{"id":105023,"date":"2015-10-09T10:46:46","date_gmt":"2015-10-09T14:46:46","guid":{"rendered":"https:\/\/today.uconn.edu\/?p=105023"},"modified":"2015-12-08T20:55:42","modified_gmt":"2015-12-09T01:55:42","slug":"a-better-way-to-read-the-genome","status":"publish","type":"post","link":"https:\/\/today.uconn.edu\/2015\/10\/a-better-way-to-read-the-genome\/","title":{"rendered":"A Better Way to Read the Genome"},"content":{"rendered":"
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UConn researchers have sequenced the RNA of the most complicated gene known in nature, using a hand-held sequencer no bigger than a cell phone.<\/p>\n

If\u00a0DNA is the\u00a0blueprint of life, RNA is the construction contractor who interprets it, so sequencing RNA tells you what’s really happening inside a cell.<\/p>\n

Genomicists Brenton Graveley from the UConn Institute of Systems Genomics, postdoctoral fellow Mohan Bolisetty, and graduate student Gopinath Rajadinakaran teamed up with UK-based Oxford Nanopore Technologies to show that the company\u2019s MinION nanopore sequencer can sequence genes\u00a0faster, better, and at a much lower cost than the standard technology.\u00a0They published their findings on Sept. 30 in Genome Biology<\/a><\/i>.<\/p>\n

If your genome was a library and each gene was a book, some genes would be straightforward reads \u2013 but some would be more like a \u201cChoose Your Own Adventure\u201d novel. Researchers often want to know which version of the gene is actually expressed in the body, but for complicated choose-your-own-adventure genes, that has been impossible.<\/p>\n

Graveley, Bolisetty, and Rajadinakaran solved the puzzle in two parts. The first was to find a better gene-sequencing technology. In order to sequence a gene using the old, existing technology, researchers first make lots of copies of it, using the same chemistry our bodies use. They then chop up the gene copies into tiny pieces, read each tiny piece, and then, by comparing all the different pieces, try to figure out how they were originally put together. The technique hinges on the likelihood that not all the copies got chopped up into exactly the same pieces. Imagine watching different scenes from a movie, out of order. If you then watched the same movie, but cut into scenes at slightly different places, you could compare the two versions and start to figure out which scenes connect to which.<\/p>\n

That technique won\u2019t work for choose-your-own-adventure genes, because if you copy them the way the body does, using RNA, each copy can be slightly \u2013 or very \u2013 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.<\/p>\n

If the gene were a movie, \u201cyou wouldn\u2019t be able to tell that scenes 1 and 2 were present together,\u201d Bolisetty says.<\/p>\n

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Then last year, the nearly impossible suddenly became possible. Oxford Nanopore, a company based in the UK, released its new nanopore sequencer, and offered one to Graveley\u2019s 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 \u2013 the \u2018letters\u2019 that spell out our genes \u2013 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.<\/p>\n

For the second part of the solution, Graveley, Bolisetty, and Rajadinakaran decided to go big. Instead of sequencing any old choose-your-own-adventure gene, they chose the most complex one known, Down Syndrome cell adhesion molecule 1<\/i> (Dscam1<\/i>), which controls the wiring of the brain in fruit flies. Dscam1<\/i> 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<\/i> looks like this: X-12-X-48-X-33-X-2-X, where X\u2019s 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).<\/p>\n

To study how many different isoforms of Dscam1<\/i> actually exist in a fly’s brain, the researchers first had to convert Dscam1<\/i> RNA into DNA. 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\u00a0of the Dscam1 <\/i>gene, while each individual Dscam1 <\/i>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.<\/p>\n

Rajadinakaran took a fruit fly brain, extracted the RNA, converted it into DNA, isolated the DNA copies of the Dscam1 <\/i>RNAs, and then ran them through the MinION\u2019s nanopores. In this one experiment, they not only found 7,899 of the 38,016 possible isoforms of Dscam1 <\/i>were expressed but also that many more, if not all versions are likely to be expressed.<\/p>\n

\u201cA lot of people said \u2018The MinION will never work,\u2019” Graveley says, “but we showed it works using the most complicated gene known.\u201d<\/p>\n

\"The<\/a>
The MinION gene sequencer in Brenton Graveley\u2019s lab is state-of-the art technology that costs about $1,000 and is roughly the same size as an iPhone. (Kim Krieger\/UConn Photo)<\/figcaption><\/figure>\n

The study demonstrates that gene sequencing technology can now be accessed by a much broader range of researchers than was previously possible, since the MinION is both relatively inexpensive and highly portable so that it requires almost no lab space.<\/p>\n

\u201cThis type of cutting-edge work puts UConn at the forefront of technology development and strengthens our portfolio of genomics research,\u201d says Marc Lalande, director of UConn\u2019s Institute for Systems Genomics. “Also, thanks to the investments in genomics through the University\u2019s 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.”<\/p>\n

Graveley will speak about the research at the Oxford Nanopore MinION Community Meeting at the New York Genome Center on Dec. 3.<\/p>\n

As for next steps, the researchers plan on going even bigger: sequencing every bit of RNA from beginning to end inside a single cell, something that cannot be done with traditional gene sequencers.<\/p>\n

\u201cThis technology has amazing potential to transform how we study RNA biology and the type of information we can obtain,” says Graveley. “Plus the fact that the MinION is a hand-held sequencer that you plug into a laptop is simply unbelievably cool!\u201d<\/p>\n","protected":false},"excerpt":{"rendered":"

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