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Identifying Breast Cancer Treatment Biomarkers Using Transcriptomics
Published in Shazia Rashid, Ankur Saxena, Sabia Rashid, Latest Advances in Diagnosis and Treatment of Women-Associated Cancers, 2022
Multiple computational methods (Scott et al., 2020) have been developed to identify cell-type-specific methylation signals using DNA methylation for whole-genome bisulfite sequencing both from reference-based and non-reference-based. This field is known as immunomethylomics and is part of the collective effort of epigenome-wide association studies (EWAS). Methods that don’t need cell sorting have also been developed (Rahmani et al., 2019).
DNA methylation analysis using bisulfite sequencing data
Published in Altuna Akalin, Computational Genomics with R, 2020
One of the most reliable and popular ways to measure DNA methylation is high-throughput bisulfite sequencing. This method, and the related ones, allow measurement of DNA methylation at the single nucleotide resolution. The bisulfite conversion turns unmethylated Cs to Ts and methylated Cs remain intact. Then, the only thing to do is to align the reads with those C- ¿ T conversions and count C- ¿ T mutations to calculate fraction of methylated bases. In the end, we can get quantitative genome-wide measurements for DNA methylation.
Investigation of DNA Methylation in Autosomal Dominant Polycystic Kidney Disease
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Popular downstream methods for analyzing DNA methylation following bisulfite conversion include methylation-specific PCR (MS-PCR), bisulfite sequencing, and methylation-based microarrays. Here, we will discuss the applications of MS-PCR, quantitative real-time methylation-specific PCR (qMS-PCR), pyrosequencing. and whole-genome bisulfite sequencing (WGBS), respectively.
TERT distal promoter GC islands are critical for telomerase and together with DNMT3B silencing may serve as a senescence-inducing agent in gliomas
Published in Journal of Neurogenetics, 2022
Naz Şerifoğlu, Begün Erbaba, Michelle M. Adams, Ayça Arslan-Ergül
The SDM sites were predicted to be the Sp1 binding sites, and it is possible that preventing the binding of Sp1 leads to promoter activity blockage. We aimed to analyze more Sp1 binding sites and also to perform bisulfite sequencing, however, we were unable to perform these tasks due to high CpG content of the region. In order to analyze the effect of methylation on TERT expression, we decided to block methylation globally; via DNMT3B siRNA and azacytidine treatment. Azacytidine (vidaza) is a hypomethylation agent, which prevents all methylation in a cell. DNMT3B, as being the de novo methyltransferase, enables addition of methyl residues. First, we wanted to compare the basal TERT expression levels in our cell lines. VA-13 cell line has been shown before to be telomerase deficient (Cerone, Autexier, Londoño-Vallejo, & Bacchetti, 2005). In our data also it had almost undetectable levels of TERT, which was as expected and SW1088 had lower levels than A172 (Figure 4(A)). Although not statistically significant, azacytidine lowers TERT expression in A172, yet increases in SW1088. When DNMT3B is silenced, TERT gene expression is reduced dramatically in both cell lines (Figure 4(B,C)). In contrast to their effect on TERT, azacytidine lowers hTR expression in all cell lines, including VA-13, whereas DNMT3B had no effect (Figure 4(D–F)). Thus, although azacytidine has differential effect on cell lines, DNMT3B silencing is consistent. Also, DNMT3B silencing acts only on TERT levels and not on TR levels.
Testing differentially methylated regions through functional principal component analysis
Published in Journal of Applied Statistics, 2022
Mohamed Milad, Gayla R. Olbricht
This research demonstrates that information from reduced representation bisulfite sequencing (RRBS) datasets can be analyzed using higher-order mathematics, specifically a functional data analysis approach. Here, a dimension reduction approach is presented, based on the Karhunen-Loève transform, to create a hypothesis test for differential methylated regions (DMRs) using functional principal components based on the spatial features of methylation profiles. This allows the investigation of dominant modes of variation in the methylation profile over a region using eigenfunctions of the covariance function. The FPCA in this study employs a few principal components that increase the power and reduce degrees of freedom in testing to make the underlying biological signals stable. An FPCA based on Fourier and B-spline functions was developed that successfully detects information from shapes of the methylation curves that cannot be identified by traditional multivariate statistics and tests for differentially methylated regions between case and control groups. An empirical comparison, using a simulation based on real data, showed a substantial increase in the true positive rate for FPCA in comparison with the M3D approach, as well as considerable robustness with respect to coverage depth and replication. In general, the simulation results were similar for FPCA-Fourier and FPCA-Bspline, with FPCA- Fourier having slightly lower type II errors across most of the simulation settings thus giving it a slight advantage.
Liquid biopsy from research to clinical practice: focus on non-small cell lung cancer
Published in Expert Review of Molecular Diagnostics, 2021
Umberto Malapelle, Pasquale Pisapia, Alfredo Addeo, Oscar Arrieta, Beatriz Bellosillo, Andres F. Cardona, Massimo Cristofanilli, Diego De Miguel-Perez, Valeria Denninghoff, Ignacio Durán, Eloísa Jantus-Lewintre, Pier Vitale Nuzzo, Ken O’Byrne, Patrick Pauwels, Edward M. Pickering, Luis E. Raez, Alessandro Russo, Maria José Serrano, David R. Gandara, Giancarlo Troncone, Christian Rolfo
Genome-wide methylation is detected mainly by measuring the content of 5mC in the genome [85,95]. Almost all of the cfDNA methylation analysis methods depend on bisulfite sequencing, which transforms non-methylated cytosines to uracils after sodium bisulfite treatment while not altering methylated cytosines [85]. Following DNA sequencing, DNA methylation sites can be detected. Sodium bisulfite treatment would cause a degree of DNA degradation which may lead to loss of some critical information [96]. For example, WGBS-based methods produce the most comprehensive and high-resolution DNA methylome maps, but typically require sequencing to 30× coverage which is still expensive for routine analysis. Additionally, optimized approaches named single-cell reduced-representation bisulfite sequencing (scRRBS) and Methylated CpG tandems amplification and sequencing (MCTA-seq) aim at capturing CpG-enriched cfDNA fragments, which would lead to loss of some critical DNA methylation sites, reducing the sensitivity of the methods.