Nanopore sequencing

The Introduction of Nanopore Sequencing

Oxford Nanopore Technologies has developed the nanopore-based DNA and RNA sequencing technology. The Nanopore sequencer is proven compatible with a variety of input material such as genomic DNA, amplified DNA, cDNA, and RNA. Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance.

Compared to the traditional workflow, the Nanopore sequencer has some highlighted advantages.

• For high molecular weight DNA (HMW-DNA) samples, ultra-long read lengths of several hundred kb may be sequenced in a single continuous read. The Nanopore sequencing data significantly improve de novo genome assemblies and structural genomic variant and transcriptome studies.
• The nanopore is nano-scale holes in nature form gateways across membranes. A nanopore passes an ionic current through nanopores and measures the changes in current. As molecules such as DNA or RNA move through the nanopores, they cause disruption in the current. The information about the change in current can be used to identify that molecule. It directly sequences the native strand of interest, without optics or amplification. Different types of library preparation protocols allow for the direct DNA/RNA sequencing with epigenetic information.

  Fig.1 How Nanopore sequencing works

• Real-time streaming of sequence data allows rapid insight into samples, on-demand sequencing, and dynamic workflows.

Our Nanopore Sequencing Service

CD genomics offers nanopore sequencing as a service. The PromethION offers on-demand use of up to 48 Flow Cells – each of which can generate up to 100Gb of sequencing data.

Our nanopore sequencing service portfolios include:

• Nanopore Full-Length Transcripts Sequencing
• Nanopore Direct RNA Sequencing

Fig.2 PromethION

Sample Requirements

DNA

• It is recommended to isolate the DNA using Qiagen DNeasy kit and treated with RNase.
• OD 260/280 of 1.8 and OD 260/230 of 2.0-2.2.
• Average fragment size, as measured by pulse-field, or low percentage agarose gel analysis >30 kb.
• Input mass, measured by Qubit, >5 µg at a concentration of 100ng/µL.
• No detergents or surfactants in the buffer, 10 mM TRIS (pH=8.0-8.4) is recommended.

RNA

• RIN, not less than 8.0.
• Input mass, as measured by Qubit RNA HS assay, >1 µg at a minimum concentration of 50 ng/µL.
• A 260:280 ratio of ~2.0. A 260:230 ratio of 2.0-2.2.
• No detergents or surfactants in the buffer.