MinION Sequencing Untangles RNA Transcripts in a Difficult Gene

By Aaron Krol

November 3, 2015 | Brenton Graveley received his first MinION shipment in April 2014, at his lab at the University of Connecticut’s Institute of Systems Genomics. His lab was among the first to unwrap one of the candy bar-sized DNA sequencers made by Oxford Nanopore Technologies, and although its accuracy was shaky and its throughput low, right away Graveley and his colleagues could see it was producing real DNA data.

“I’m still amazed to this day that it works at all,” Graveley says. “It’s like Star Trek.”

A lot of buzz around the MinION has focused on its tiny size: early adopters have plotted to take MinIONs into outbreak zones and species-hunting tromps through the rainforest, working with bare-bones labs and laptop computers. But for Graveley, the size of the DNA strands the MinION reads is just as exciting as the size of the sequencer itself. That’s because most other sequencers rely on picking up chemical reactions that become more error-prone over time, meaning DNA can only be read in short fragments. The MinION, which reads genetic material by observing single molecules of DNA as they pass through extremely narrow “nanopores,” keeps producing data for as long as DNA is moving through the pore.

“You get the read length of whatever fragment you put into the MinION,” he says. “We’ve gotten reads that are over 100 kilobases,” hundreds or even thousands of times longer than researchers can expect with most other technologies.

Now, in a paper published in Genome Biology, Graveley and two of his lab members, post-doc Mohan Bolisetty and PhD student Gopinath Rajadinakaran, have shown how these read lengths can help explain the cellular behavior of Dscam1, one of the most difficult-to-study genes known to science. Related to a gene in humans that has been linked to Down syndrome ― the name stands for “Down Syndrome Cell Adhesion Molecule” ― Dscam1 plays a fundamental role in forming the architecture of insect brains. This single gene can produce thousands of subtly different proteins, an ability that makes it both a fascinating subject of research, and almost impossible to understand using standard sequencing technology.

The Tangled Transcriptome

Graveley’s lab studies the transcriptome, the mass of RNA molecules in living cells whose job is to translate DNA into proteins. The transcriptome is a sort of snapshot of which parts of the genome are active at a given time and place. Which genes are transcribed into RNA, and in what quantities, changes from organ to organ and even cell to cell, and can vary over an organism’s lifetime or in response to environmental changes.

Of particular interest to Graveley are those RNA molecules than can take different shapes, or “isoforms,” depending on random chance or what the cell needs at a particular time. RNA isoforms are distinct versions of the same gene. Through a process called alternative splicing, the different subunits, or “exons,” that make up a gene can be reshuffled in new combinations. Many genes have two or more mutually exclusive exons, and which ones are actually expressed as RNA and protein can have big effects on cellular behavior ― in effect, expanding the protein arsenal of the genome.

“For the entire field of transcriptomics and gene function, knowing what isoforms are expressed is critical,” says Graveley. “Most genes are complicated, especially in humans, and have alternative splicing that occurs at multiple places.”

That brings us to the challenge of Dscam1, the world record holder for alternative splicing. In fruit flies, a particularly well-studied model organism, Dscam1 is made up of 115 exons, only 20 of which are always transcribed into RNA. The other 95 exist in four “clusters” of mutually exclusive exons, and as a result, over 38,000 possible isoforms of Dscam1 have been predicted.

“This is by far, an order of magnitude, more than any other gene,” Graveley explains. This flexibility makes sense in light of Dscam1’s function. The protein it makes helps to “identify” single neurons in the insect brain, making them distinct enough from their neighbors for these cells to assemble a neural circuit on principles of like avoiding like. In experiments where Dscam1 has been altered to make fewer RNA isoforms, the neural wiring breaks down during development, sometimes severely enough to kill the flies.

Dscam1 also plays a role in the insect immune system, another reason for it to produce a huge variety of isoforms. Each of these molecules might be more or less effective at fighting certain pathogens.

It’s frustratingly hard, however, to figure out exactly which isoforms are in a specific sample. Graveley has been working on Dscam1 in fruit flies for more than a decade, but very basic questions remain unanswered: are some isoforms more common, or more important, than others? Are all the theoretical isoforms expressed? Do the isoforms have different behaviors, or are they just arbitrary ways of tagging neurons?

Size Matters

The trouble is the current state of the art in sequencing technology, which reads just a couple of hundred DNA bases at a time. That works great for identifying which exons are present in the transcriptome, but it’s no good for saying which mix of exons any specific strand of RNA is carrying. Different exons can lie thousands of bases apart on the RNA molecule, and there’s no way to bridge the gap between reads.

Graveley has tried a lot of solutions. He’s used the outdated Sanger sequencing method, which is much slower and more labor-intensive than modern sequencers, but does span longer reads. His lab also worked out a roundabout way of reconstructing RNA transcripts with contemporary Illumina sequencers, through a combination of chemistry and computational approaches.

“It worked,” he says, “but it was complicated by a lot of library preparation artifacts, and you basically had to jury-rig a genome analyzer to do something it was not supposed to do.”

Graveley’s preferred method is to use a sequencer produced by Pacific Biosciences, which, like the MinION, is built on long-read, single-molecule technology. PacBio sequencing is much better established than nanopores, and its results are known to be reliable; it also has the high throughput typical of modern instruments. For researchers working on alternative splicing, it’s clearly the technology to beat.

Unfortunately, it’s also very expensive. So Graveley’s team set out to learn whether the MinION, a low-throughput but extremely cheap alternative, could be an adequate substitute.

For the Genome Biology paper, the team focused on a 1.8-kilobase region of Dscam1 RNA that covers 93 of the gene’s 95 alternatively spliced exons. To get their samples, they crushed fruit fly heads, isolated Dscam1 RNA from the sample using a polymerase, and reverse-transcribed it into cDNA for sequencing. They also sequenced transcripts of three other alternatively spliced genes, Rdl, MRP, and Mhc.

The biggest concern for new applications of the MinION is its shaky accuracy. While most sequencers can achieve comfortably over 99% consensus with reference sequences, Graveley’s group has seen only about 90% identity with the MinION. That’s actually a little better than most MinION users have managed, although the device’s accuracy has been steadily improving. Users have had to pick their projects carefully to account for this: the device is pretty reliable in resequencing studies that map DNA reads to known references, but it’s still a dubious choice for sequencing unknown genetic material from scratch (although it’s been tried).

To accurately pin down the exact isoforms in the transcriptome, the MinION didn’t have to read every RNA molecule perfectly, but it did have to come close enough to decisively tell one exon from another ― and in Dscam1, those exons could be as much as 80% identical.

In fact, Graveley and his co-authors found that the MinION was very capable of this. Out of around 33,000 high-quality Dscam1 reads pulled off the sequencer, almost 29,000 were a strong match for one and only one combination of exons. To further check their accuracy, the team also sequenced the same sample on Illumina technology. While the Illumina sequencer could not give whole isoforms, it did show the same proportions of different exons, suggesting that the MinION gave a complete and unbiased picture of the sample.

“Alternative splicing, it turns out, is probably one of the ideal applications for this platform,” Graveley says. “Even with a gene as complicated as this one, we’re able to accurately distinguish the isoforms from one another. Unless you have very, very small exons, or two exons that are almost identical to each other, the accuracy is good enough.”

Make Way for PromethION

The results are good news for researchers studying the transcriptome, but the MinION probably won’t push out other methods for dealing with alternative splicing just yet. Its low throughput means that at best it can cover a very small portion of the transcriptome with each run ― and that means isolating targeted RNA transcripts, a process that can introduce new biases into the data.

“You need a lot of reads to get the whole transcriptome, and what happens is you end up sequencing boring genes like actin and tubulin, the really abundantly expressed things,” Graveley explains. Still, his data from this experiment was good enough to replicate a few earlier findings: for instance, that Dscam1 does appear to make every predicted isoform. In this experiment, his lab observed almost half the possible isoforms, containing 92 of 93 possible exons.

Meanwhile, Oxford Nanopore Technologies is working on a new instrument, the PromethION, which will contain 48 MinION-style flow cells in a battery. Graveley has already signed on to be one of the first recipients, in an access program that is likely to start in the winter.

Judging by studies like this one, the PromethION stands a good chance of becoming the instrument of choice for large-scale RNA sequencing. With Dscam1, Graveley hopes to reach high enough throughput to do functional studies, seeking to learn whether different combinations of isoforms give rise to physical or behavioral differences. He also wants to look at human genes with high levels of alternative splicing, and to test whether the MinION can accurately count total numbers of RNA isoforms.

“The fact that you can use this technology to characterize whole isoforms is very exciting,” Graveley says. “It’s going to help us start characterizing the transcriptome in ways that have been very difficult.”