Nanopore Sequencing

What is 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.

How Does Nanopore Protein Sequencing Work?

Nanopore sequencing, as the term implies, fundamentally operates by exploiting a nanopore, to which a molecular adaptor is covalently attached. Upon securing the nanopore protein onto a resistive film, motor proteins are employed to guide nucleic acids through the nanopore. As nucleic acids traverse the nanopore, charge alterations take place, provoking changes in the current on the resistive film. Given the highly diminutive diameter of the nanopore, only single nucleic acid polymers are permitted passage. As each nucleic acid base - adenine (A), thymine (T), cytosine (C), and guanine (G) - possesses unique charge characteristics, they induce distinctive disruptions in the current when channeled through the protein nanopore. By real-time monitoring and interpreting these current signals, the base sequence can be determined, thereby enabling sequencing. For a more in-depth exploration of the underlying principles, please refer to the article "Principle of Nanopore Sequencing".

Fig.1 How Nanopore sequencing works

Advantages of Nanopore Sequencing

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

Application of Nanopore Sequencing

Nanopore Sequencing in Whole Genome Assembly

In historical short-segment genome assembly practices, the complexities of some animal and plant genomes—characterized by polyploidy, high repetition, and high heterozygosity—have proved to be profoundly challenging for successful genomic assembly. Nevertheless, Nanopore sequencing technology, with its inherent long-read characteristic, is advantageous in fostering the assembly of large genomes. This can substantially augment the integrity of the assembled genome.

Nanopore Sequencing For Full-length Transcriptome

Previous transcriptome analyses could not directly sequence RNA, often requiring fragmentation of mRNA, followed by reverse transcription into cDNA, thereby not capturing and analyzing the full-length transcript. The long-read feature of Nanopore sequencing can accurately identify multiple isoforms of each gene, simplifying the process while maintaining precision. Moreover, this technique permits direct RNA sequencing, thereby identifying RNA base modifications directly.

Nanopore Sequencing In Detecting Large Structural Variations

Numerous large-structure variations (like deletions, inversions, and translocations etc.), often associated with human diseases, occur in the genome. Short-read sequencing is incapable of detecting these variations accurately. However, Nanopore sequencing, with its longer-read length, is more apt for detecting these large structural variations, demonstrating promising prospects in disease research and beyond.

Nanopore Sequencing For Rapid Identification Of Microbiota

The Nanopore sequencing technology's expeditious and real-time attributes have expedited the identification of microbiota. This technological innovation enables field-based, point-of-collection sequencing, which has streamlined the process of sequence information acquisition. Subsequently, this allows for taxonomic classification as well as identification of various microbial species with increased efficiency and pacing. Thus, Nanopore sequencing has significantly contributed to the rapid elucidation of microbial identities.

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 full-length transcripts sequencing captures entire RNA molecules, providing insights into transcript structures and splice variants. This technique allows for comprehensive transcriptome analysis without the need for amplification. CD Genomics offers this service to help researchers gain detailed insights into gene expression and transcript diversity.

• Nanopore Direct RNA Sequencing

  • CD Genomics provides cutting-edge direct RNA sequencing services, enabling researchers to read RNA molecules directly and preserve the native transcript sequences and modifications. This method eliminates the need for cDNA conversion, reducing potential biases, and facilitating accurate gene expression profiling and RNA modification analysis.

• Nanopore metagenomics sequencing

  • Nanopore metagenomics sequencing enables the analysis of complex microbial communities by sequencing all DNA present in a sample. This approach helps in identifying and characterizing diverse microorganisms and their functions. CD Genomics offers comprehensive metagenomics sequencing solutions to uncover the intricacies of microbial ecosystems.

• Nanopore de novo whole genome requencing

  • CD Genomics provides advanced de novo sequencing services to support groundbreaking genomic research. Nanopore de novo whole genome resequencing allows for the assembly of entire genomes from scratch, providing detailed genomic information without relying on reference genomes. This technique is particularly useful for studying novel or non-model organisms.

We aslo provide long read sequencing service By taking advantage of PacBio SMRT long reads sequencing technology.

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, >2 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.

Full-length transcript characterization of SF3B1mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

SF3B1 is the encoding gene for the pivotal spliceosomal U2 snRNA, and mutations in this gene have been associated with the development of diseases such as chronic lymphocytic leukemia (CLL), breast cancer, and myelodysplastic syndromes, with the highest prevalence in CLL patients. In this study, three samples of CLL patients with no SF3B1 mutation (CLL SF3B1WT), three samples of CLL patients with SF3B1K700E mutation (CLL SF3B1K700E), and three samples of normal B lymphocytes were selected as research subjects. Whole transcriptome sequencing was conducted using the Nanopore technology platform, and the FLAIR workflow was developed to identify high-confidence transcripts. This led to the discovery of changes in splice sites associated with SF3B1 mutations, changes in retained intron transcripts related to SF3B1 mutations, and variations in both effective and ineffective isoforms.

Fig.2 PromethION

Reference:

1. Tang, A.D., Soulette, C.M., van Baren, M.J. et al. Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns. Nat Commun 11, 1438 (2020).