CD Genomics Perspective: the Principles of Nanopore Array for Direct RNA Sequencing

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Posted on: 30 September 2021 by chen shanshan

CD Genomics Perspective: the Principles of Nanopore Array for Direct RNA Sequencing

Introduction to RNA sequencing

The transcriptome of a cell includes rich information, such as gene structure (such as splice variants and fusion genes), various transcript tiers of expression, and transcription of antisense. A technique to best obtain this data is one that is effective, strand-specific, quantitative across a wide diverse spectrum, does not require previous sequence information, can expose the existence and individuality of altered bases, and can identify transcripts of antisense without worrying that these are library preparation artifacts. Ideally, constant sequence reads spanning any splice junctions would also be generated by the technique. Based on the high sequencing of complementary DNA (cDNA), existing sequencing-based transcriptomic analyses (RNA-seq) have allowed us to create a more precise view of the active transcriptional trends within organisms. The most used RNA-seq approach includes whether in priming of polydeoxythymidine (poly(dT)) or fragmentation of RNA and random hexamer priming, preceded by the production of cDNA. PCR amplifies these cDNA strands, which can incorporate bias3, like decreased cDNA library complexity, disruption of relative cDNA abundances, and relapse of some RNA species. These problems would be sidestepped by an amplification-free library preparation 4. Consequently, any alterations to the RNA are lost during PCR amplification.

 

For RNA-seq library preparation, FRT-seq5, and the DRS method on the Helicos framework, two existing techniques do not involve PCR amplification, but both techniques create short sequence reads, which can make it hard to properly determine substitute splicing in eukaryotes. By integrating a long-read sequencing innovation with a library preparation technique that retains the integrity of the RNA being assessed, this issue should be resolvable. The products of a synthesis reaction are detected by all prior RNA-seq techniques rather than directly identifying the RNA molecule. Sequences produced with these techniques are therefore subject to the restrictions of reverse transcription processivity and error rate and either cannot identify base alterations or cannot differentiate homopolymers.

 

Nanopore sequencing

Our potential to sequence genomes and transcriptomes has been pioneered by the introduction of third-generation sequencing (TGS) innovations. TGS can generate long sequencing reads in comparison to second-generation sequencing innovations, ignoring the inconvenience of fragmenting the RNA or DNA molecules into smaller parts to reconfigure them together again. In addition, without a PCR amplification phase, TGS techniques can sequence DNA and RNA, thus enabling direct identification of DNA and RNA alterations, with single-nucleotide resolution and in specific molecules.

 

The framework provided by Oxford Nanopore Technologies can be used to obtain direct sequencing of native RNA molecules (dRNA-seq) ONT. This framework focuses on the usage of protein nanopores that are confined to an electric field, integrated into a membrane. As the RNA molecule moves through the pore, characteristic disturbances in the electric current are assessed, allowing one single molecule to be observed. The RNA molecule's low translocation velocity is attained by combining motor proteins that control the nucleic acid molecule's translocation, and the generated existing intensity readings can, in turn, be transformed to sequence data using initially trained base-calling methodologies.

 

Principles of nanopore-based platform

The framework contains single nanopores integrated on one single flowcell in an array of hundreds of individual synthetic polymer membranes. An electrical potential fuels DNA toward the nanopores and into them. When a single DNA molecule is collected in a pore and ramped by an engineered motor protein through the pore at a sustained frequency, it produces nanopore current disruptions, which are converted into base sequences by a recurrent neural network (RNN).

 

But there are still limitations

Nanopore sequencing allows immediate assessment without conversion to cDNA of RNA molecules, thus opening the doors to a new era for RNA biology. The absence of direct RNA nanopore sequencing databases for molecular barcoding has a serious impact on the suitability of this innovation to biological specimens, where the accessibility of RNA is often restricted.

 

About CD Genomics

CD Genomics provides the research community with high-quality next-generation sequencing, third-generation sequencing, genotyping, microarray, and population genetics services. CD Genomics has become an influential company in the industry and continues to innovate, keeping up with the forefront of scientific research and leading the latest and most comprehensive genomics technical support.

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howar dellis posted 04 October 2021

RNA research is also a process that interests me.  Instead of directly identifying the RNA molecule, all previous RNA-seq techniques detected the fusion products. 

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Gabriella gabriella123 posted 14 October 2021

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