Release date: 2018-01-02
The output of high-throughput sequencing is amazing, but the long read is often criticized. However, from 100 bp to 1 kb, the read length is always improving. Recently, an exciting news came from the sequencing community. Researchers at the Garvan Institute in Australia used Oxford Nanopore sequencing technology to achieve a read length of > 1 Mb, a milestone in sequencing history.
Dr. Martin Smith of the Kinghorn Clinical Genomics Center announced the news on Twitter. Under his leadership, the team first sequenced a DNA fragment of more than 1 Mb in length, from chromosome 19, with a length of 1.015 Mb.
This sequencing process is portrayed: If the nanopore is compared to the size of your fist, the 1 Mb DNA strand through the nanopore is equivalent to 3.2 km. Wow, it’s almost equivalent to running a mini marathon.
The data provided by the traditional short-reading and long-sequencing technology is difficult to assemble into a complete genome or data set. This is like a 1000-piece puzzle, which is very challenging to assemble. Strictly speaking, nanopore sequencing technology does not have the concept of long reading because it can measure a DNA fragment from the beginning to the end. However, due to sample preparation, researchers mostly sequenced 10 kb to 100 kb fragments.
When sequencing a 1 Mb fragment, Dr. Smith undoubtedly encountered many challenges, but DNA extraction and sample preparation were the most difficult steps. Even with simple pipetting, DNA molecules longer than 100 kb may be cleaved. High molecular weight DNA samples form a large piece of gel-like material that is no longer a liquid, so measuring concentrations is also difficult. In addition, data analysis is also a challenge, as most software tools are developed for short read lengths.
However, after overcoming the challenges, the researchers finally completed the sequencing of the ultra-long DNA fragments. They said that the quality of the reading was not affected by the length. The uncorrected sequence is 90% identical to the human reference sequence. It is important to note that natural DNA molecules contain methylated nucleotides which are not taken into account in the conversion of the original electronic signal into a nucleotide sequence and may therefore result in erroneous base detection.
Now, with these long sequences, people can assemble genomes more easily, parsing complex areas, and even areas that were previously unseen. Dr. Smith believes that nanopore sequencing is inherently different from optical sequencing, easier to obtain, and more interesting. Of course, he believes that other types of sequencing will still play a big role, and the various technologies can complement each other and need to be combined.
The research team led by Dr. Smith focuses on evaluating emerging technologies, including single-cell sequencing, epigenetics, and metagenomics. The 1 Mb DNA fragment was also sequenced to resolve the sequence and structure of the cancer-related neochromosome (abnormal chromosome), which includes an abnormal genomic sequence in which hundreds of DNA fragments are spliced ​​together. In the future, he believes that the sequencing of longer fragments (such as 2 Mb) will also be just around the corner.
Source: Biopass
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