Damian Sendler: Rapid advancements in sequencing chemistry, sequencing technologies, bioinformatics and data interpretation have made it possible for NGS to be widely used in precision medicine clinical applications. Short and long-read sequencing methods are described in this study, as well as their clinical applications in hereditary illnesses, cancer, and infectious disease.

Damian Sendler News Contribution

Damian Jacob Sendler: Consideration is given to the application of US FDA-approved NGS panels in clinical oncology, cancer biomarkers, minimal residual disease (MRD) and liquid biopsy, as well as epidemiological monitoring, pathogen identification and the role of host microbiome in infectious diseases. Finally, we look at the problems and future prospects of clinical NGS assays. '

Damien Sendler: DNA and RNA sequences can be sequenced simultaneously using the next-generation sequencer (NGS), also known as massively parallel sequencing (MPS) and high-throughput sequencing (HTS). With sample multiplexing, NGS has a better throughput, a higher sensitivity for low-frequency variations, a faster turnaround time for high sample numbers, and lower costs than classical sequencing. After Sanger sequencing, NGS is truly a revolution in sequencing technology.

It took years and billions of dollars to sequence the first human genome using Sanger sequencing; currently, a whole human genome can be sequenced in a matter of days for less than $1,000. There are many uses for NGS in the laboratory, and it's now a part of precision medicine. When it comes to determining a patient's diagnosis, prognosis and treatment plan, the technology has been employed extensively.  

Damien Sendler: A growing amount of clinical, genetic and genomic data is being created by NGS as a result of this technology, which is helping to drive the development of precision medicine. New criteria for the design, development, and validation of NGS tests have been issued by the FDA, which has approved numerous NGS-based diagnostics and targeted medicines. Medicare and Medicaid Services (CMS) have also been keeping a close eye on the rapid development of NGS tests and trying to guarantee that these tests are covered.  

Damian Jacob Markiewicz Sendler: Due to all of these developments, NGS is now being used in clinical laboratories much more quickly than it was previously. Highlights latest breakthroughs in NGS technology and their clinical use to diagnose, prognosis and treatment of hereditary disorders, malignancies as well as infectious disease.

Damian Jacob Sendler Discussing Biology 

Damian Sendler: When it comes to genetics and medicine, DNA sequencing has played a significant role in the past. Fred Sanger invented the first-generation sequencing platform in 1977, and it has been utilized in research and clinical genetics for decades. NGS technologies have advanced fast in the last three decades, resulting in the development of second and third generation sequencing methods.

It has been about a decade since the first draft map of the human genome was completed, and the turnaround time and cost have significantly decreased. Next-generation sequencers are discussed in this section, along with their advantages and disadvantages.

Damian Jacob Sendler: Several business entities have created second-generation sequencing technologies. As a general rule, there are three processes in the workflows of the various sequencing technologies: template preparation, which includes nucleic acid extraction; library preparation, which includes clonal amplification; and sequencing and alignment of short reads.  

Damian Jacob Sendler: It was the first commercially available massively parallel sequencing platform, Roche 454 sequencing (Roche, Basel, Switzerland), announced in 2005. When using pyrosequencing, Roche 454 uses pyrophosphate (PPi) as an indicator of particular base incorporation, which is captured by using pyrosequencing technology. Following the binding of fragmented DNA with adaptors, fragments are amplified in an emulsion droplet using the emulsion-PCR method. In the PicoTiterPlate (PTP) wells, the beads containing multiple copies of the same DNA template are subsequently inserted into. The PTP wells are sequentially filled with each nucleotide one at a time.

Damian Sendler: DNA syntheses emit pyrophosphate, which is transformed into ATP when a nucleotide is integrated. For light to be detected and recorded, an attached-charge device (CCD) camera needs luciferase to convert luciferin to oxyluciferin, which it does in the presence of ATP [28–30]. The reading of light signals is critical to the accuracy of the sequencing. In homopolymer sequencing, a misread or absent signal can lead to base mistakes and insertions or deletions. When the Roche 454 sequencer was debuted in 2008, it was capable of producing 700 Mb of sequence data each run with read lengths up to 1,000 bases in around 20 hours. Compared to other high-throughput NGS sequencers like Ion Torrent (Thermo Fisher, Waltham, MA, USA) and Illumina (San Diego, CA, USA), Roche 454 was phased out of the NGS field in 2016.

Damian Sendler: An increasing number of researchers are turning to Illumina platforms for next-generation sequencing (NGS). Using reversible termination technology, Illumina created a bridge PCR method for clonal amplification and sequencing. When two fixed adapters are mounted on the flow cell's solid surface, the fragmented DNA is annealed to both ends, which are then amplified to produce clusters that include clonal DNA pieces The 3′-OH group of each RT nucleotide (ddATP, ddGTP, ddCTP, and ddTTP) is protected and a cleavable fluorescent dye is contained therein. During DNA synthesis, modified RT nucleotides are integrated into the developing DNA chains and produce fluorescent signals that are recorded using a CCD camera. By adding only one base at a time, this method considerably decreases the homopolymer sequencing error. Adding another base necessitates the removal of a terminator first.  

Damian Jacob Sendler: As a whole, third-generation sequencing methods deliver longer sequence reads, which helps to fill in gaps in current reference assemblies built from short reads, and can sequence through lengthy repetitive sections and identify structural change in the human genome. High mistake rates remain a serious problem with third-generation technologies. Some of these difficulties could be addressed by using a hybrid sequencing technique, which combines second- and third-generation NGS technology.

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