Archive for the 'Technology' Category

How emerging targeted mutation technologies could change the way we study human genetics

Mari Niemi

This is a guest post from Mari Niemi at the Wellcome Trust Sanger Institute. Mari is a graduate researcher whose research combines the results of human genetic studies with zebrafish models to study human disease.

The turn of the year 2012/13 saw the emergence of a new and exciting – and some may even say revolutionary – technique for targeted genome engineering, namely the clustered regularly interspaced short palindromic repeat (CRISPR)-system. Harboured with the cells of many bacteria and archaea, in the wild CRISPRs act as an adaptive immune defence system chopping up foreign DNA. However, they are now being harnessed for genetic engineering in several species, most notably in human cell lines and the model animals mouse (Mus musculus) and zebrafish (Danio rerio). This rapid genome editing is letting us to study the function of genes and mutations and may even help improve the treatment of genetic diseases. But what makes this technology better than what came before, what are its downsides, and how revolutionary will it really be?

Genetic engineering – then and now

Taking a step backward, the ability to edit specific parts of an organism’s genetic material is certainly not novel practice. In the last decade or two, zinc finger nucleases (ZFNs) and more recently employed transcription activator-like endonucleases (TALENs) saw the deletion and introduction of genetic material, from larger segments of DNA to single base-pair point mutations, at desired sites become reality. ZFNs and TALENs are now fairly established methods, yet constructing these components and applying them in the laboratory can be extremely tedious and time-consuming due to the complex ways in which they binding with DNA. Clearly, there is much room for improvement and a desire for faster, cheaper and more efficient techniques in the prospect of applying genome engineering in treatment of human disease.

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Crowd-funding personalized bioscience

Human_Microbiome_Project_logoHere at Genomes Unzipped we love genomes. But there is more to the world of biology than genomics, there is more to understanding your own body than personal genetic tests. To understand the human body, you have to look not just at the DNA present, but also at what genes are turned on in what tissues, what cells are being produced in what numbers, what compounds are circulating in your blood, and even what other organisms are also living on your body. However, for the interested consumer the non-genetic aspects of personalized medicine have generally been less readily accessible than the genetic aspect. This post discusses a few companies that are trying to fill this gap, and who are looking to the general public to crowd-source funding for their products.

A quick note: I have not investigated these companies in detail, and, as with all crowd-sourcing, you should be aware that the company may not manage to produce the product as they describe it (or even get to make it at all).

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My genome, unzipped

As part of the Personal Genome Project (PGP), my genome was recently sequenced by Complete Genomics. My PGP profile, including the sequence, is here, and their report on my genome is here. As I play around with the best ways to analyze these data, I’ll write additional posts, but for now I’ve noticed only one thing: I’m almost surprised by how unsurprising my full genome sequence is.

According the the PGP’s genome annotator, I have two variants of “high” clinical relevance. The first is the APOE4 allele, which Luke had already reported that I carry. The second is a variant that causes alpha-1-antitrypsin deficiency, which is also typed by 23andMe.

Of course, this is all quite reassuring. Long-time readers will remember that last year I was briefly worried that I might have Brugada syndrome. I do not carry any of the known pathogenic mutations (modulo worries about false negatives); this of course is now unsurprising, but would have been really nice information to have, say, when I was talking with a cardiologist last year.

Making sequencing simpler with nanopores

The Advances in Genome Biology and Technology (AGBT) conference, one of the main go-to destinations for those who get excited by DNA sequencing technology, is currently going down in Florida. Sadly, no-one from GNZ could make it this year, but we are keeping up with the various announcements about new genomics tech as best we can. One that caught our attention was the announcement of a brand new sequencing machine from a company that has previously kept very quiet about its technology.

Oxford Nanopore, who we have written about before, today announced two new sequencing machines to come out this year. The announcement has caused quite a buzz amoungst, well, everyone. Nature, New Scientist, GenomeWeb, BioIT World and Forbes all have reported on it, and bloggers Nick Loman and Keith Robison have also had a chance to talk to some of the Oxford Nanopore peeps about their new toys.

A lot of the interest has come from the (very cool) MinION, a tiny, disposable USB-key sequencer (shown in the picture above) that can sequence about a billion base pairs of DNA, and cost around $500-$900 each. The applications of this are endless – the ability to pick up a bit of biological matter, mix it with a few chemicals, and read whatever DNA is in it, could help with diagnostics, epidemiology, ecology, forensics. It is also (though not quite) the price where hobbyists could consider having a play; perhaps in a few years plug-and-play DIY genetics could be a possibility.

Less immediately striking, but still just as interesting, is the GridION sequencing machine. This is the work-horse of the nanopore sequencing world, made for reading lots of DNA, and scaling up to massive sequencing centers. Obviously, many scientists are going to be very interested in many of the features (notably, the ability to read very long pieces of DNA, a trick that has previously been more-or-less impossible to do reliably). However, what will this announcement mean for those of us who are interested in personal genomics?

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Next-Gen Sequencing Heading to Madison Avenue?

For companies seeking to make their mark in the ultra-competitive next-generation sequencing (NGS) market, new technology and lower prices may no longer be enough.

As the size of the NGS sequencing market grows, and an increasing number of NGS purchasers evaluate an expanding array of providers and technologies (see William Blair’s Next-Generation Sequencing Survey), NGS companies are beginning to look beyond price points and product specs in an attempt to stand out.

Ion Torrent on the Offensive. Consider Ion Torrent, an NGS newcomer recently acquired by Life Technologies, which launched its first product (the Personal Genome Machine) a scant four months ago. Since then, Ion Torrent has announced improvements to the PGM’s output, read length and sample prep (coverage from Matthew Herper of Forbes here and here).

As it seeks to distinguish the PGM from its competitors’ products, particularly Illumina’s offerings (see J.P. Morgan’s Next Gen Sequencing Survey), Ion Torrent has added a new dimension to its PGM campaign. Ion Torrent recently launched several creative online advertisements, with its side-by-side comparison of the PGM and Illumina’s MiSeq system—modeled after Apple’s popular “I’m a Mac/I’m a PC” campaign—raising the most eyebrows.
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Cluster Sequencing with Oxford Nanopore’s GridION System

More on nanopore sequencing this week. I mentioned in my Genetic Future post that the UK sequencing company Oxford Nanopore is somewhat of a dark horse, and an agreement with Illumina has required complete silence about their potential DNA sequencing machines. However, this wasn’t strictly true; Illumina has signed an agreement for the exonuclease sequencing technology, and on that we aren’t likely to hear anything until it is ready.

However, Oxford Nanopore still can, and does, talk about other aspects of their technology. And today, they have released information on their website on the GridION platform, which will be used to run all their nanopore technology (including DNA sequencing and protein analysis). In effect, these are details about the sequencing machine, but with no new specifics about the sequencing process itself.

Here are a few first impressions.

Sequencing in Clusters

The machines are small and low-cost; I expect they will cost the same or less than an Ion Torrent machine. Like the Ion Torrent, MiSeq and GS Junior, the Nanopore machines should be suitable to sit on the bench of a small lab, running small projects and with small budgets and floorspace.

However, this isn’t the full story. Each individual machine is rocking the VCR-machine-circa-1992 look, and the reason for this becomes clear when you see many of them together. The boxes are designed to fit together in standard computing cluster racks, and Oxford Nanopore refer to each of the individual machines as “nodes”. The nodes connect together via a standard network, and can talk to each other, as well as reporting data in real time through the network to other computers. When joined together like this, one machine can be designated as the control node, and during sequencing many nodes can be assigned to sequence the same sample.

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Sequencing DNA with Nanopores

As we have already mentioned, Daniel has recently moved his blog Genetic Future over to the Wired science blogging network. While Daniel is off flying around Europe introducing his newborn to mozzarella and skiing, I have written a guest post for Genetic Future entitled An Introduction to Nanopore Sequencing.

I have been meaning to write about nanopore sequencing for quite a while (if you don’t know what nanopore sequencing is at all, go read the post!). What prompted me to write this was a CGI video made by Oxford Nanopore that managed to sum up nicely some of the basic theory behind nanopore sequencing:

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A Googol of Genomes?

[Editor's Note: this was originally posted over at the Genomics Law Report but we'd like to survey Genomes Unzipped readers as well. How many complete genomes do you think will be sequenced in 2011? Poll is at bottom.]

Earlier this week we took a look back at 2010 and offered our projections for the coming year in personal genomics. Topic #1, just as it was last year: the $1,000 genome.

In hindsight, it might have been ill-advised to offer predictions about the near-term future of genome sequencing during the same week in which one of the year’s major industry conferences (the JP Morgan annual Healthcare Conference) is taking place.

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Saturday Links

Due to a communication breakdown, no-one wrote a Friday Links post yesterday, so today we have a Saturday Links to make up for it.

Steve Hsu has a very appropriately named post, News from the future, about the Beijing Genomics Institute. The BGI is the largest genome sequencing center in China, and one of the largest in the world, and is growing faster than any other, and loading up on a shedload of high-tech HiSeq machines.

Steve reports that the BGI are claiming that their sequencing rate will soon be at 1000 genomes per day, with a cost of about $5k (£3.2k) each. To put a slight downer on these amazing numbers, he clarifies that this might be referring to 10X genomes, which would realistically mean ~300 high quality genomes a day, at $15k (£9.6). Either way, if you want to keep an eye on how fast whole-genome sequencing is progressing, perhaps with an eye to when you’re ready to shell out to get your own done.

A question for the comments: how cheap would a whole-genome sequence have to get before you’d order one?

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Basics: Second-Generation Sequencing

This is an edited repost of a year-old article from my blog Genetic Inference. It explains how the state-of-the-art Second Generation sequencing works, and how it is being used to sequence thousands of genomes per day. I also try to explain some of the distinctions between First, Second and Third Generation sequencing.

This post follows on from an even older post that explained First Generation sequencing; the tech that was used in the Human Genome Project.

Recap: What are we trying to do?

In a previous post, we saw how DNA is made up of little strings of nucleotides, and we used different shapes to represent different base pairs (A = triangle, C = diamond, G = circle, T = pentagon). For instance, stage20_1 is GCAT.

We looked at how the DNA polymerase enzyme can be used to amplify up DNA, using the Polymerase Chain Reaction, and how we can determine the sequence of DNA using ddNTPs; nucleotides that, when incorporated into DNA, stop the polymerase working.

In First Generation (Sanger) sequencing, we run a PCR reaction in the presence of a bunch of ddNTPs, with each different base pair dyed a different colour. We then measure the length and colour of the resulting fragments of DNA, and use that to work out the sequence; a bit of DNA 35 base pairs long ending in a blue ddNTP tells us that the sequence has a “C” at the 35th position.

The problem with this method is that it requires a lot of space; you need a place to run the reaction, and then you need a capillary tube or a gel to determine the length of the DNA. As a result, you could only run perhaps a hundred of these reactions at any one time. There are 3 billion base pairs of DNA in the human genome, meaning about 6 million 500-base pair fragments of DNA; it would take a very long time to sequence all of these if you had to do them one hundred at a time.

Second Generation sequencing techniques overcome this restriction by finding ways to sequence the DNA without having to move it around. You stick the bit of DNA you want to sequence in a little dot, called a cluster, and you do the sequencing there; as a result, you can pack many millions of clusters into one machine. Sequencing a strand of DNA while keeping it held in place is tricky, and requires a lot of cleverness. I’ll explain how Illumina‘s Second Generation technology achieves this, as it is the most similar to Sanger sequencing.

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