Nanopore Direct RNA Sequencing

CD Genomics offers state-of-the-art Nanopore Direct RNA Sequencing services for accurate detection of RNA modifications. Our platform provides high-throughput analysis, delivering detailed insights into RNA structure and function. This service supports research in gene expression, transcriptomics, and molecular biology studies.

The Introduction of Nanopore Direct RNA Sequencing

Current the high-throughput sequencing of complementary DNA (cDNA) (NGS-RNA-seq) have enabled transcriptome-wide studies of RNA biology, These RNA-seq methods detect the products of a synthesis reaction rather than directly detecting the RNA molecule. New third-generation sequencing are being developed to overcome limitations such as amplification biases, lack of single-molecule sensitivity and isoform ambiguity. This improves the ease and speed of RNA analysis, while yielding richer biological information. Furthermore, NGS-RNA-seq is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples.

Here we CD Genomics provide nanopore direct single molecule RNA sequencing, a highly parallel, real-time, single-molecule method that without prior conversion of RNA to cDNA. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA, and provides a path to high-throughput and low-cost direct RNA sequencing, achieves the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.

Oxford Nanopore Technologies' nanopore-based platform detects single molecules of DNA, RNA, proteins and small molecules as they traverse through a nanopore, without the need for an enzymatic synthesis reaction. The platform consists of single nanopores embedded in an array of thousands of individual synthetic polymer membranes on a single flowcell. An electric potential drives DNA toward and into the nanopores. When a single DNA molecule is captured in a pore and ratcheted through the pore at a consistent rate by an engineered motor protein, it creates perturbations of the nanopore current which a recurrent neural network (RNN) converts into base sequences. This technology offers long sequencing reads (up to 2Mb) and detection of epigenetic markers.

Principle of Nanopore Direct RNA Sequencing

In nanopore sequencing, the motor proteins that sequence RNA strands progressively move along the native RNA strand. These enzymes operate in the opposite direction to DNA helicases, resulting in RNA strands translocating through the nanopore in the 3'–5' direction, contrasting with the 5'-3' direction of DNA signals. The motor protein in RNA sequencing adapters is specifically designed to control RNA passage through the nanopore, regulating RNA translocation at an average speed of 70 nucleotides per second. Compared to motor proteins used in cDNA-based sequencing, those employed in direct RNA sequencing significantly reduce speed, resulting in a notable decrease in throughput.

Key Features and Advantages of Nanopore Direct RNA Sequencing

• Low input amounts combined with rapid, streamlined workflows enable highly sensitive gene expression analysis.
• Full-length transcripts-The high yields of long, full-length reads delivered by nanopore sequencing allow unambiguous identification of splice variants and gene fusions
• Accurate transcript and isoform quantification
• Eliminate PCR bias using direct RNA sequencing
• Detect base modifications alongside nucleotide sequence using direct RNA
• Easy identification of anti-sense transcripts
• Accurately detect transcript poly(A) tail lengths.

Applications of Nanopore Direct RNA Sequencing

• Gene function: Focus on samples carrying specific functions to reveal the main causes of different functions
• Gene structure: Alternative splicing, APA, fusion gene, SSR, CDS prediction, TSS/TES identification, etc.
• Full-length transcript quantification: Find comprehensive and effective differential transcripts and identify functional genes
• RNA methylation: Direct full-length transcriptome sequencing can detect base modifications at the RNA level, such as m6A/m5C
• Virus RNA research
• Sequencing native RNA
• Exploring RNA modifications

Nanopore Direct RNA Sequencing Workflow

Direct RNA sequencing was performed using the nanopore sequencing platform, with 500-700 ng of poly(A) RNA (to ensure sufficient material remained for sequencing) used as input for library preparation. The RNA was ligated to a poly(T) adaptor using T4 DNA ligase. Following adaptor ligation, the products were purified by RNAClean beads. Sequencing adaptors preloaded with motor protein were then ligated onto the overhang of the previous adaptor using T4 DNA ligase, and the final library was cleaned up once more. The RNA library was quantified using a Qubit fluorometer. The final RNA libraries were added to flowcells and carried out on the nanopore sequencing platform.

Figure 1. Library preparation steps for direct RNA sequencing.

Service Specifications

	Sample Requirements Total RNA ≥ 15 μg RIN>8, 28S/18S≥1.5 OD260/280 ~2.0, OD260/230: 2.0-2.2 All RNA samples are validated for purity and quantity Note: Sample amounts are listed for reference only. For detailed information, please contact us with your customized requests.
Click	Sequencing Strategy ONT Platforms, Library: 8K, 2G/per sample
	Bioinformatics Analysis We provide multiple customized bioinformatics analyses: Raw data quality control RNA base modification detection Poly(A) tail length estimation Reference transcriptome alignment Gene function and transcript structure annotation Quantification of differential genes/transcript isoforms Functional enrichment and protein interaction analysis Note: Recommended data outputs and analysis contents displayed are for reference only. For detailed information, please contact us with your customized requests.

Analysis Pipeline

Deliverables

• The original sequencing data
• Experimental results
• Data analysis report
• Details in Nanopore Direct RNA Sequencing for your writing (customization)

Discover RNA modification capabilities with CD Genomics' Nanopore Direct RNA Sequencing service. We provide direct sequencing of RNA along with thorough bioinformatics analysis to reveal detailed genetic information. Contact us to elevate your research endeavors.

References

1. Garalde D R , Snell E A , Jachimowicz D , et al. Highly parallel direct RNA sequencing on an array of nanopores. Nature Methods, 2018, 15(3).
2. A I S , A K A C D S , C J M B , et al. Evaluating the potential of direct RNA nanopore sequencing: Metatranscriptomics highlights possible seasonal differences in a marine pelagic crustacean zooplankton community. Marine Environmental Research, 153.
3. Feng, Jiang, Jie, et al. Long-read direct RNA sequencing by 5'-Cap capturing reveals the impact of Piwi on the widespread exonization of transposable elements in locusts. Rna Biology, 2019.
4. Lorenz D A , Sathe S , Einstein J M , et al. Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base specific resolution. RNA, 2019, 26(1): rna.072785.119.