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 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.

Metabolon Validates a Method for Metabolomic Profiling in Blood Microsamples

Recently, the Neoteryx Mitra devices from Trajan 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.

Metabolon’s validation of Mitra with VAMS revealed reproducible results within an acceptable relative standard deviation (RSD). For the Metabolon Global Discovery Panel, 20 µL sample volume is optimal to identify the most metabolites using the smallest amount of sample. More than 90% of metabolites assessed had good precision between four technical replicates.

GenTegra Develops an RNA Stabilizer for Mitra Microsampling

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.

Promega Automates RNA Purification from Blood on Mitra Devices Treated with the GenTegraRNA-NEO Stabilizer

Promega researchers published an application note, which demonstrated a method for purifying Total RNA, including miRNA, from whole blood collected on Mitra microsampling devices that had been treated with the GenTegraRNA-NEO solution.

The researchers processed the samples using their Promega Maxwell^® RSC simplyRNA Blood Kit and Maxwell^® RSC Instrument. 

Cellecta Detects Up- & Down-Regulated Genes in Blood Microsamples Collected on Pre-Treated Mitra Devices 

Using Mitra devices that were pre-treated with the GenTegraRNA-NEO product before blood collection, Cellecta researchers analyzed the blood microsamples using the DriverMap™ Targeted RNA-Seq Expression Profiling Technology 19K Panel and the DriverMap™ T/B Immune Markers Panel to effectively detect up- and down-regulated Genes in blood microsamples.

Cellecta published a research and development poster explaining how they found that analysis of 30 μL of blood using Mitra microsampling devices provided overall satisfactory performance and produced transcriptome profiles comparable to those from larger volumes of blood in Tempus tubes.

These recent developments show that blood microsampling is amenable to RNA/DNA isolation and purification using advanced instruments and central lab kits. The remote blood collection using Mitra devices 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. 

April 2024: Article updated to include new advancements in RNA research, method development and validation.