Adventures with MinION Part I: Understanding ONT’s DNA sequencing technology
The 50th anniversary of the historic double helix DNA model came three years after the completion of the Human Genome Project (HGP). Providing industrialized methods of DNA sequencing, this project lead the way for the development of Next-Gen sequencing (NGS). The development of high through-put modes of DNA sequencing increased affordability, but still remains costly. Recognizing this problem, companies like Oxford Nanopore Technologies (ONT) have made it their mission to take DNA sequencing affordability to the next level. With the goal of “enabling the analysis of any living thing, by any person, in any environment,” the UK based company has set a high bar. Visit their website.
What ONT Does:
Offering devices both small enough to plug into your smart phone and those suitable for a core facility, this company delivers on their promise of portability and scalability. The second smallest of these sequencing devices, the MinION, is a handheld device that connects to a computer through a USB cable. Currently, the MinION and flow cell each cost ~$1000. This is a far cry away from the Illumina’s iSeq with a price tag of $19,000.
In addition, ONT provides library prep kits for approximately $700. One of which boasts a 10-minute prep time! Another benefit to using ONT’s technology is their online forum, containing massive amounts of useful information from expert users. Purchasing any of the ONT sequencers also gives you access to their sequencing software, MinKNOW.
How the MinION works:
DNA libraries, after prepping, are loaded through the SpotOn port which places the samples on top of the sensory array. This compact sensor array houses an electrically resistant membrane scaffold containing all the nanopores. When sequencing begins, an electric potential is applied to the membrane and an ionic current is generated across the nanopore. Adaptors, which are attached to the DNA fragments during the library prep, guide the DNA into the nanopores (learn more about adaptors here). An exonuclease guards the pore’s entrance, cleaving individual nucleotides from the strand before entry (Figure 1). The passing of nucleotides through the opening of the pore disrupts the ion flow, causing a characteristic disruption in the current. This disruption, unique to every class of nucleotide, is the bass of the signal. For Nanopore community members, check out an in-depth video explaining how the Minion functions, here.
MinION Flowcell Design:
Each MinION device functions with the addition of flowcells, which contain a sensor array (Figure 2). The array houses 512 channels, each containing four wells. Each well is embedded in the membrane scaffold and houses a nanopore, for a total of 2048 nanopores. Each channel is controlled and measured individually by an electrode which is a part of the Application Specific Integrated Circuit (ASIC). While each channel has four embedded nanopores, the channel only functions when exactly one nanopore is relaying sequencing information. This limitation arises from the sensor array design where a single electrode is responsible for relaying information from 4 wells. Learn more about how the Flow cell works, here.
Requirements for a sequencing run:
There are a number of stringent requirements that are necessary to produce a successful sequencing run. First, the technological capabilities of your lab are a crucial requirement (figure 3). Second, starting with high quality, high molecular weight DNA is paramount. Low quality DNA greatly effects downstream processes, data quality, and data analysis (see figure 4).
In addition to isolating high quality DNA, deciding on the optimal
DNA kit for your specific application is crucial. The kits are optimized for a variety applications, so it is important to choose the kit that fits your DNA sequencing needs (for new users: find descriptions of the kits here. If you are a member of the Nanopore community, access more selective options here).
Base Calling Accuracy: Quality Score
Accurate DNA sequencing requires high quality data. NGS technologies utilize the Phred quality score(Q) when judging the accuracy of base calls. Moreover, the quality score (QS) indicates the specific
likelihood of error for a specific base, where P is the base calling error probability. Q is a property that is logarithmically related to P, shown in figure 5.
With the Illumina Phred Quality scoring system, a base with a quality score of 10 indicates there is a 1 in 10 probability the base was called incorrectly (accuracy of 90%). Furthermore, a QS of 20 indicates a 1 in 100 probability of an incorrect base call ( accuracy of 99%). Obviously, a higher Phred score is more desirable and gives researchers more confidence that the called base is the ‘true’ base.
Clearly, a higher QS is most desirable.
ONT asserts that a QS of 10 relays a base-calling accuracy of 90%. However, some data produced by users in the Nanopore Community tells a different story. Based on their analysis (found here), a quality score of 15.5 indicates 90% accuracy. MinION Q-scores produced by the live base-calling software, MinKNOW, are based on a Phred scoring method but are also encrypted with an ASCII 33 Sanger format, denoted Phred+33
Now you are equipped with the basic knowledge of Oxford nanopore’s MinION. For more information on the Peccoud Lab’s experience with the technology, stay tuned for the rest of the blog series.