New technology developed to sequence genes: faster, better and cheaper

If DNA is called the blueprint of life, RNA is the construction contractor who interprets it. Therefore, it is quite important to sequence RNA for it shows you what is happening inside a cell.

Recently, researchers from UConn Institute of Systems Genomics have sequenced the RNA of the most complicated gene known in nature. What they use is a hand-held sequencer which is not as big as a cell phone.

Genomicists Brenton Graveley from the UConn Institute of Systems Genomics, postdoctoral fellow Mohan Bolisetty, and graduate student Gopinath Rajadinakaran together with UK-based Oxford Nanopore Technologies to show that the company's MinION nanopore sequencer can sequence genes faster, better, and at a much lower cost than the standard technology. Their findings were published in Genome Biology on Sept. 30.

If your genome was a library and every gene was a book, some genes would be straightforward reads, but some would be more like a "Choose Your Own Adventure" novel. Researchers tend to know which version of the gene is actually expressed in the body, but for complicated choose-your-own-adventure genes, it has been impossible. The researchers solved the problem in two parts.

The first was to find a better gene-sequencing technology. They used the old existing technology to sequence genes. This technique won't work for choose-your-own-adventure genes, because if you copy them the way the body does, using RNA, each copy can be slightly or very different from the next. Such different versions of the same gene are called isoforms. When the different isoforms get chopped up and sequenced, it becomes impossible to accurately compare the pieces and figure out which versions of the gene you started with.

Last year, the impossible things became possible. Oxford Nanopore, a company based in the UK, released its new nanopore sequencer, and offered one to Graveley's lab. The nanopore sequencer, called a MinION, works by feeding a single strand of DNA through a tiny pore. The pore can only hold five DNA bases - the 'letters' that spell out our genes - at a time. There are four DNA bases, G, A, T, and C, and 1,024 possible five-base combinations. Each combination creates a different electrical current in the nanopore. GGGGA makes a different current than AGGGG, which is different again than CGGGG. By feeding the DNA through the pore and recording the resulting signal, researchers can read the sequence of the DNA.

In the second part, the researchers chose the most complex one known, Down Syndrome cell adhesion molecule 1 (Dscam1), which controls the wiring of the brain in fruit flies. Dscam1 has the potential of making 38,016 possible isoforms, and every fruit fly has the potential to make every one of them, yet how many of these versions are actually made remains unknown.

Dscam1 looks like this: X-12-X-48-X-33-X-2-X, where X's denote sections that are always the same, and the numbers indicate sections that can vary (the number itself shows how many different options there are for that section).

The researchers had to convert Dscam1 RNA into DNA to study how many different isoforms of Dscam1 actually exist in a fly's brain. If DNA is the book or set of instructions, RNA is the transcriber that copies the book so that it can be translated into a protein. The DNA includes the instructions for all 38,016 isoforms of the Dscam1 gene, while each individual Dscam1 RNA contains the instructions for just one. No one had yet used a MinION to sequence copies of RNA, and though it was likely it could be done, demonstrating it and showing how well it worked would be a substantial advance in the field.

Rajadinakaran took a fruit fly brain, extracted the RNA and converted it into DNA, isolated the DNA copies of the Dscam1 RNAs, and then ran them through the MinION's nanopores. In this one experiment, they not only found 7,899 of the 38,016 possible isoforms of Dscam1 were expressed but also that many more, if not all versions are likely to be expressed.

The study shows that gene sequencing technology can now be accessed by more researchers than before, since the MinION is both relatively inexpensive and highly portable.

"This type of cutting-edge work puts UConn at the forefront of technology development and strengthens our portfolio of genomics research," says Marc Lalande, director of UConn's Institute for Systems Genomics. "Also, thanks to the investments in genomics through the University's Academic Plan, Brent Graveley can leverage his expertise so that faculty and students across our campuses will successfully compete for grant dollars and launch bioscience ventures."

The researchers are planning to sequence every bit of RNA from beginning to end inside a single cell. This can't be done by traditional gene sequencers. This new technology is supposed to transform the way we study RNA biology and the type information.

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