MLC-Seq: de novo Sequencing of Full-Length tRNAs and Quantitative Mapping of Multiple RNA Modifications

2021 ◽  
Author(s):  
Shenglong Zhang ◽  
Xiaohong Yuan ◽  
Yue Su ◽  
Xudong Zhang ◽  
Spencer Turkel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
De Novo ◽  
Direct Sequencing ◽  
Rna Modifications ◽  
Rna Sequences ◽  
Trna Modifications ◽  
Quantitative Mapping ◽  
Sequencing Method ◽  
Direct Sequencing Method ◽  
Generation Sequencing

Abstract Despite the extensive use of next-generation sequencing of RNA, simultaneous sequencing and quantitative mapping of multiple RNA modifications remain challenging. Herein, we develop MLC-Seq, a mass spectrometry-based direct sequencing method allowing for simultaneously unravelling the RNA sequences and quantitatively mapping different tRNA nucleotide modifications site-specifically. Importantly, MLC-Seq reveals the stoichiometric changes of tRNA modifications upon treatment with the dealkylating enzyme AlkB, and led to the discovery of new nucleotide modifications.

scholarly journals Clinical utility of the HCV drug resistance assay using the direct sequencing method and the cycleave PCR method in the dual therapy with daclatasvir and asunaprevir

Kanzo ◽  
2015 ◽  
Vol 56 (10) ◽  
pp. 533-535 ◽  
Author(s):  
Hideyuki Kudoh ◽  
Yoko Nagasawa ◽  
Michiru Ito ◽  
Nobuko Watanabe ◽  
Isao Naruse ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Utility ◽  
Direct Sequencing ◽  
Dual Therapy ◽  
Sequencing Method ◽  
Direct Sequencing Method ◽  
Pcr Method

scholarly journals It’s the Little Things (in Viral RNA)

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Jiří František Potužník ◽  
Hana Cahová
Keyword(s):  
Mass Spectrometry ◽  
Viral Rna ◽  
Detection Methods ◽  
Scientific Methodology ◽  
Rna Modifications ◽  
Chemical Modifications ◽  
Viral Protein Synthesis ◽  
Antibody Assays ◽  
Rna Export ◽  
Generation Sequencing

ABSTRACT Chemical modifications of viral RNA are an integral part of the viral life cycle and are present in most classes of viruses. To date, more than 170 RNA modifications have been discovered in all types of cellular RNA. Only a few, however, have been found in viral RNA, and the function of most of these has yet to be elucidated. Those few we have discovered and whose functions we understand have a varied effect on each virus. They facilitate RNA export from the nucleus, aid in viral protein synthesis, recruit host enzymes, and even interact with the host immune machinery. The most common methods for their study are mass spectrometry and antibody assays linked to next-generation sequencing. However, given that the actual amount of modified RNA can be very small, it is important to pair meticulous scientific methodology with the appropriate detection methods and to interpret the results with a grain of salt. Once discovered, RNA modifications enhance our understanding of viruses and present a potential target in combating them. This review provides a summary of the currently known chemical modifications of viral RNA, the effects they have on viral machinery, and the methods used to detect them.

scholarly journals A Functional Polymorphism in the Promoter Region of Interleukin-12B Increases the Risk of Colorectal Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
Yabin Liu ◽  
Binghui Li ◽  
Lili Wang ◽  
Dexian Kong
Keyword(s):  
Colorectal Cancer ◽  
Direct Sequencing ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Normal Tissues ◽  
Sequencing Method ◽  
Direct Sequencing Method ◽  
Healthy Control ◽  
Polymerase Chain ◽  
Interleukin 12B

Objective. To investigate whether the polymorphisms of interleukin-12B (IL-12B) were associated with the risk of developing colorectal cancer (CRC). Patients and Methods. Genotypes of rs17860508 and rs3212227 were determined by polymerase chain reaction with a direct sequencing method in 329 CRC patients and 342 matched healthy control subjects. The expression of IL-12B mRNA was determined by RT-qPCR in 50 pairs of CRC tissues and their adjacent normal tissues. Results. Compared with TTAGAG/TTAGAG genotype of rs17860508, the GC/GC and TTAGAG/GC genotypes may significantly increase the risk of CRC (OR = 1.81, 95% CI = 1.18–2.78; OR = 1.46, 95% CI = 1.01–2.12, respectively). Furthermore, the mRNA levels of IL-12B were significantly higher in the CRC tissues from patients with the rs17860508 GC/GC genotype than those with the TTAGAG/GC (P=0.009) and TTAGAG/TTAGAG (P=0.001) genotypes. Conclusion. These data suggested that the rs17860508 GC/GC genotype might upregulate IL-12B expression at the transcriptional level and thus increase the risk of CRC.

scholarly journals Emerging approaches for detection of methylation sites in RNA

Open Biology ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 180121 ◽  
Author(s):  
Anna Ovcharenko ◽  
Andrea Rentmeister
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Direct Detection ◽  
False Negative ◽  
Detection Methods ◽  
Rna Modifications ◽  
Negative Results ◽  
Third Generation Sequencing ◽  
False Negative Results ◽  
Generation Sequencing

RNA methylations play a significant regulatory role in diverse biological processes. Although the transcriptome-wide discovery of unknown RNA methylation sites is essential to elucidate their function, the development of a bigger variety of detection approaches is desirable for multiple reasons. Many established detection methods for RNA modifications heavily rely on the specificity of the respective antibodies. Thus, the development of antibody-independent transcriptome-wide methods is beneficial. Even the antibody-independent high-throughput sequencing-based methods are liable to produce false-positive or false-negative results. The development of an independent method for each modification could help validate the detected modification sites. Apart from the transcriptome-wide methods for methylation detection de novo , methods for monitoring the presence of a single methylation at a determined site are also needed. In contrast to the transcriptome-wide detection methods, the techniques used for monitoring purposes need to be cheap, fast and easy to perform. This review considers modern approaches for site-specific detection of methylated nucleotides in RNA. We also discuss the potential of third-generation sequencing methods for direct detection of RNA methylations.

scholarly journals Detected EGFR mutation in cerebrospinal fluid of lung adenocarcinoma patients with meningeal metastasis

Open Medicine ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. 93-96 ◽  
Author(s):  
Jiang Rong ◽  
Ma Chunhua ◽  
Lv Yuan ◽  
Mu Ning ◽  
Li Jinduo ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Lung Adenocarcinoma ◽  
Gene Mutation ◽  
Direct Sequencing ◽  
Gene Mutations ◽  
Egfr Mutations ◽  
Egfr Gene ◽  
Sequencing Method ◽  
Direct Sequencing Method ◽  
Egfr Gene Mutation

AbstractObjectiveTo discuss the application of ARMS method to detect EGFR gene mutation in cerebrospinal fluid of lung adenocarcinoma patients with meningeal metastasis.Methods5 cases of lung adenocarcinoma were identified with meningeal metastasis that were cleared EGFR gene mutation by gene sequencing method. From each patient 5ml cerebrospinal fluid was obtained by lumbar puncture. ARMS method was used to detect EGFR mutations in cerebrospinal fluid.Results5 samples of cerebrospinal fluid were successfully detected by ARMS method, 3 samples found that EGFR gene mutations, the mutations in line with direct sequencing method.ConclusionARMS method can be used to detect EGFR gene mutations of cerebrospinal fluid samples in lung adenocarcinoma with meningeal metastasis. But cerebrospinal fluid specimens from histological specimens, blood samples need to be confirmed by further comparative study whether there is advantage.

scholarly journals Variants of Interleukin-22 Gene Confer Predisposition to Autoimmune Thyroid Disease

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Rong-hua Song ◽  
Qian Li ◽  
Wen Wang ◽  
Qiu-ming Yao ◽  
Xiao-qing Shao ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Thyroid Disease ◽  
Gene Polymorphisms ◽  
Autoimmune Thyroid Disease ◽  
Direct Sequencing ◽  
Autoimmune Thyroid ◽  
Interleukin 22 ◽  
Sequencing Method ◽  
Thyroid Associated Ophthalmopathy ◽  
Direct Sequencing Method

As there are no previous studies on the interleukin-22 (IL-22) variants in autoimmune thyroid disease (AITD), the present study aimed to explore the association between polymorphisms of IL-22 and the predisposition to AITD. The study had 975 AITD patients, including 639 Graves’ disease (GD) and 336 Hashimoto’s thyroiditis (HT) individuals and 851 healthy cohorts. Ligase detection reaction (LDR) and direct sequencing method were used for genotyping the IL-22 gene polymorphisms at rs2046068, rs2227478, rs2227485, rs11611206, and rs1179251. In comparison to female controls, genotype CC of rs1179251 was increased in the female AITD patients. Alleles C at rs2046068, C at rs2227478, and C at rs1179251 linked to the susceptibility of HT males. Genotype CC in rs1179251 was higher in male HT. Variants at rs2046068, rs2227478, and rs1179251 were associated with the AITD teenagers. Besides, genotype GG in rs11611206 was correlated with thyroid-associated ophthalmopathy (TAO). Moreover, allele G at rs11611206 was associated with decreased risk for TAO by 28.9%. Similarly, genotype CC of rs1179251 and genotype GG of rs11611206 were associated with Graves’ ophthalmopathy (GO). Allele G in rs11611206 increased people with HT towards the predisposition of hypothyroidism. In conclusion, genetic variants of IL-22 are associated with the occurrence of AITD.

The analysis of EGFR expression and EGFR mutations in advanced pancreatic cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e15629-e15629 ◽  
Author(s):  
M. Ueno ◽  
S. Ohkawa ◽  
Y. Sakamoto ◽  
K. Miyakawa ◽  
Y. Miyagi
Keyword(s):  
Lung Cancer ◽  
Pancreatic Cancer ◽  
Cancer Patients ◽  
Direct Sequencing ◽  
Advanced Pancreatic Cancer ◽  
Phase Iii ◽  
Egfr Mutations ◽  
Egfr Expression ◽  
Sequencing Method ◽  
Direct Sequencing Method

e15629 Background: The standard chemotherapy of advanced pancreatic cancer is still gemcitabine and recently gemcitabine + EGFR tyrosine kinase inhibitor (TKI)is noted to be positive on Phase III study. In lung cancer, EGFR mutations (the deletion of exon 19, the point mutation of exon 18, 21) have been reported to be correlated with the effect of EGFR TKI. On the other hand, such EGFR mutations were not reported to be recognized by the direct sequencing method in pancreatic cancer. This time we examined EGFR expressions and EGFR mutations in advanced pancreatic cancer. Methods: We examined EGFR expressions immunohistochemically and EGFR mutations by Loop-Hybrid Mobility Shift Assay (LH-MSA) which is more sensitive than the direct sequencing method in the tissue obtained from percutaneous biopsies in advanced pancreatic cancer patients. In addition we examined the correlation between EGFR expressions and survivals by the log-rank test. Results: The subjects were 31 inoperable pancreatic cancer patients. Patients received chemotherapy (gemcitabine: 10, S-1: 8, gemcitabine+S-1: 12, no treatment: 1).The UICC stages were as follows:stage II; 2, stage III; 6, stage IV; 23. The tissues were obtaind from liver; 12, pancreas; 19. EGFR expressions were positive; 15, negative; 16. EGFR expressions were not correlated with survival (p=0.386). Although LH-MSA were performed successfully in all patients, the same EGFR mutations as lung cancer were not detected. Conclusions: EGFR expressions were not correlated with survivals and the same EGFR mutations as lung cancer were not detected in inoperable advanced pancreatic cancer. No significant financial relationships to disclose.

scholarly journals Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications

2016 ◽  
Vol 213 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Konstantin Licht ◽  
Michael F. Jantsch
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Rna Editing ◽  
Messenger Rna ◽  
Gene Expression Regulation ◽  
Rna Modification ◽  
Rna Modifications ◽  
Transcriptome Regulation ◽  
Affect Gene Expression ◽  
Generation Sequencing

Advances in next-generation sequencing and mass spectrometry have revealed widespread messenger RNA modifications and RNA editing, with dramatic effects on mammalian transcriptomes. Factors introducing, deleting, or interpreting specific modifications have been identified, and analogous with epigenetic terminology, have been designated “writers,” “erasers,” and “readers.” Such modifications in the transcriptome are referred to as epitranscriptomic changes and represent a fascinating new layer of gene expression regulation that has only recently been appreciated. Here, we outline how RNA editing and RNA modification can rapidly affect gene expression, making both processes as well suited to respond to cellular stress and to regulate the transcriptome during development or circadian periods.

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