"Why I Don’t Want to Know My Genome Sequence"

"Why I Don't Want to Know My Genome Sequence"

Kind of interesting to read this as someone who at least has his own exome sequenced and has learned a lot from it. It's kind of interesting to read it in contrast to my own post explaining why I wanted to sequence myself.

It ends with a funny statement:

An osteoarthritis mutation manifested itself as an inability to play an F chord at age 33. A p53 mutation and then another that bloomed in response to years of orthodontia X-rays gave me thyroid cancer a few years after I gave up the guitar. And I don’t need a genetic test to know I didn’t inherit my father and grandfather’s psychotic depression. 

Ron Crystal, even though he’s among the sequenced, has the right idea: don’t smoke, exercise, eat a healthy diet, and don’t worry about DNA sequences. That’s good enough for me – at least for now.

This is one of those statements used to make one's self feel better about not doing something he or she wants to do. That's my feeling, at least.

To be more specific, let's use myself.

Sure, I could have lived my life thinking my borderline migranes were from caffeine withdrawel and my mother and not bothered to find out the exact mutations likely to cause it and therefore which drugs may actually have a beneficial effect on me. I could have. But I didn't.

I could have lived my life thinking my intestinal issues were due to a bad diet or food allergies. Because clearly copious amounts of salad, low fat, low carb, low salt is a bad diet, right? And clearly my wife who eats the same things and has zero gastrointestinal issues is just lucky. Oh no, it must be food allergies. It couldn't possibly be that I inherited two mutations, one from each parent, that damage a particular gene already known to be causative for gastrointestinal problems (among a number of other things that I happen to have that most general practitioners wouldn't link together).

I could have survived without knowing I have asthma. Maybe.

I mean, go ahead and live in ignorance if that's your thing. If you're not interested in your own genetics, then fair enough. You're not alone. But it was damn reassuring to me, personally, to figure out exactly what genes are mutated and how those are causing conditions that negatively effect me.

And I would say not only am I healthier, but I'm also more aware of my health. And that's a good thing.

And that genetic information isn't going to expire.

Anyway, let's not devalue our genetics this way. The fact that we don't know everything yet doesn't make the data itself less valuable. Yes, it will take effort to understand but then again, so does nearly everything about your health and life.

I see it as a great thing.

Sequencing My Exome: Why?

“Why do you want to do this?”

My wife, immediately after I tell her I'm going to sequence my own exome.

There are a few times in life where you want to do something so badly, but find it difficult to convey to others why. This was one of those times. Such a simple question, but so many different answers. And each answer as valid as all the others. All of them coming together to explain why I would want to do something so, well, unusual.

I could frame a whole dissertation on the reasons behind wanting to sequence my own exome. (And I will.) But first, the simple answer:

I’m curious about myself.

I want to see if I can figure out why I am the way I am. And by that I mean both physically and mentally. For some people, this isn’t something they’ll ever think about. For others, they might see very clearly that they are this way because God made them this way, or because their parents raised them like this, or because they had bad luck. They may simply accept that they have specific features that make them who they are and aren’t concerned with why.

For me, those answers are not good enough.

I know that there are mysteries to be solved in my genetic code. It comes with the territory of being a geneticist. That said, though, almost everyone thinks this way, usually without even realizing it. We can all look at our own families as a proxy for genetics. If your mom had type 2 diabetes and your sister has type 2 diabetes and your uncle has type 2 diabetes, you’re pretty sure you have a higher chance of getting type 2 diabetes. You’ll hear all sorts of people saying that—“guess I got my mom’s bad genes” and “guess he took after his father” and so forth. If you’ve ever known somebody who got old enough, you might have heard her tell you about her mother lived to a ripe, old age and her mother before her, and so on. That’s what I call thinking genetic.

The difference for me is that I’m thinking genetic at a different level. I actually want to look at my genetics to try to explain these types of things. I don’t believe in fate without reason. If my mom lives to be 90, and my grandmother lived to be 90, I want to know if I got the mutations that helped them get there. Could I just shrug my shoulders, say, “I probably did,” and move on? Absolutely. But that’s just not good enough for me.

And really, it’s not good enough for anyone. We’re no longer entering the era where we can do better than that. We’re already there. Exome sequencing represents that first major step into the era.

I think, to make a case for exome sequencing (and by the way, whole genome sequencing is basically just an expansion and improvement upon exome sequencing—more on that later), I first need to explain what we can learn from it.  And to do that, first you’ll need to know what an exome is. For those readers who already know all about this, feel free to skip down.

What is an exome, anyway?

To understand what the exome is, you first have to understand what the genome is. There are massive tomes on the details of the subject, but to describe the genome succinctly:

The genome is the blueprint for every cell in your body.

Every single protein in every one of your cells is encoded on this massive blueprint. In order to create and maintain a cell (and therefore, your body and very being), your cell quite literally reads the genome and generates certain amounts and types of various proteins to fit the particular cell it’s trying to become or to fulfill a particular function.

The exome is a subset of the genome that contains the instruction to create the proteins themselves. The exome makes up about 1-2% of the whole genome. If the genome is the blueprint, then:

The exome is the instructions for making every protein in your body.

Therefore, being able to read those instructions means we can figure out if differences in them will result in different protein structures.

What can I learn from the exome?

Identifying variations in the exome that lead to differences in proteins (which we call mutations) can give us a direct way of determining if a protein might have altered function in us compared to other people. Significant protein mutations will manifest themselves as traits. To bring up an example from a previous post, the earwax trait is a result of a variation in the exome that leads to a mutation in a protein that results in determining if your earwax will be wet or dry. But it goes far beyond that type of “interesting” trait. Mendelian disorders (which this blog derives its title from) are disorders resulting from mutations in a single gene, which we can detect in the exome (and, in fact, quite a few Mendelian disorders have been “solved” through exome sequencing now).

By sequencing the exome, we can directly assess every line of the “instructions” and identify those lines that differ from the norm.

But that’s not the only way to use this information. We can hunt for mutations that damage our proteins, and that is the first obvious thing to do when looking at the exome. But we don’t know how every mutation will affect a person. To the contrary—there are very few mutations for which we understand the effect.

In fact, the current standard in personal genomics testing (such as that from DTC companies like 23andMe or through-physician companies like Navigenics) is actually an approach that dominated the field for about a decade before next-generation sequencing really became a reality. Using microarray technology, these approaches measure specific sites known to harbor variants in the genome that are associated with a trait or disease but typically not causative for the trait or disease.

For example, right now if you were to do a standard 23andMe test, you’d have genetic variations assessed at about a million sites across your genome. These variations would then be compared to a database that tells how strongly particular variations associate with particular traits or diseases. So 23andMe can tell me that I have a collection of variants that associate with type 2 diabetes, and it can calculate how that increases my risk of getting the disease compared to the average person.

This is more of a science than people often think. This is thinking genetic at a slightly more advanced level. I could simply turn to my family history and guess that I’m at an increased risk for type 2 diabetes. However, the fact that my genetics confirm the increased risk makes it much more “real” to me. Not only do I have a family history, I actually inherited some of those genetic factors. My risk is real.

Knowing my exome sequence takes that to the next level. Rather than simply having associations, I may be actually able to go into the regions of association and identify mutations causing these problems.

Moreover, as more and more information regarding the genetic causes of various traits and diseases are discovered, my exome sequence will always be at hand for me to cross-reference. Imagine that tomorrow a study is released identifying a gene that tells you with complete confidence whether or not you’ll get type 2 diabetes. I would check that gene in my own exome for mutations immediately!

That may sound unrealistic, but when it comes to conditions like cancer, these kinds of studies come out all the time. I may identify a random mutation in a gene that pre-disposes people to getting a particular type of cancer in my own genome, and then I will know that I need to have my doctor monitor for that. Having worked closely on brain cancer for a few years, it struck me that the reason it’s the deadliest type of cancer is because by the time we detect it, it’s already at a very advanced stage. But if we have a gene or set of genes that we know predisposes people to get malignant brain tumors, we could look in our own exomes for mutations in those genes and then get ourselves MRIs starting at a particular age to try to detect them earlier and hopefully allow effective, long-term treatement.

I think anyone can see how powerful that type of diagnostic and predictive tool can be.

And that brings up a major reason to sequence one’s genome: This information is immutable. Your exome is not changing. On the day you die, you’ve got pretty much the same exome and genome you had when you were born. If a major discovery is made tomorrow, I’ll have my exome to look at for it. If another discovery is made in ten years, I can take that same exome sequence and look for it. There’s no “expiration date” on that information.

And that’s what really sold me on the whole thing, actually. My intimate knowledge that my exome is always going to be a part of me, and that our understanding of genetics and diseases will always be expanding. That means my investment now is going to pay off for my whole life. Or at least until I sequence my whole genome.

I hope that conveys my major reasoning behind why I would want to do this. Of course there are other factors as well. For one thing, I am a geneticist. Genetics is not just my job, it’s my hobby. I love it. And over the years I’ve become increasingly interested in my own genetics. But that’s honestly not the only reason. At this point, I see it as a choice that will help me keep myself healthy throughout my life. 

I think there will be a shift generally towards that thinking in the medical community at large in the very near future as well. It may only be a couple years before your doctor suggests you get your exome sequenced as well. In a society where I feel most of us already think genetic, I think it's only a matter of time before we stop simply guessing that it's genetic and instead actually prove it. And beyond that, we actually figure out that there's something we can do about it. That is empowering right there.

Enter the Exome

My Exome kit from 23andMe has arrived! In a few short weeks, I will have my exome sequence in hand and ready to analyze. I've looked at hundreds of exomes over the past year, but only now, when I'm about to look at my own, have I started to really think about how to extract meaning from a healthy individual's exome. All of the work I've done has either been to assess exome sequencing as a science or to hunt for mutations causing specific conditions (Mendelian Disorders and novel genetic syndromes).

Now I'm going to have my own sequence in hand and have a very basic yet exceedingly complex question to answer: What does this all mean?

And with that question comes other questions:

Why do I care about my own exome?

What can I learn from it?

What justifies the cost?

Is it safe?


In the coming days, I will be posting about my answers to these questions and more. I came to a realization a couple of days ago (right after I ordered this kit) that even questions that seem simple to me as a geneticist are not so simple for most people.

"Why do you want to do this?" is harder to answer than people may think. Off the cuff I might say, "I'm a geneticist, it's what I do!" but that isn't at all the whole answer. So I am going to make it a goal to explain why anyone would want to have his or her exome (or genome) sequenced in terms that hopefully anyone can understand.

Exome Sequencing Q&A

In case you missed it, the most recent issue of Genome Biology is focused on exome-sequencing. Included among a number of exome-seq platform comparison papers (like this one and this one, which I intend to discuss in comparison with my own recent publication in Nature Biotech in a future post) is a fun Q&A article entitled "Exome sequencing: the expert view". The article asked three experts (Dr. Leslie G. Biesecker of NHGRI, Dr. Kevin V. Shianna of Duke University, and Dr. Jim C. Mullikin of NHGRI) a series of questions related to exome-seq ranging from what we've learned to whether exome-seq will be around in a couple years.

Since I like to think of myself as a bit of an expert on the topic (mostly because I dedicated the past year of my life to it), I thought it might be fun to answer the questions myself and discuss a bit what I think of the answers in the article.

How can exome sequencing contribute to our understanding of the dynamic nature of the genome?
Exome sequencing is quite literally the best way we currently have to assess the most interpretable part of the genome: the protein-coding sequence. While it accounts for only about 1% of the whole genome, that 1% is the part we understand the best, because it's the part that we can assess through central dogma. Basically, when we see a mutation in an exon, we can then determine how the RNA will be affected, and subsequently how the protein will be mutated. We can determine precisely which amino acids will be mutated, and which amino acids they will turn into. And thanks to decades of work on proteins and amino acids, we can predict fairly reasonably how damaging a particular amino acid change will be. We can also predict nonsense mutations, which almost always result in loss of the protein as well as frameshifts (which typically lead to a nonsense mutation), splice site variations, and regulatory site mutations (assuming the UTRs/upstream and downstream regions are enriched).

I put it like this to someone not long ago: What would you look at if you sequenced someone's genome? Exons. That is literally the first thing, because it's the low hanging fruit. If you find nothing there, you might zoom out and look at splice sites (which exome-seq generally captures), micro RNAs (which exome-seq generally captures), and regulatory elements (which are becoming recognized as increasingly important--be ready for regulatory element/transcription factor binding site enrichment kits in the near future). As for the intergenic regions, they're such a mystery with regards to function that I feel secure saying we basically don't know what to do with variants in those regions.

At the very least, I have no doubt exome-sequencing will drive a revolution in Mendelian disorder research (it already is). We've already seen a number of them published and as an increasing number of samples are sequenced, that will only improve. Because Mendelians are most often caused by changes in the protein coding region of genes, exome-seq is a prime way of solving them. The one major barrier I see for solving Mendelians is the (perhaps surprising) prevalance of dominant low-penetrance disorders. Mendelian disorders with this mode of inheritance are inherently difficult to solve, and will require a large number of samples sequenced. That said, this again primes exome-seq as a major method, and I feel secures it as a major technology for at least a few more years.

In the paper, Kevin Shianna mentions some shortcomings. In particular, he mentions that structural variations (SVs) are difficult to detect by exome-seq, which is certainly true. I would say there is some light at the end of that tunnel, at least with regards to copy-number variations (CNVs), as some successful work is already showing positive results with detecting CNVs in exome-seq.

How much has exome sequencing been driven by cost alone?
Certainly I think the increasing prevalence of exome-seq has been almost completely driven by cost. Targeted sequencing (and subsequently, exome-seq) were developed precisely because labs wanted to assess particular regions by sequencing without paying for an entire genome sequence (most of which they wouldn't even look at the data from). That said, were the exome-seq and WGS assays the same cost, there would still be significantly higher cost both in terms of time and money associated with the bioinformatics, analysis and storage of WGS data compared with exome-seq data. Even at equivalent assay cost, exome-seq is cheaper. Will that ever change? Unlikely, I think.

Consider the value of an exome versus the value of a whole genome currently. One could certainly argue that because the information in the exome is so much more meaningful (please don't write me letters for saying that), every exome base is worth significantly more than intergenic bases. In the paper, LGB and JCM make the point that an exome costs about 1/6th what a whole genome costs. That seems pretty accurate to me. And that does demonstrate how much more valuable we consider the exome compared to the other regions. Because of that I would not say that exome-seq has been driven by cost alone, but also because it is considered more valuable per base than whole genome.

What are the major limitations of exome sequencing?
Obviously, any meaningful variation outside of the exome is missed by exome-seq, and that's a major drawback. Just because they're less interpretable does not mean that non-exonic variations are meaningless. Missing SVs is another large drawback, although I feel it's important to point out that SV detection does not require a deep whole genome sequencing experiment--a low depth WGS that is not adequate for small variant calling is more than adequate for SV calling. Therefore, exome-seq could be supplemented by low depth WGS for SV calling without going as far as doing a full WGS. I'd estimate the cost of an exome-seq plus low-depth SV-WGS to still be less than 1/4th the cost of a full WGS.

The other major issue, which is also brought up in the paper, is the fact that exome-seq misses almost everything it doesn't target. If the mutation causing a particular disorder is in an exon that doesn't happen to be on your target list, you're going to miss it. WGS, on the other hand, would likely catch it. That's a major limitation. However, if the exon is poorly annotated and that's why it's not on the exome-seq design, there's a good chance you'll miss it with WGS as well. So while this is a major limitation, it's as much a major limitation of sequencing in general. Specifically, that mutational analysis is so heavily reliant on annotation, which is incomplete at best.

What lessons from exome sequencing studies can be applied to whole genome sequencing studies?
Most of what we've learned from exome sequencing studies is directly applicable to whole genome sequencing studies. Currently, the field is in a state where exomic variation is highly detectable and interpretable, and that's due in no small part to exome-seq. Coding region annotation in particular has become significantly more powerful as a result of all the exome-seq work that's been done. We have a much stronger appreciation for the limitations of sequencing as well. Although we are very sensitively detecting variants, even at miniscule false positive rates we have trouble detecting disease-causing variants with single samples. As far as I'm aware, not a single Mendelian disorder has been solved by sequencing of a single sample (exome or otherwise). Because we need some way to eliminate false positives, and we use family members and additional samples for that.

And that brings up the other major lesson we've learned that we'll need to apply to WGS in the future: Small sample groups are simply not adequate for most purposes. Even for monogenic conditions, numerous samples and multiple families are required. Much like GWAS and linkage studies, we're going to need to sequence a lot of people to find meaning. This has a lot to do with sensitivity and specificity. A false positive rate of 1% sounds okay in a lot of fields, but in genomics, that's 400 false positives in the exome and 30,000 in the whole genome. Good luck finding your one disease causing mutation in there! But we're doing it through clever decision-making with regards to which family members to sequence, which individuals to sequence, et cetera. And that is perhaps the most useful lesson from exome-seq so far.

How do exome sequencing studies contribute to our mechanistic understanding of disease?
The most obvious answer is that exome-seq is very likely to be how we find the genetic etiology of the vast majority of Mendelian disorders. How this will translate to complex disease has yet to be seen, though it's certainly being pursued, particularly in the field of autism.

I would like to respond to a statement made by Kevin Shianna in the paper:

However, exome studies will have very limited power to identify causative variants in regulatory regions spread across the genome (transcription binding sites, enhancers, and so on). Implementing a WGS approach would allow detection of variants in these regions, thus increasing our knowledge of disease beyond the coding region of the genome.

While it is certainly true that exome-seq may be unable to identify mutations in the regulome (yes, regulome), WGS is not the only alternative to it. Why not instead supplement the exome-seq with a regulome-seq? I guarantee you companies are going to create such a thing, and even if you don't want to wait for them to be commercially available, one could create a custom target enrichment that covers the regulome as well.

Back to the cost issue, at about 1/6th the cost of WGS each, exome-seq + regulome-seq would still only come to about 1/3rd the cost of WGS. Outside the exome and regulome, is there much more in the genome that we can even comprehend by sequencing at this moment? Structural variation, perhaps, but as I've already mentioned, SVs can be detected with a low depth WGS. So for half the cost, we may be able to obtain the same biological meaning. That's something to seriously consider when deciding how to budget your sequencing. If you can squeeze out double the samples and obtain practically the same info by doing exome-seq + regulome-seq + low depth WGS, wouldn't you?

I would also add that while it may not be mechanistic, the rise of exome-seq has led to a realization that that we are not adequately prepared from a policy standpoint yet. More than anything, I think it has led to a general realization that medical genetics, genetic counselors and DTC genomics are arriving before society is really ready for them. And that's an issue that I think is going to need increased attention as we researchers charge forward with sequencing everybody.

Does exome sequencing have a limited 'shelf life'?
Not as limited as many people think. Even in the $1000 genome world, exome-seq will still have a place. I'll discuss this a bit more shortly, but there are genomic elements that are more sensitively determined by exome-seq. I feel strongly that once sequencing is sufficiently cheap, exome-seq will become the de facto standard for genomic information much as microarrays were for a good decade. This is due to a combination of factors--the majority of the genome being uninterpretable, the storage cost of genomes versus exomes, the bioinformatic challenges of whole genome versus whole exome, et cetera. The article goes into good depth on many of those issues. The one I'd focus on, however, is that until WGS is actually cheaper than exome-seq, it will always have a place in our field.

I have to disagree strongly with the following statement:

Yes, as soon as the difference in cost between exome and whole genome diminishes (which will be soon) and issues with data management and storage are resolved, whole genome sequencing will be the method of choice.

First of all, he knows as well as the rest of us that "issues with data management and storage" are not trivial to resolve. But beyond that, this is the stance that says, basically, "what's a thousand dollars for a whole genome compared to $300 for just the exome"? I state these numbers because that's where we're heading. At some point exome-seq will stop getting cheaper because the enrichment assay will always cost something. Same for WGS. And my answer to that question is simple: You'll still be able to get three exomes for the price of one whole genome.

The real thing that will limit exome-seq's lifespan is the interpretability of the rest of the genome. If we can utilize the intergenic data to a greater degree, then those bases become more valuable. Then WGS becomes more appealing. Until then, it's all exome (and regulome--I swear, it is coming soon!).

How much do you think that future research will be restricted by the IT-related costs of the analysis?
A great deal. Clouds are not cheap. I'm not convinced they're even the answer at this point. But are clusters? I'm not so sure there, either. I'm not convinced we have the IT solution to this yet because, frankly, I don't think the hardware manufacturers have realized what a lucrative market it's going to be.

I had a conversation about this with somebody not long ago. I said, we've gone from producing gigabytes to terabytes to petabytes over the span of four years in the sequencing world. By now I think there's a good chance we're at or nearing a worldwide exabyte of genomic information. As we start sequencing more and more people, we're going to start hitting the limit for storage capacity worldwide. Sounds crazy, right? But is it?

I also had another rather humorous thought that evolved from that one. How much energy will the number of hard drives needed to store the entire human population's genomic data require? How much heat will those drives produce? In the future, could sequencing be a major cause of global warming? Think about it!

Frankly, I've started to take the issue seriously. One of the tasks on my plate is a thorough assessment of alternative storage formats for genomic data. But that's a post for another day.

Are there any advantages of whole exome over whole genome sequencing?
Yes. Let me say that in less uncertain terms:
YES!
And by that I don't mean what Leslie Biesecker and Jim Mullikin said in their response in the paper. There are literally exonic regions that are better resolved by exome-seq than WGS. We demonstrate that in our recent Nature Biotech paper. A typical WGS will miss a small but meaningful number of exonic variations that are detected by exome-seq. To be fair, the opposite is also true: WGS will detect some exonic variations missed by exome-seq. To me, that says one thing: To be truly comprehensive at this point in time, we need to do both. Naturally, budgets prevent such a thing, but it's important to recognize that targeted enrichment can allow sequencing of regions missed by WGS, and that this is an advantage of exome-seq.



Reference:

Biesecker LG, Shianna KV, Mullikin JC. Exome sequencing: the expert view. 2011. Genome Biol. Sep 14; 12(9):128 [Epub]