6533b82efe1ef96bd1292806
RESEARCH PRODUCT
High-throughput sequencing for 1-methyladenosine (m1A) mapping in RNA
Mark HelmLyudmil TserovskiFlorence Blanloeil-oilloYuri MotorinVirginie MarchandRalf Hauenschildsubject
0301 basic medicineAdenosineLibrary preparationSequencing dataBiologyGeneral Biochemistry Genetics and Molecular BiologyDNA sequencingPrimer extension03 medical and health sciencesComplementary DNANucleotideRNA Processing Post-Transcriptional[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biochemistry [q-bio.BM]Molecular BiologyComputingMilieux_MISCELLANEOUSGene LibraryGeneticschemistry.chemical_classificationRNAHigh-Throughput Nucleotide Sequencing[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry Molecular Biology/Molecular biologyReverse transcriptase030104 developmental biologychemistryRNA[SDV.MHEP]Life Sciences [q-bio]/Human health and pathologydescription
Abstract Detection and mapping of modified nucleotides in RNAs is a difficult and laborious task. Several physico-chemical approaches based on differential properties of modified nucleotides can be used, however, most of these methods do not allow high-throughput analysis. Here we describe in details a method for mapping of rather common 1-methyladenosine (m1A) residues using high-throughput next generation sequencing (NGS). Since m1A residues block primer extension during reverse transcription (RT), the accumulation of abortive products as well as the nucleotide misincorporation can be detected in the sequencing data. The described library preparation protocol allows to capture both types of cDNA products essential for further bioinformatic analysis. We demonstrate that m1A residues produce characteristic arrest and mismatch rates and combination of both can be used for their detection as well as for discrimination of m1A from other modified A residues present in RNAs.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2016-09-01 |