Aims: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma. In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques and demonstrated its clinical applicability.
Methods: We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification.
Results: Nanopore sequencing displayed a strong correlation (R = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm's classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island.
Conclusions: This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted.
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http://dx.doi.org/10.1111/nan.12984 | DOI Listing |
BMC Genomics
December 2024
Grapevine Improvement, Bragato Research Institute, Lincoln, New Zealand.
Nanopore sequencing enables detection of DNA methylation at the same time as identification of canonical sequence. A recent study validated low-pass nanopore sequencing to accurately estimate global methylation levels in vertebrates with sequencing coverage as low as 0.01x.
View Article and Find Full Text PDFGenome Res
December 2024
Institute for Integrative Systems Biology, Spanish National Research Council;
While the production of a draft genome has become more accessible due to long-read sequencing, the annotation of these new genomes has not been developed at the same pace. Long-read RNA sequencing (lrRNA-seq) offers a promising solution for enhancing gene annotation. In this study, we explore how sequencing platforms, Oxford Nanopore R9.
View Article and Find Full Text PDFUnlabelled: Caviid gammaherpesvirus 1 (CaGHV-1), formerly known as the guinea pig herpes-like virus, is an oncogenic gammaherpesvirus with a sequenced genome but an as-yet uncharacterized transcriptome. Using nanopore long-read RNA sequencing, we annotated the CaGHV-1 genome and constructed a detailed transcriptomic atlas. Our findings reveal diverse viral mRNAs and non-coding RNAs, along with mapped promoter elements for each viral gene.
View Article and Find Full Text PDFFront Microbiol
December 2024
Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea.
This study investigated age-related changes in the gut microbiota and metabolome of Sapsaree dogs through metagenomic and metabolomic analyses. Using Illumina (short-read) and Nanopore (long-read) sequencing technologies, we identified both common and unique bacterial genera in the dogs across different age groups. In metagenomic analysis, Firmicutes were predominant at the family level.
View Article and Find Full Text PDFAnim Genet
February 2025
Key Laboratory of Genetic Evolution & Animal Models and Yunnan Key Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China.
The feralization of domestic chicken makes the conservation and management of red jungle fowl (Gallus gallus) more complicated and challenging. We collected two Sulawesi feral chickens, located east of the Wallace Line, for whole-genome sequencing and de novo genome assembly. Phylogenetic and f4-statistics analyses indicated that the Sulawesi feralized domestic chickens (G.
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