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