DNA sequencing is a common tool in many biological and bioinformatic fields. Since the Sanger sequencing, these technologies have been improving. Nowadays, third-generation methods are gaining popularity and surpassing second-generation in some parameters. In this topic, we are going to discuss the third-gen technology Oxford Nanopore.
Sequencing concept
Oxford Nanopore belongs to the third-gen sequencing (TGS) method. In contrast to NGS, TGS technologies detect signals from a single DNA molecule and produce much longer reads at a cost of accuracy. The core of Nanopore sequencing technology is protein nanopore, which creates holes in biological membranes. These proteins are incorporated into a synthetic membrane and submerged in an electrophysiological solution. A potential is applied across the membrane, creating an ionic current through the nanopore. When a single molecule passes through the aperture of the nanopore it causes alteration in ionic current. These alterations are specific to various molecules. We can detect ionic current changes in the nanopore and therefore identify molecules. Since nucleotide bases have different structures, we can distinguish them too.
The nanopore protein is designed to allow only single-stranded DNA through. During sample preparation, a single-stranded adapter sequence is added to the end of a DNA molecule, and a specific motor protein binds to this adapter sequence. The motor protein unzips double-stranded DNA and passes it through the nanopore one base at a time.
As was said before, different molecules cause specific changes in the ionic current. Here we measure ionic current alterations in the narrowest part of the nanopore channel, induced by a combination of k nucleotides (called k-mer). The alterations are specific to a particular k-mer. Since the ionic current is measured every time DNA shifts by one base, we can identify single nucleotides and determine the order of the bases. Thus, the technology converts ionic current alterations caused by a passing DNA molecule into a nucleotide sequence. This approach allows not only the sequencing of one strand but also of both strands in a run. We can add a hairpin sequence to one end of double-stranded DNA. After the DNA helix unzips, strands connected through the oligonucleotide sequence become the same strand. In such a way, we can sequence both strands in one run as one.
Nanopore advantages
The Nanopore sequencing technology has several advantages over NGS technologies. It produces much longer reads than NGS platforms, like Illumina. Nanopore does not require preliminary DNA cutting and theoretically could process a whole DNA molecule presented to it. However, read length is still limited by the sample preparation step, which can't preserve too long intact DNA pieces. There are also difficulties in delivering long DNA molecules to the nanopore. Nanopore's typical read length accounts for 10-100 Kb compared to Illumina's 50-300 bp. The technology has a special sequencing mode that produces ultra-long reads of 100-300 Kb in length.
Unlike NGS technologies, Nanopore sequencing does not require template amplification and therefore is free from amplification bias. Amplification bias emerges because various DNA templates have different amplification efficiency. Thus, after cDNA library creation, we are more likely to sequence one type of template but not the other. However, the amplification step can be performed if needed.
Nanopore allows real-time sequencing. The user can get sequence information as soon as the detector recognizes a nucleotide in the nanopore and sends the information to the computer. Therefore, a researcher can detect the presence of a particular mutation in real time and not wait for the end of the sequencing process.
The technology performs direct sequencing of native DNA or even RNA without converting it to a cDNA library. Nucleotides in native DNA or RNA are sometimes modified. These modifications have biological meanings. Nanopore detects modified nucleotides and notes this information in resulted sequence.
Portability is another advantage of this technology. Nanopore offers small convenient handheld units. They make sequencing available in any location, not only in a lab. There are also benchtop devices for extensive lab experiments. Handheld units and benchtop devices both use the same core technology.
Nanopore limitations
Nanopore sequencing also has its drawbacks. It has a much higher error rate than Illumina. Nanopore struggles with homopolymer regions in DNA. The speed of DNA translocation through the nanopore is not constant over time, but there are minor variations in speed. It causes certain challenges in determining the exact length of homopolymer when the device detects a prolonged monotonous signal from the sequence. As a result, the technology is prone to insertion and deletion types of errors. Nanopore error rates correspond to 1.6–2.7% for deletions, 1.2–2.2% for mismatches, and 1.1–2.4% for insertions. For comparison, Illumina has around 0.1-1% substitution rates and lower than 0.01% insertion or deletion rates. Remember that these numbers are approximate and partly depend on the sample type and preparation method.
Nanopore loses to Illumina in terms of price. The average sequencing cost per gigabase of Nanopore platforms ranges from 50 to 2000 USD per Gb. For comparison, Illumina's average price is 40-63 USD per Gb, with the lowest cost of 10-35 USD per Gb for the NovaSeq 6000 platform. The large-scale Nanopore platform, PromethION, has a comparable cost to Illumina of 21-42 USD per Gb.
Applications
Long-read sequencing is widely used in genome assembly. It can resolve repetitive regions, which poses a challenge to Illumina short reads data. In the field of metagenomics, long reads are handy to describe microbial diversity in a sample. Nanopore also makes it possible to detect structural variants. Indeed, structural variants are large genomic alterations that may reach up to the megabase scale, and can not be spanned by short reads.
As we are able to sequence whole RNA molecules at a time, long-read sequencing allows more accurate RNA isoform characterization. The opportunity of direct RNA sequencing allows dynamic tracking of virus evolution during a pandemic.
Conclusion
Nanopore technology determines the order of DNA bases by detecting a sequence of nucleotide-specific ionic current alterations induced by DNA translocation through the protein nanopore. The technology produces much longer reads than Illumina but suffers from a higher error rate. These features determine Nanopore application fields. Nanopore long reads are used in genome assembly, RNA isoform characterization, species identification in metagenomics, etc.