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The Role of Epigenetics in Breast Cancer: Implications for Diagnosis, Prognosis, and Treatment
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
Amy M. Dworkin, Tim H.-M. Huang, Amanda E. Toland
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
Published in Jinghua Hu, Yong Yu, 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
Companion Diagnostics and Precision Medicine for Colorectal Cancer
Published in Il-Jin Kim, Companion Diagnostics (CDx) in Precision Medicine, 2019
Seung Eun Yu, Ji Yeon Baek, Sang Myung Woo, Kyun Heo, Byong Chul Yoo
There is no preferred assay for the detection of PIK3CA mutations. The literature suggests that NGS platforms, gel electrophoresis, and PCR sequencing all have excellent sensitivity, while Sanger sequencing is less sensitive (Ney et al., 2012; Arsenic et al., 2015; Li et al., 2016; Ang et al., 2017). High-resolution melting (HRM) analysis has been shown to have a sensitivity similar to that of Sanger sequencing, whereas pyrosequencing appears to be more sensitive than Sanger sequencing (Ihle et al., 2014).
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.
The meconium microbiota shares more features with the amniotic fluid microbiota than the maternal fecal and vaginal microbiota
Published in Gut Microbes, 2020
Qiuwen He, Lai-Yu Kwok, Xiaoxia Xi, Zhi Zhong, Teng Ma, Haiyan Xu, Haixia Meng, Fangqing Zhao, Heping Zhang
The accuracy of profiling microbial communities in low microbial biomass samples (e.g., placenta, amniotic fluid, meconium) has been hindered by our ability to distinguish the authentic signals beyond the level of background contamination. The placental microbiome was firstly characterized metagenomically by Aagaard et al. (2014) using whole-genome shotgun sequencing and 454 pyrosequencing technologies.17 The 454 pyrosequencing technology produced sequences of medium read length (~450 bp).28 By analyzing samples collected from multiple human body sites of pregnant and nonpregnant subjects, Aagaard et al. found that the placental microbiome comprised nonpathogenic commensals most akin to the oral microbiome.17 In contrast, de Goffau et al. (2019) failed to identify distinguishable signals between the placental samples and contaminant controls by sequencing relatively short reads covering the V1-V2 hypervariable regions of the 16S rRNA (~260 bp).27 The conflicting inferences could be resulted from the chosen technologies that relied on different sub-regions and read lengths of 16S rRNA genes, resulting in different power of taxonomic resolution. Moreover, the long-read sequencing technology was potentially advantageous in reducing the contamination risk for metagenomic profiling of low microbial biomass samples, and employing thoughtful filtering settings and vigorous contaminant controls might further ‘decontaminate’ the putative contaminant amplicon sequence variants.22