Genetic sequencing is one the major methods to look into the genetic information. It is of great significance in studies of genomics, medicine, drug development and agricultural breeding. In 1977, Sanger proposed the Chain Termination Method (also known as Sanger sequencing) using 4 radioactive dideoxynucleosides (ddNTP), and sequenced the first genome (Phage PhiX-174, 5375 bp). This marked the beginning of a sequencing era. By then, the base sequence could not be read directly from the original nucleotide but could only be determined in the synthesis of new DNA strand. Sanger sequencing has decent read length and accuracy, but complicated sample processing and low throughput, making parallel-sequencing of multiple DNA impossible.
In the 2010s, the Next-Generation Sequencing (NGS) entered market, featured of Miseq platform by Illumina Incorporation and 454 Pyrosequencing by Roche. Compared with Sanger sequencing, the cost and required time of NGS are significantly reduced. Previously, it requires 3 years to sequence a complete human genome, now it is only a week [1]. Meanwhile the output has expanded by 1000 times, for example, using a few microliters sample, NGS can be massively parallel and generate up to 1000 giga bases per round, therefore NGS is also known as High-Throughput Sequencing. The biggest limitation lies in the compromised read length, for only 100-250 bp.
The 3rd generation sequencing, featured of SMRT by PacBio Incorporation and Nanopore by Oxford Nanopore Technologies (ONT), targets single-molecule sequencing. In Nanopore sequencing, nucleotide molecules are placed in an electric field to direct them to pass through a pore, which is in nanometers and allows one DNA/RNA molecule to pass through at a time. When passing through the nanopore, 4 different bases provide 4 different electric signals and therefore by detecting such signals, the sequencer can identify which base it is [2]. This technique does not include an amplification step (i.e. PCR) so will not introduce amplification bias [3]. Theoretically there is no upper limit of the read length as long as the DNA strand remains intact, whereas in actual use, the read length can be affected by the reading process, e.g. the enzyme activity. Still, the actual read length can meet almost all requirements.
The most widespread nanopore platform is MinION, which is portable because it is as big as a USB flash driver. Apart from PCR-free and excellent read length, it may complete a round of sequencing within hours, and the output is real-time readable on PC/laptop. Here we introduce its application in 3 fields. (1) Rapid pathogen identification, e.g. the identification of Ebola virus in the 2015 outbreak. (2) Non-invasive prenatal testing, to screen genetic diseases of embryo e.g. Down syndrome, using free DNA in the maternal peripheral plasma. (3) Microbial identification in extreme environments, e.g. bacteria in the Antarctic lake and in the spaceship [4-6]. Recently ONT has launched new sequencers GridION and PromethION. These two desktop platforms bring higher output and improved accuracy (92% for MinION and 99.999% for PromethION) [6]. The critical factor that keeps nanopore sequencing from prevailing seems to be the high cost of platform and supplies.
[1] https://blog.csdn.net/weixin_30484739/article/details/96935797
[3] https://www.sohu.com/a/241673470_100199392
[4] Eisenstein, M. (2017). An ace in the hole for DNA sequencing. Nature, 550(7675), 285–288. doi: 10.1038/550285a
[5] https://card.weibo.com/article/m/show/id/2309404313522807913960?_wb_client_=1
[6] https://mp.weixin.qq.com/s/8xdq7bW5c5fENPAKrLYFvQ