Nanopore sequencing
How does nanopore sequencing work in a nutshell:
Nanopores are tiny synthetic protein pores (think of them as little tunnels) attached to a membrane, that enable the passage of DNA through them. Take into account that there is a constant internal electrical current caused by the passage of ions (i.e electrically charged atoms) through the nanopore. When a DNA molecule passes through the nanopore, each nucleotide will cause a small disruption of the pores internal ionic current. This disruption is then measured by a chip with at a 5 kHz frequency. The measured electrical current disruption is then outputted as a “squiggle” sgnal which is then saved in fast5 or pod5 format computer files. As specific combinations of nucleotides result in characteristic disruptions of the ionic current, this squiggle signal can be translated into genetic code using special algorithms such as neural networks. In this case, the production of squiggle and genetic sequence data is referred to as “sequencing” while the translation from squiggle signal to DNA sequence data alone is generally referred to as “basecalling”. Nanopore technology allows for real- time basecalling, reducing the time gap between sample sequencing and data production. Basecalling can also be done afterwards which is usually done when using the more accurate but computationally demanding basecalling algorithms. These algorithms are referred to as: Fast, High Accuracy (HAC) and Super High Accuracy (SUP) algorithms, representing a tradeoff between sequence accuracy and the computational resources needed to run them.
Nucleotide refers to one any of the 4 molecular building blocks, or bases that build the so-called genetic code in DNA: these are adenine (A), cytosine (C), guanine (G), and thymine (T))
In this case, a sequencing read is and inferred sequence of squiggle or more commonly base pairs (i.e basecalled squiggle signal) that corresponds to all or part of a single DNA fragment.
Read accuracy: Then and now
Historically nanopore sequencing has had a higher error rate than other DNA sequencing technologies. Some studies in recent years reported an inflated sequencing read error rate of up to 8% which arose mainly from difficulties of the nanopores to accurately distinguish homopolymers.
However, thanks to improvements in flow cells, reaction chemistry, and bioinformatics together with duplex sequencing (basecalling the forward and reverse‐complementary DNA strands in tandem), this sequencing read error rate has decreased to 0.6%. Several studies have shown that nanopore can produce DNA barcodes that are >99.99% identical to Sanger barcodes, at a fraction of the cost.
Homopolymer: A molecule made from a single repeated unit. In DNA sequences this would be one of the 4 bases (i.e A, C, G, or T) repeated several times in a row.
DNA barcode: A relatively short (<1kb) DNA sequence that contains enough variation to distinguish between species. The name barcodes derives from it fulfiling a very similar function than normal warehouse inventory barcodes.
Read length and throughput
Nanopore sequencing is one of the only two sequencing technologies capable of producing accurate long reads. When using regular sequencing kits most reads tend to be around 10-100 kb in length while using ultra long read sequencing kits will lead to an increase in reads of 100 to 300 kb in length provided that the DNA is of good quality. In some instances extremely long reads, called “whales” of several Megabases (Mb) in length have been reported.
The amount of data produced by a nanopore sequencing will depend on the model. For example: while a MinION will produce on average 10-20 Gb per flowcell, while the bigger PromethION can produce up to 200 Gb per flow cell.
Kilobase (kb) = 1000 bases (nucleotides) Megabase (Mb) = 1,000,000 bases Gigabase (Gb) = 1,000,000,000 bases
How can we benefit from nanopore sequencing for biodiversity monitoring
Perhaps the most attractive features of nanopore sequencing when it comes to conservation and biodiversity monitoring, is the portability and low investment cost of its devices. The MinION, the smallest of the sequencers can easily fit in your pocket and can be acquired for around ~2,000$. This essentially gives you the power to sequence DNA anywhere with an initial investment cost order of magnitude below that of other technologies.
Being able to sequence on the study site or in-situ has several advantages. It allows to employ novel genetic biodiversity monitoring approaches in highly biodiverse countries which very frequently lack the necessary infrastructure, equipment and expertise to do so. Thus, portable sequencing can democratize these methods through local capacity building. Furthermore, sequencing on site can cut the gap between sample collection and data production, by reducing sample transportation time as well as reduce the costs and paperwork often required for sample export.
Coupled with environmental DNA sampling approaches, nanopore portable sequencing offers a new way of monitoring biodiversity. The data indicating the presence of one or several species can be produced and analyzed close to the sample source. If you want to learn more about how we are using nanopore sequencing for eDNA based in situ monitoring of Zambian wildlife please read our related blog article.
