Nanopore Direct RNA Sequencing

Nanopore direct RNA sequencing is a third generation sequencing (TGS)-based method to sequence the whole transcriptome of cells or tissues. We provide nanopore direct RNA sequencing to help you resolve structural variants and repetitive regions, enhance metagenomic identification of closely related species, sequence entire microbes in single reads-in real-time.

Overview

Nanopore direct RNA sequencing is one kind of long-read sequencing, emerged in 2011, provided by Oxford Nanopore Technologies (ONT). Nanopore direct RNA sequencing provides a direct, real-time sequencing of long RNA fragments. Nanopore direct RNA sequencing using nanopore inserted in an electrical resistant membrane, there are 2048 nanopores on the membrane. As the RNA fragments pass through the nanopores, the bound polymerase or enzyme can attach to the RNA, the electrical current change was measured, indicating a specific RNA sequence. Nanopore direct RNA sequencing allows ultra-long reads, the longest fragment is close to 1 million bp. Advantages of Nanopore direct RNA sequencing include: 1) direct, real-time analysis; 2) allowing ultra-long kilobases in a continuous read; 3) pocket-sized sequencers are portable with low cost.

We provide Nanopore direct RNA sequencing services for identifying new genes, transcripts, and alternative splicing events, resolving structural variants and repetitive regions, enhancing metagenomic identification of closely related species, sequencing entire microbes in single reads-in real-time. Nanopore direct RNA sequencing also performs rapid pathogen detection and strain characterization, for example, it can be used to screen Ebola and Lassa virus outbreak in Africa.

Features

High throughput Real-time Analysis High Resolution Multiple Applications
This method allow long reads for accurate analysis with a short sequencing time. Direct, real-time sequencing of long RNA fragments. Discovery of more cellular differences based on high resolution analysis. Identify new genes, transcripts and alternative splicing events.

Project Workflow

Sample Preparation

1. Sample Preparation

RNA purification; quality assessment and quantification.

Library Preparation

2. Library Preparation

DNA library preparation.

Sequencing

3. Sequencing

Real-time analysis, takes as little as 10 minutes to perform.

Data Analysis

4. Data Analysis

Visualize and preprocess results, and perform custom bioinformatics analysis.

Nanopore direct RNA sequencing

Data Analysis Workflow

In-depth data analysis:

  1. Identification of new genes
  2. Identification of new transcripts
  3. Alternative splicing events comparation between species
  4. Metagenomic identification of closely related species
  5. Sequencing entire microbes in single reads-in real time
  6. Pathogen detection and strain characterization

Sample Requirements

RNA sample (quantity ≥ 100 pg), 1.8 ≤ OD260/280 ≤ 2.2, OD260/230≥2.0, RIN ≥ 6.5, 28S:18S≥1.0. Please make sure that RNA is not significantly degraded.

Sample storage: The sample should be stored at -80°C. Avoid repeated freezing and thawing.

Shipping Method: When shipping the sample, it is stored in a 1.5 mL Eppendorf tube, sealed with a sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable: FastQ, raw data, coverage summary, QC report, experiment results, custom bioinformatics analysis.

References:

  1. Xiao T, Zhou W. The third generation sequencing: the advanced approach to genetic diseases. Translational Pediatrics, 2020, 9(2): 163–173.
  2. Schatz MC. Nanopore sequencing meets epigenetics. Nature Methods, 2017, 14: 347-348.
* For Research Use Only. Not for use in diagnostic procedures.


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