Since the deoxyribonucleic acid (DNA) was discovered by Oswald Avery in 1944 and its double helical strand structure composed of four bases was determined by Watson and Crick in 1953, our knowledge about genome structure and function has tremendously increased. This knowledge and its multiple applications, such as molecular cloning, breeding, finding pathogenic genes, comparative and evolution studies, would not exist without sequencing technologies.
First sequencing techniques were developed in 1977 by Frederic Sanger and Walter Gilbert. Sanger sequencing, based on the chain termination method, was adopted as a primary technique in the “first generation” of laboratory and commercial sequencing applications. At that time, DNA sequencing was a labor consuming and required radioactive materials. A decade later, in 1987, Applied Biosystems (AB) introduced the first automatic sequencing machine (AB370), utilizing capillary electrophoresis, which made the sequencing process much faster and accurate. Thus, the capillary sequencing machines and Sanger sequencing technology became the main tools for the completion of Human Genome Project in 2003.
This project and X-PRIZE competition stimulated the development of the next or second-generation of sequencing applications (NGS) that exhibit massively parallel analysis, high throughput, and reduced cost. Massive parallel sequencing utilizes simultaneous reading of a large amount (1-100 million) of short, 50-400 bases, DNA fragments. Years of evolution yielded three major sequencing systems: i) Roche 454 System- detection of pyrophosphate released during nucleotide incorporation; ii) AB sequencing by Oligo Ligation Detection (SOLiD), iii) Illumina GA/HiSequ System that is based on Solexa’s Genome Analyzer (GA) – sequencing by synthesis (SBS). These highly effective sequencing systems demonstrate their own advantages in terms of read length, accuracy and cost. Briefly, the Illumina HiSeq 2000 features the biggest output and lowest reagent cost, the SOLiD system has the highest accuracy and the Roche 454 system has the longest read length. Other advanced sequencing systems include compact Personal Genome Machine (PGM) and MiSeq, targeted for clinical applications and small laboratories.
While the second generation of sequencing became widely popular in basic research, new improvements in sequencing technologies open era of the third generation sequencers which have two main characteristics: i) PCR is not needed before sequencing which shortens DNA preparation time to several hours; ii) the signal, the fluorescence (PacBio) or the electric current (Nanopore), is captured in real time and get monitored during the enzymatic reaction.
Single-molecular real-time (SMRT) is the third-generation of sequencing method developed by Pacific Bioscience (PacBio, Menlo Park, CA, USA). This method has a shorten sample preparation time, longer average read length (1300pb), which is quite useful for the clinical laboratories and microbiological research and allows predicting epigenetic modifications such as DNA methylation due to differential intensity of the fluorescent signal.
Nanopore sequencing method represents another of state-of-the-art bioengineering model. Nanopore is a tiny biopore, a ion-exchange channel within a trans-membrane protein α-haemolysin (α-HL) isolated from Staphylococcus aureus. The unique property of this protein is its tolerance to the extraordinary voltage up to 100mV with current 100pA. In nanopore sequencing, the ionic flow is applied continuously while the single-stranded DNA thread moves through the pore and disrupts the current. Detected by the electrophysiological technique fluctuations in current depend on the size of dNMPs and comprise the readout. The outstanding Oxford Nanopore technology allows sequencing your genome “before dinner”.
Among the broad roles gene expression profiling has played in life sciences, it recently has been utilized to functionally characterize biological systems in basic research and discover biomarkers for disease and treatment management in clinical settings. The NuGEN company offers a fast, simple, robust RNA sample preparation solutions broaden the usable sample types for analysis on all leading microarray and NGS analysis platforms. Whether samples are limited and degraded, or ample and well-preserved, NuGEN’s RNA sample preparation technologies provide solutions of superior sensitivity for rapid analysis of the transcriptome. Whether the focus is RNA-Seq, 3′ or whole transcriptome analysis requiring single-stranded or double-stranded product, there are systems that meet those requirements.