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

Bhat, Smitha; Mallya, Sandeep; Varghese, Vinay Koshy; Jayaram, Pradyumna; Chakrabarty, Sanjiban; Joshi, Kalpana; Nesari, Tanuja Manoj; Satyamoorthy, Kapaettu

doi:10.1016/j.gene.2016.09.019

Cited by 5 publications

(8 citation statements)

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“…The simplicity of the approach, test time, and affordable cost make the prepared bioplatforms competitive from a practical point of view against other reported single-base resolution methodologies. These involve previous chemical modification of the target epimark , and/or the use of nanopores , or bisulfite, , and next-generation sequencing. ,, Another interesting feature of the constructed bioplatforms is their versatility, which makes them applicable to the determination of single-base methylation events involving any epigenetic alteration in any target DNA or RNA sequence by simply changing the synthetic biotinylated capture probe and assuming that a selective antibody to the epimark of interest is available. In fact, the possibility of applying the developed methodologies to the determination of another closely related cytosine variant (5-mC) at single-base resolution is confirmed in the following section.…”

Section: Resultsmentioning

confidence: 99%

“…It is worth noting also that the complex studies that examine the connection among the environment, the epigenome, and disease states require DNA (hydroxy)methylation profiling across the genome with single-nucleotide resolution, which is particularly difficult with conventional techniques. Moreover, it is important to keep in mind that, although the difference in DNA methylation of genes is sometimes >50% in cancer-related genes compared to control studies, this difference falls below 5% in other noncommunicable diseases . Therefore, the development of methodologies able to perform the challenging determination of 5-mC and 5-hmC DNA methylation levels at such low resolution and to reveal locations of these epimarks in the genome envisages to shed considerable light on human disease etiology and biomarker discovery. ,, …”

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confidence: 99%

“…These can be roughly categorized into four types including bisulfite sequencing, endonuclease digestion, affinity enrichment, and emerging single-molecule techniques . They involve the use of nanopores, , bisulfite, , and real-time DNA sequencing strategies. ,, Sometimes, they involve chemical modification of the target epimark, , cannot differentiate 5-hmC from 5-mC and use long and expensive protocols, which makes them impractical. Indeed, the lack of simple, fast, and affordable methods for high-throughput, strand-specific, base resolution, and selective sequencing of 5-hmC and 5-mC has hindered a fast advance in the understanding of complicated regulatory mechanisms of 5-mC and 5-hmC underlying tumorigenesis.…”

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confidence: 99%

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Amperometric Bioplatforms To Detect Regional DNA Methylation with Single-Base Sensitivity

et al. 2020

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This work reports the first bioplatform able to determine electrochemically 5-hydroxymethylcytosine (5-hmC) methylation events at localized sites and single-base sensitivity. The described bioplatform relies on a specific antibody (anti-5-hmC), further conjugated with commercial bioreagents loaded with multiple horseradish peroxidase (HRP) molecules, recognizing the epimark in a target DNA, captured through hybridization onto streptavidin–magnetic microbeads (Strep–MBs) modified with a complementary DNA capture probe. The electrochemical detection is performed by amperometry (−0.20 V vs Ag pseudoreference electrode) at disposable screen-printed carbon electrodes (SPCEs) in the presence of H2O2/hydroquinone (HQ) upon magnetic capture of the modified MBs onto the SPCE. The use of the commercial bioreagents ProtA–polyHRP80 and Histostar, very scarcely explored so far in electrochemical biosensors, provides high sensitivities for a synthetic target DNA sequence with a unique 5-hmC in the promoter region of MGMT tumor suppressor gene. Amplification factors of 43.6 and 55.2 were achieved using ProtA–polyHRP80 or Histostar, respectively, compared to the conventional secondary antibody labeling. This amplification was crucial to detect methylation events at single-nucleotide resolution achieving limits of detection (LODs) of 23.0 and 13.2 pM, respectively, without any target DNA amplification. The ProtA–polyHRP80-based bioplatform, selected as a compromise between sensitivity and cost per determination, exhibited full discrimination toward the target 5-hmC against the closely related 5-mC. In addition, the bioplatform detected 5-hmC at the regional level (MGMT promoter region) in just 10 ng of genomic DNA (gDNA, ∼2700 genomes) extracted from cancer cells and tissues from colorectal cancer (CRC) patients within 60 min.

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Section: Resultsmentioning

confidence: 99%

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Amperometric Bioplatforms To Detect Regional DNA Methylation with Single-Base Sensitivity

et al. 2020

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“…3d). Several other BSAS variations include using restriction enzymes (Varley and Mitra 2010), use of microdroplets for the PCR (Komori et al 2011), further evolutions of the library preparation (Bernstein et al 2015;Bhat et al 2016), and the addition of analyzing hydroxymethylation (Chen et al 2017). These approaches provide base-specific absolute quantitation in up to 96 samples of 1-10 kb targeted regions, making this method highly valuable for hypothesisdriven aging research.…”

Section: Amplicon Sequencingmentioning

confidence: 99%

Analysis of DNA modifications in aging research

et al. 2018

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As geroscience research extends into the role of epigenetics in aging and age-related disease, researchers are being confronted with unfamiliar molecular techniques and data analysis methods that can be difficult to integrate into their work. In this review, we focus on the analysis of DNA modifications, namely cytosine methylation and hydroxymethylation, through nextgeneration sequencing methods. While older techniques for modification analysis performed relative quantitation across regions of the genome or examined average genome levels, these analyses lack the desired specificity, rigor, and genomic coverage to firmly establish the nature of genomic methylation patterns and their response to aging. With recent methodological advances, such as whole genome bisulfite sequencing (WGBS), bisulfite oligonucleotide capture sequencing (BOCS), and bisulfite amplicon sequencing (BSAS), cytosine modifications can now be readily analyzed with base-specific, absolute quantitation at both cytosine-guanine dinucleotide (CG) and non-CG sites throughout the genome or within specific regions of interest by next-generation sequencing. Additional advances, such as oxidative bisulfite conversion to differentiate methylation from hydroxymethylation and analysis of limited input/single-cells, have great promise for continuing to expand epigenomic capabilities. This review provides a background on DNA modifications, the current state-of-theart for sequencing methods, bioinformatics tools for converting these large data sets into biological insights, and perspectives on future directions for the field.

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“…To elucidate the biology of DNA methylation in chondrocyte dedifferentiation, the first step is to evaluate the DNA methylation level of chondrocytes, which so far has proven challenging. Bisulfite genomic sequencing is the most widely used technique to analyze DNA methylation 7,8 . In this assay, bisulfite conversion causes DNA degradation, and thus a substantial amount of sample must be provided for the assay.…”

Section: Introductionmentioning

confidence: 99%

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation <em>In Vitro</em>

Jia

Liang

et al. 2017

JoVE

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The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

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

Cited by 5 publications

References 57 publications

Amperometric Bioplatforms To Detect Regional DNA Methylation with Single-Base Sensitivity

Amperometric Bioplatforms To Detect Regional DNA Methylation with Single-Base Sensitivity

Analysis of DNA modifications in aging research

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation <em>In Vitro</em>

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