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DNA methylation detection at single base resolution using targeted next generation bisulfite sequencing and cross validation using capillary sequencing. 2016

Smitha Bhat, and Sandeep Mallya, and Vinay Koshy Varghese, and Pradyumna Jayaram, and Sanjiban Chakrabarty, and Kalpana S Joshi, and Tanuja M Nesari, and Kapaettu Satyamoorthy
Department of Biotechnology, School of Life Sciences, Manipal University, Manipal 576104, Karnataka, India.

With a purpose of accurate and simultaneous determination of DNA methylation from multiple loci in multiple samples, here, we are demonstrating a method to aid rapid DNA methylation detection of genomic sequences. Using genomic DNA of peripheral blood from 14 healthy individuals, DNA methylation in 465 CpG sites from 12 loci of genes (ADAM22, ATF2, BCR, CD83, CREBBP, IL12B, IL17RA, MAP2K2, RBM38, TGFBR2, TGFBR3, and WNT5A) was analysed by targeted next generation bisulfite sequencing. Analysed region for three genes, BCR, IL17RA and RBM38 showed an absolute mean DNA methylation of 25.6%, 89.2% and 38.9% respectively. Other nine gene loci were unmethylated and exhibited <10% absolute mean DNA methylation. Two genes, IL17RA and RBM38 were technically validated using direct capillary sequencing and results were comparable with positive correlation (P=0.0088 & P<0.0001 respectively) in the CpG sites for DNA methylation. All CpG sites analysed from RBM38 genes locus displayed 95% limits of agreement for DNA methylation measurements from the two methods. The present approach provides a fast and reliable DNA methylation quantitative data at single base resolution with good coverage of the CpG sites under analysis in multiple loci and samples simultaneously. Use of targeted next generation bisulfite sequencing may provide an opportunity to explore genes in the discovery panel for biomarker identification and facilitate functional validation.

UI MeSH Term Description Entries
D008297 Male Males
D005260 Female Females
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000328 Adult A person having attained full growth or maturity. Adults are of 19 through 44 years of age. For a person between 19 and 24 years of age, YOUNG ADULT is available. Adults
D017422 Sequence Analysis, DNA A multistage process that includes cloning, physical mapping, subcloning, determination of the DNA SEQUENCE, and information analysis. DNA Sequence Analysis,Sequence Determination, DNA,Analysis, DNA Sequence,DNA Sequence Determination,DNA Sequence Determinations,DNA Sequencing,Determination, DNA Sequence,Determinations, DNA Sequence,Sequence Determinations, DNA,Analyses, DNA Sequence,DNA Sequence Analyses,Sequence Analyses, DNA,Sequencing, DNA
D056426 Genetic Loci Specific regions that are mapped within a GENOME. Genetic loci are usually identified with a shorthand notation that indicates the chromosome number and the position of a specific band along the P or Q arm of the chromosome where they are found. For example the locus 6p21 is found within band 21 of the P-arm of CHROMOSOME 6. Many well known genetic loci are also known by common names that are associated with a genetic function or HEREDITARY DISEASE. Genetic Locus,Loci, Genetic,Locus, Genetic
D059014 High-Throughput Nucleotide Sequencing Techniques of nucleotide sequence analysis that increase the range, complexity, sensitivity, and accuracy of results by greatly increasing the scale of operations and thus the number of nucleotides, and the number of copies of each nucleotide sequenced. The sequencing may be done by analysis of the synthesis or ligation products, hybridization to preexisting sequences, etc. High-Throughput Sequencing,Illumina Sequencing,Ion Proton Sequencing,Ion Torrent Sequencing,Next-Generation Sequencing,Deep Sequencing,High-Throughput DNA Sequencing,High-Throughput RNA Sequencing,Massively-Parallel Sequencing,Pyrosequencing,DNA Sequencing, High-Throughput,High Throughput DNA Sequencing,High Throughput Nucleotide Sequencing,High Throughput RNA Sequencing,High Throughput Sequencing,Massively Parallel Sequencing,Next Generation Sequencing,Nucleotide Sequencing, High-Throughput,RNA Sequencing, High-Throughput,Sequencing, Deep,Sequencing, High-Throughput,Sequencing, High-Throughput DNA,Sequencing, High-Throughput Nucleotide,Sequencing, High-Throughput RNA,Sequencing, Illumina,Sequencing, Ion Proton,Sequencing, Ion Torrent,Sequencing, Massively-Parallel,Sequencing, Next-Generation
D018899 CpG Islands Areas of increased density of the dinucleotide sequence cytosine--phosphate diester--guanine. They form stretches of DNA several hundred to several thousand base pairs long. In humans there are about 45,000 CpG islands, mostly found at the 5' ends of genes. They are unmethylated except for those on the inactive X chromosome and some associated with imprinted genes. CpG Clusters,CpG-Rich Islands,Cluster, CpG,Clusters, CpG,CpG Cluster,CpG Island,CpG Rich Islands,CpG-Rich Island,Island, CpG,Island, CpG-Rich,Islands, CpG,Islands, CpG-Rich
D019175 DNA Methylation Addition of methyl groups to DNA. DNA methyltransferases (DNA methylases) perform this reaction using S-ADENOSYLMETHIONINE as the methyl group donor. DNA Methylations,Methylation, DNA,Methylations, DNA

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