Elucidation of bacterial genome complexity using next-generation sequencing

Kim, Jungkon; Lee, Sooin; Shin, HyeonSeok; Kim, Sun Chang; Cho, Byung-Kwan

doi:10.1007/s12257-012-0374-x

Cited by 2 publications

(2 citation statements)

References 116 publications

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“…Additionally, in multiplex genotyping assays, the identification and quantification of the amplified signals, as well as the specific amplification itself, are challenging. Three major identification methods are available: parallel sequencing , microarray , and CE . Although parallel sequencing and microarray analysis provide superior multiplexing compared to CE, CE has several advantages over the others regarding the suitability for routine clinical use.…”

Section: Introductionmentioning

confidence: 99%

Multiplex ligase‐based genotyping methods combined with CE

Shin

Chung

Jung³

et al. 2013

Electrophoresis

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In this genomic era, the ability to assay multiple genomic hot spots that have strong clinical implications is greatly desired. Conventional PCR-based methods suffer from frequent false-positive detections, particularly when a multiplex analysis is desirable. As an alternative to the error-prone conventional methods, multiplex ligase-based genotyping methods combined with CE have a strong potential. In this review, both previously developed methods and emerging methods are described to reveal the specificity, sensitivity, and simplicity of the ligase-based methods. For each step (ligation, amplification, and separation), the principles of several alternative methods are discussed along with their applications to explore the future development of ligase-based diagnostic methods.

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Section: Introductionmentioning

confidence: 99%

Multiplex ligase‐based genotyping methods combined with CE

Shin

Chung

Jung³

et al. 2013

Electrophoresis

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“…This difference can be analyzed later by several methods, including methylation-specic PCR (MSP), 12 Sanger sequencing, and pyrosequencing. [13][14][15] However, bisulte conversion is not always efficient, especially when the DNA quality is poor, 11 and errors can potentially be introduced in quantitative analyses due to artifacts from bisulte conversion, such as incomplete bisulte conversion and biased PCR efficiency. In particular, bisulte conversion generates G/C-poor sequences, creating a bias in PCR efficiencies between the products of methylated and non-methylated DNA.…”

Section: Introductionmentioning

confidence: 99%

A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

Shin

Jung³

et al. 2013

Analyst

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Aberrant DNA methylation is a potential diagnostic marker for complex diseases, such as cancer. With the increase in the number of genes known to exhibit disease-associated aberrant methylation, the need for accurate multiplex assays for quantifying DNA methylation has increased. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is one method that has been highlighted in this context. However, two limitations make the custom design of MS-MLPA assays impractical: the need for long probes containing stuffer sequences and a reliance on only one restriction enzyme. Here, we developed a variation of MS-MLPA that employs a simpler probe-design process. To overcome the above-mentioned limitations, we used stuffer-free MS-MLPA probes that are subsequently analyzed using high-resolution capillary electrophoresis-based single-strand conformational polymorphism (CE-SSCP) instead of conventional length-dependent CE. Moreover, multiple methylation-sensitive restriction enzymes (HhaI, HpaII, and AciI) were used simultaneously; thus, probes satisfying desired criteria were available for all targets. Using this assay concept, we analyzed 17 genes associated with hepatocellular carcinoma. Our results showed that the custom-designed assay based on MS-MLPA-CE-SSCP provided robust multiplex quantification of DNA methylation levels.

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Elucidation of bacterial genome complexity using next-generation sequencing

Cited by 2 publications

References 116 publications

Multiplex ligase‐based genotyping methods combined with CE

Multiplex ligase‐based genotyping methods combined with CE

A robust and simple-to-design multiplex DNA methylation assay based on MS-MLPA-CE-SSCP

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