Sequencing by ligation variation with endonuclease V digestion and deoxyinosine-containing query oligonucleotides

Ho, Antoine; Murphy, Maurice H.; Wilson, Susan M.; Atlas, Susan R.; Edwards, Jeremy S.

doi:10.1186/1471-2164-12-598

Cited by 16 publications

(8 citation statements)

References 32 publications

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“…To cleave the terminal fluorophore, reverse PRICKLi probe incorporates ribonucleotides (rK, rR, or rC) that are nicked by RNase H2 (Fig. 1d) at the 5' side (24), while the forward PRICKLi probe incorporates inosine for Endonuclease V-dependent cleavage (25). Because the cleavage position of Endonuclease V is variable, phosphorothioate modifications are used to block undesired cleavage sites ( Fig.…”

Section: Resultsmentioning

confidence: 99%

In Situ Transcriptome Accessibility Sequencing (INSTA-seq)

Fürth

Hatini

Lee

2019

Preprint

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Subcellular RNA localization regulates spatially polarized cellular processes, but unbiased investigation of its control in vivo remains challenging. Current hybridization-based methods cannot differentiate small regulatory variants, while in situ sequencing is limited by short reads. We solved these problems using a bidirectional sequencing chemistry to efficiently image transcript-specific barcode in situ, which are then extracted and assembled into longer reads using NGS. In the Drosophila retina, genes regulating eye development and cytoskeletal organization were enriched compared to methods using extracted RNA. We therefore named our method In Situ Transcriptome Accessibility sequencing (INSTA-seq). Sequencing reads terminated near 3' UTR cis-motifs (e.g. Zip48C, stau), revealing RNAprotein interactions. Additionally, Act5C polyadenylation isoforms retaining zipcode motifs were selectively localized to the optical stalk, consistent with their biology. Our platform provides a powerful way to visualize any RNA variants or protein interactions in situ to study their regulation in animal development.

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Section: Resultsmentioning

confidence: 99%

In Situ Transcriptome Accessibility Sequencing (INSTA-seq)

Fürth

Hatini

Lee

2019

Preprint

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“…This is a DNA sequencing technique, which determines the DNA sequence by utilizing the mismatch sensitivity of DNA ligase enzyme (Ho et al 2011). Applied Biosystems, USA marketed this technology in 2008.…”

Section: Sequencing By Ligation Technologymentioning

confidence: 99%

Next-Generation Sequencing and Its Application: Empowering in Public Health Beyond Reality

Gupta

Verma

2019

Microorganisms for Sustainability

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Next-generation sequencing has the ability to revolutionize almost all fields of biological science. It has drastically reduced the cost of sequencing. This allows us to study the whole genome or part of the genome to understand how the cellular functions are governed by the genetic code. The data obtained in huge quantity from sequencing upon analysis gives an insight to understand the mechanism of pathogen biology, virulence, and phenomenon of bacterial resistance, which helps in investigating the outbreak. This ultimately helps in the development of therapies for public health welfare against human pathogen and diagnostic reagents for the screening. This chapter includes the basic of Sanger's method of DNA sequencing and next-generation sequencing, different available platforms for sequencing with their advantages, and limitations and their chemistry with an overview of downstream data analysis. Furthermore, the breadth of applications of high-throughput NGS technology for human health has been discussed.

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“…Sequencing by ligation is a DNA sequencing method that harnesses the mismatch sensitivity of DNA ligase to determine the underlying sequence of nucleotides in a given DNA sequence (Ho et al, 2011). Platforms based on this method use a pool of oligonucleotide probes of varying lengths, which are labeled with fluorescent tags, depending on the nucleotide to be determined.…”

Section: Sequencing By Ligationmentioning

confidence: 99%