Dataset Information

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

ABSTRACT: Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ?50% were detected in defined amplicons, and this threshold can be lowered to ?10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.

SUBMITTER: Werner S

PROVIDER: S-EPMC7913672 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Werner Stephan S Galliot Aurellia A Pichot Florian F Kemmer Thomas T Marchand Virginie V Sednev Maksim V MV Lence Tina T Roignant Jean-Yves JY König Julian J Höbartner Claudia C Motorin Yuri Y Hildebrandt Andreas A Helm Mark M

Nucleic acids research 20210201 4

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6 ...[more]

PMID: 33313868

Similar Datasets

Project description:Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R²?=?0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R²?=?0.97-0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R²??=?0.96) and whole transcriptome RNA-sequencing following "traditional" library preparation using Illumina NGS kits (R²?=?0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10?-fold range was reduced more than 10,000-fold, from 2.3×10? to 1.4×10? sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

Project description:High-throughput amplicon sequencing is a critical tool for studying microbiota; however, it results only in relative abundance data. Thus, changes in absolute abundance of microbiota cannot be determined, which hinders further microbiology research. We have therefore established a gradient internal standard absolute quantification (GIS-AQ) method to overcome this issue, which can simultaneously obtain the absolute abundances of bacteria and fungi. Deviations from the quantitative equations of microbes and internal standards were eliminated through calibration. Compared with traditional quantitative real-time PCR and microscopy quantifications, this method is reliable (R2average = 0.998; P < 0.001) and accurate (Pinternals versus microscopy > 0.05). The GIS-AQ method can be adapted to any amplicon primer choice (e.g., 336F/806R and ITS3/ITS4), rendering it applicable to ecosystem studies including food, soil, and water samples. Crucially, when using solid-state fermentation samples from various temporal dimensions, the results obtained from the relative and absolute abundance are different. The absolute abundance can be used to study the difference in communities between different samples, and the GIS-AQ method allows this to be done rapidly. Therefore, combining the absolute abundance with relative abundance can accurately reflect the microbiota composition.IMPORTANCE To solve the problem of amplicon sequencing cannot discern the microbiota absolute abundance, we proposed a gradient internal standard absolute quantification method. We used Chinese liquor fermentation as a model system to demonstrate the reliability and accuracy of the method. By comparing the relative and absolute abundances of microbiota in various temporal dimensions, we found dynamic changes in the absolute abundance of communities under various temporal dimensions from the relative abundance. Based on its design principle, this method can be widely applied to different ecosystems. Therefore, we believe that the GIS-AQ method can play an immeasurably useful role in microbiological research.

Dataset Information

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Publications

NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets