The Journey through the Gene: a Focus on Plant Anti-pathogenic Agents Mining in the Omics Era
Mahendra Rai, Chistiane M. Feitosa in Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
The SGS term describes platforms generating a huge amount (even billions of reads) of short nucleotide sequences (25 to 400 bp), increasing the high-throughput production, and reducing the cost/million bases by orders of magnitude (Mardis 2008). The pyrosequencing system developed by 454 Life Sciences, after acquired by Roche®, was the first successful NGS/SGS platforms. Other successful SGS platforms comprise the Illumina® Genome Analyzer (GA) II/IIx, the Applied Biosystems SOLiD™ (Sequencing by Oligo Ligation Detection), the Helicos HeliScope™, among others (Mardis 2008). Further optimization has led to innovative third-generation (long-read sequencing) platforms as single-molecule real-time sequencing by PacBio®, nanopore sequencing, etc. (Bleidorn 2015; Xiao and Zhou 2020). The sequencing of entire genomes gives rise to a new genetics sub area, the genomics.
The Role of Epigenetics in Breast Cancer: Implications for Diagnosis, Prognosis, and Treatment
Brian Leyland-Jones in Pharmacogenetics of Breast Cancer, 2020
MassARRAY and pyrosequencing, two additional methods of detecting methylation, are currently being developed for use in clinical settings. MassARRAY is a highly sensitive technique that uses base-specific cleavage and matrix-assisted laser desorption/ionization time-to-flight mass spectrometry (MALDI-TOF MS). It is capable of detecting DNA methylation levels as low as 5%. It is suitable for testing methylation patterns on various sources, including archival tissues and laser capture microdissected specimens (1). Pyrosequencing is a locus-specific quantitative method that utilizes the detection of pyrophosphate, which is liberated from incorporated nucleotides by DNA polymerase during strand elongation. Free pyrophosphates are converted to adenosine triphosphate (ATP), which provides energy for the oxidation of luciferin to then generate light. Nucleotides are added sequentially to enable base calling. Pyrosequencing has two major advantages over MS-PCR. First, the data are actual nucleotide sequences rather than fluorescence data. Second, pyrosequencing can detect partially methylated sequences that are outside of the priming sites (30).
Investigation of DNA Methylation in Autosomal Dominant Polycystic Kidney Disease
Jinghua Hu, Yong Yu in Polycystic Kidney Disease, 2019
Pyrosequencing relies on light generation after nucleotides are incorporated in a growing chain of DNA. When the first one of the four deoxynucleotides (dNTPs) is added to the sequencing reaction, DNA polymerase catalyzes its incorporation into the DNA strand, in case there is complementarity. With each incorporation event, a phosphodiester bond is formed between the dNTPs, releasing pyrophosphate (PPi). Unincorporated nucleotides are degraded by apyrase before the next nucleotide dispensation occurs. In the presence of APS, ATP sulfurylase utilizes the PPi to produce ATP, which is used to drive the conversion of luciferin to oxyluciferin by luciferase. The intensity of light produced and detected by this reaction is proportional to the amount of ATP used and reflects the amount of nucleotide incorporated at specified sequences surrounding CpG sites; this is translated as a peak in a pyrogram, with the height of each peak representing the number of nucleotides incorporated. From these pyrograms, methylation percentages can be calculated.74–76
Helicobacter pylori serology is associated with worse overall survival in patients with melanoma treated with immune checkpoint inhibitors
Published in OncoImmunology, 2022
Marion Tonneau, Alexis Nolin-Lapalme, Suzanne Kazandjian, Edouard Auclin, Justin Panasci, Myriam Benlaifaoui, Mayra Ponce, Afnan Al-Saleh, Wiam Belkaid, Sabrine Naimi, Catalin Mihalcioiu, Ian Watson, Mickael Bouin, Wilson Miller, Marie Hudson, Matthew K. Wong, Rossanna C. Pezo, Simon Turcotte, Karl Bélanger, Rahima Jamal, Paul Oster, Dominique Velin, Corentin Richard, Meriem Messaoudene, Arielle Elkrief, Bertrand Routy
Fecal samples were obtained at the initiation of the ICI treatment for 44 of the 97 patients. We also examined fecal samples from patients with NSCLC (n = 28) (CRCHUM IRB 18.085) from a cohort published in Oster et al.18 Feces were collected according to International Human Microbiome Standards (IHMS) guidelines (SOP 03 V1). Isolated DNA was analyzed using shotgun sequencing to investigate the microbial composition in fecal samples.22 DNA was extracted following Suau et al.’s protocol.23 The genetic material was subsequently sequenced using pyrosequencing. Resulting reads were then filtered using AlienTrimmer to both remove low quality reads as well as sequencing adapters.24 The resulting cleaned data was further processed to remove human and other potential DNA contaminants. This was performed by removing any sequences matching to the human, Bos taurus and Arabidopsis thaliana genome with an identity score threshold of 97% using Bowtie 2.25
Application of next-generation sequencing in the diagnosis of gastric cancer
Published in Scandinavian Journal of Gastroenterology, 2022
Narges Moradi, Solmaz Ohadian Moghadam, Siamak Heidarzadeh
Roche 454 sequencing system functions in exclusive steps including library preparation, DNA amplification and pyrosequencing. While constructing the library, different DNA samples are broken into 300–800bp fragments. Specific primers are used to amplify denaturized DNA and clonal amplifications take place and eventually, library of single stranded DNA is constructed. The amplification system in Roche 454 fixes DNA strands in emulsion overwhelmed beads. This emulsion PCR approach is beneficiary due to its capacity for independent reactions and various beats are separated using emulsion characteristics. The system amplifies all of the fragments about one million times. The last step of pyrosequencing is based on identifying the emitted light of a chain reaction. Molecular mechanism of pyrosequencing is depicted in Figure 4. A high average read length of 400 bp and inaccuracy in assessing homopolymer length are relatively the significant advantage and disadvantage of Roche 454 sequencing system (www.creative-biogene.com). New molecular markers can lead us to develop personal treatments and faster and more accurate diagnosis of GC.
Oral mycobiome identification in atopic dermatitis, leukemia, and HIV patients – a systematic review
Published in Journal of Oral Microbiology, 2020
Camila Stofella Sodré, Paulo Matheus Guerra Rodrigues, Mayra Stambovsky Vieira, Alexandre Marques Paes da Silva, Lucio Souza Gonçalves, Marcia Gonçalves Ribeiro, Dennis de Carvalho Ferreira
In the present review, only the works of Mukherjee et al. [44], Mukherjee et al. [53], Fukui et al. [45], Li et al. [47] and Vijendran et al. [46] identified genera other than Candida in HIV-infected patients. This can be explained due to the techniques used by their studies: Mukherjee et al. [44] used the Multitag 454 Pyrosequencing; Mukherjee et al. [53] used the Ion-Torrent sequencing platform; and Fukui et al. [45] used the Illumina Miseq. Next-generation sequencing provides the identification and typing applications of all pathogens in a single protocol, which contrasts with Sanger sequencing [93]. This advantage is of great use in medical microbiology and also for preventive measures against infection [93]. However, next-generation sequencing requires basic knowledge in bioinformatics, which makes its use more difficult [94]. Thus, when comparing the advantages and differences between sequencing techniques, we can see that the Ion Torrent platform, used by Mukherjee et al. [53] is the simplest and also has a low cost, and consequently, it is widely available, especially in Europe and the USA [74]. The Multitag 454 Pyrosequencing that was used in the work of Mukherjee et al. [53] is more efficient than the Sanger method presenting greater sequence coverage capacity, precision, flexibility, parallel processing, easy automation potential, and instantaneous sequencing response [95]. However, its use requires the use of numerous reagents, in addition to presenting a high cost and an error rate that is considered high (0.0098) [96].
Related Knowledge Centers
- DNA Polymerase
- DNA Sequencing
- Enzyme
- Nucleoside Triphosphate
- Nucleotide
- Pyrophosphate
- Streptavidin
- DNA Sequencing
- DNA Polymerase
- Solid Phase Sequencing
- Luciferase
- Sulfate Adenylyltransferase