scholarly journals Whole human genome 5'-mC methylation analysis using long read nanopore sequencing

2021 ◽  
Author(s):  
Catarina Silva ◽  
Miguel Machado ◽  
José Ferrão ◽  
Sebastião Rodrigues ◽  
Luís Vieira
Keyword(s):  
Human Genome ◽  
Cpg Island ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Attractive Alternative ◽  
Nanopore Sequencing ◽  
Coverage Depth ◽  
Genomic Regions

DNA methylation is a type of epigenetic modification that affects gene expression regulation and is associated with several human diseases. Microarray and short read sequencing technologies are often used to study 5'-methylcytosine (5'-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustable results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulfite and the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5'-mC since the long reads produced by this technology allow to resolve those genomic regions more easily. Moreover, it allows direct sequencing of native DNA molecules using a fast library preparation procedure. In this work we show that 10X coverage depth nanopore sequencing, using DNA from a human cell line, produces 5'-mC methylation frequencies consistent with those obtained by methylation microarray and digital restriction enzyme analysis of methylation. In particular, the correlation of methylation values ranged from 0.73 to 0.90 using an average genome sequencing coverage depth <2X or a minimum read support of 17X for each CpG site, respectively. We also showed that a minimum of 5 reads per CpG yields strong correlations (>0.89) between sequencing runs and an almost uniform variation in methylation frequencies of CpGs across the entire value range. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns according to genomic annotations, including a majority of unmethylated and methylated sites in the CpG islands and inter-CpG island regions, respectively. These results demonstrate that low coverage depth nanopore sequencing is a fast, reliable and unbiased approach to the study of 5'-mC in the human genome.

scholarly journals Expression of Sclerostin in Osteoporotic Fracture Patients Is Associated with DNA Methylation in the CpG Island of the SOST Gene

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yanming Cao ◽  
Bin Wang ◽  
Ding Wang ◽  
Dongxiang Zhan ◽  
Caiyuan Mai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Osteoporotic Fracture ◽  
Cpg Island ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Gene Promoter ◽  
Primary Osteoporosis ◽  
Sost Gene ◽  
Fracture Bone

Purpose. SOST gene is one of the key factors in regulating bone absorption. Although there are reports showing diverse transcription factors, epigenetic modification could be responsible for regulating SOST gene expression. There is still little exploration on promoter methylation status of SOST gene in osteoporotic bone tissues. The aim of this study is to investigate the involvement of CpG methylation in regulation of SOST expression in patients with primary osteoporosis. Methods. The diagnosis of osteoporosis was established on the basis of dual energy X-ray absorptiometry to measure BMD. All femoral bone tissues were separated in surgeries. After extracting total RNA and protein, we checked the relative expression levels of SOST by quantitative real-time PCR and western blot. Also, immunohistochemical staining was performed to observe the expression of SOST protein in the bone samples. The genomic DNA of non-OPF (non-osteoporotic fracture bone tissues) and OPF (osteoporotic fracture bone tissues) were treated by bisulfite modification, and methylation status of CpG sites in the CpG island of SOST gene promoter was determined by DNA sequencing. Results. SOST gene expression in the non-OPF group was lower than that in OPF group. Bisulfite sequencing result showed that SOST gene promoter was slightly demethylated in the OPF group, as compared with non-OPF group. Conclusion. Our study demonstrated that DNA methylation influenced the transcriptional expression of SOST gene, which probably may play an important role in the pathogenesis of primary osteoporosis.

Purification of circular YACs from yeast cells for DNA sequencing

Genome ◽  
2008 ◽  
Vol 51 (2) ◽  
pp. 155-158 ◽  
Author(s):  
S.-H. Leem ◽  
Y.-H. Yoon ◽  
S. I. Kim ◽  
V. Larionov
Keyword(s):  
Dna Sequencing ◽  
Human Genome ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Genome Project ◽  
New Method ◽  
Yeast Cells ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
The Human Genome Project

We describe a method for the purification of circular yeast artificial chromosome (YAC) DNA 120–150 kilobases (kb) in size that is of sufficient quantity and quality for restriction enzyme analysis and DNA sequencing. This method preferentially enriches for circular YAC DNA and avoids the time-consuming step of centrifugation in CsCl – ethidium bromide (EtBr) gradients. We applied this method to the purification of circular YACs carrying DNA segments that are extremely unstable in E. coli, including those that correspond to GAP2 and GAP3 on human chromosome 19. We showed that YAC DNA (GAP2 and GAP3) purified using this new method is clearly resolved in EtBr-stained gels. The sequence of YAC-GAP3 was obtained, representing the first GAP clone sequenced in YAC form. At present, it is estimated that there are more than 1000 gaps in the human genome that cannot be cloned using bacterial vectors. Thus, our new method may be very useful for completing the last stage of the human genome project.

scholarly journals A Scan for Linkage Disequilibrium Across the Human Genome

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1711-1722 ◽  
Author(s):  
Gavin A Huttley ◽  
Michael W Smith ◽  
Mary Carrington ◽  
Stephen J O’Brien
Keyword(s):  
Linkage Disequilibrium ◽  
Human Genome ◽  
Pedigree Analysis ◽  
Exact Test ◽  
Genomic Scale ◽  
Genomic Regions ◽  
Formal Test ◽  
Scale Data ◽  
Short Tandem ◽  
Tandem Repeat Polymorphism

Abstract Linkage disequilibrium (LD), the tendency for alleles of linked loci to co-occur nonrandomly on chromosomal haplotypes, is an increasingly useful phenomenon for (1) revealing historic perturbation of populations including founder effects, admixture, or incomplete selective sweeps; (2) estimating elapsed time since such events based on time-dependent decay of LD; and (3) disease and phenotype mapping, particularly for traits not amenable to traditional pedigree analysis. Because few descriptions of LD for most regions of the human genome exist, we searched the human genome for the amount and extent of LD among 5048 autosomal short tandem repeat polymorphism (STRP) loci ascertained as specific haplotypes in the European CEPH mapping families. Evidence is presented indicating that ∼4% of STRP loci separated by &lt;4.0 cM are in LD. The fraction of locus pairs within these intervals that display small Fisher’s exact test (FET) probabilities is directly proportional to the inverse of recombination distance between them (1/cM). The distribution of LD is nonuniform on a chromosomal scale and in a marker density-independent fashion, with chromosomes 2, 15, and 18 being significantly different from the genome average. Furthermore, a stepwise (locus-by-locus) 5-cM sliding-window analysis across 22 autosomes revealed nine genomic regions (2.2-6.4 cM), where the frequency of small FET probabilities among loci was greater than or equal to that presented by the HLA on chromosome 6, a region known to have extensive LD. Although the spatial heterogeneity of LD we detect in Europeans is consistent with the operation of natural selection, absence of a formal test for such genomic scale data prevents eliminating neutral processes as the evolutionary origin of the LD.

scholarly journals Exploring Differentially Methylated Genes in Vulvar Squamous Cell Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3580
Author(s):  
Shatavisha Dasgupta ◽  
Patricia C. Ewing-Graham ◽  
Sigrid M. A. Swagemakers ◽  
Thierry P. P. van den Bosch ◽  
Peggy N. Atmodimedjo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Dna Sequences ◽  
Cpg Island ◽  
Epigenetic Modification ◽  
Vulvar Squamous Cell Carcinoma ◽  
Differentially Methylated Genes ◽  
Methylation Profiling

DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC (n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies (n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ± 0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

Association Between Cyclin D1 Polymorphism with CpG Island Promoter Methylation Status of Tumor Suppressor Genes in Gastric Cancer

2010 ◽  
Vol 55 (12) ◽  
pp. 3449-3457 ◽  
Author(s):  
Tomomitsu Tahara ◽  
Tomoyuki Shibata ◽  
Masakatsu Nakamura ◽  
Hiromi Yamashita ◽  
Daisuke Yoshioka ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Tumor Suppressor ◽  
Cyclin D1 ◽  
Promoter Methylation ◽  
Tumor Suppressor Genes ◽  
Cpg Island ◽  
Methylation Status ◽  
Suppressor Genes ◽  
Promoter Methylation Status

scholarly journals A molecular characterization ofClostridium difficileisolates from humans, animals and their environments

1993 ◽  
Vol 111 (2) ◽  
pp. 257-264 ◽  
Author(s):  
G. O'Neill ◽  
J. E. Adams ◽  
R. A. Bowman ◽  
T. V. Riley
Keyword(s):  
Fragment Length Polymorphism ◽  
Hospital Environment ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Direct Transmission ◽  
Chromosomal Dna ◽  
Veterinary Clinic ◽  
Enhanced Chemiluminescence ◽  
Typing Methods ◽  
Rflp Typing

SummaryIt is generally accepted that most patients withClostridium difficile-associated diarrhoea acquire the organism from the environment. Recently we demonstrated that household pets may constitute a significant reservoir ofC. difficilethrough gastrointestinal carriage in up to 39% of cats and dogs. These findings suggested that direct transmission from household pets, or contamination of the environment by them, may be a factor in the pathogenesis ofC. difficile-associated diarrhoea. To investigate this possibility, we examined isolates ofC. difficilefrom humans, pets and the environment by restriction enzyme analysis (REA) and restriction fragment length polymorphism (RFLP) typing using enhanced chemiluminescence. Both REA and RFLP typing methods usedHindIII digests of chromosomal DNA. A total of 116 isolates ofC. difficilefrom pets (26), veterinary clinic environmental sites (33), humans (37) and hospital environmental sites (20) was examined. REA was far more discriminatory than RFLP typing and for all isolates there were 34 REA types versus 6 RFLP types. There was good correlation between the REA types found in isolates from pets and from the veterinary clinic environment, and between isolates from humans and from those found in the hospital environment. There was, however, no correlation between REA type ofC. difficilefound in pets and isolates of human origin. We conclude that there may still be a risk of humans acquiringC. difficilefrom domestic pets as these findings may be the result of geographical variation.

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