling devices have also been used by GenTegra to develop their GenTegraRNA-NEO product, which is an Active Chemical Protection™ (ACP) technology for RNA preservation and stabilization in blood microsamples. Pre-treating Mitra devices with GenTegraRNA-NEO before blood collection was shown to keep the RNA stable in the samples for up to 7 days at room temperature.

The 7-day sample stability enables remote RNA-based studies and field work where the samples can be shipped back to a central lab for analysis using standard mail versus expensive cold shipping.

These recent developments show that blood microsampling is amenable to RNA/DNA isolation and purification using central lab kits. The remote blood collection with a simple finger-stick is convenient to both study participants and field researchers. Finally, the samples are compatible with Next Generation Sequencing platforms.

The main advantages of NGS over traditional Sanger sequencing include speed, cost, accuracy, and the sample size. In addition, NGS requires less RNA/DNA than Sanger sequencing.

To learn more about applying blood microsampling to Omics Research visit out resources page.