Nanopore Direct RNA Sequencing

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.

Key Features and Advantages

• 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

Project 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.

Service Specifications

	Sample Requirements Total RNA ≥ 1µg, Tissue: 500mg-1g; Cell ≥108 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
	Sequencing Strategy ONT Platforms, Library: 8K, 2G/per sample
	Application 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

References:
[1] Garalde D R , Snell E A , Jachimowicz D , et al. Highly parallel direct RNA sequencing on an array of nanopores[J]. 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[J]. 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.[J]. 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[J]. RNA, 2019, 26(1):rna.072785.119.