aDNA in the Life

Ancient DNA (aDNA)

I heard the news today oh boy,

DNA retrieved from ancient human skeletons shows that migrants ultimately originating from modern-day Turkey were mainly responsible for the introduction of farming across Europe 8000 years ago,

And though the news was rather sad,

I just had to laugh.

There are possibly a few too many syllables here to fit the verse, and I reckon my version is probably catchier on the whole (verbosity is the soul of wit, I think the saying goes), but most importantly this is the kind of statement archaeologists can now make with some certainty thanks to recent breakthroughs in the analysis of human ancient DNA.

I work as a bioarchaeologist (an archaeologist that specialises in the study of organic materials – in my case, human remains) at London’s Natural History Museum on a project that is attempting to track the genetic development of humans in Britain from around 10,000 years ago to the present day. We are primarily focused on looking at how the human genome in Britain has adapted through natural selection to changes in lifestyle, such as the switch from hunter-gathering to farming. However, we are also investigating genetic change resulting from arrivals of new people. We do this by sampling the DNA of British ancient skeletons from varied time periods and looking to see how it has changed over time.

This kind of study has only been possible in the last 5 years or so after a major leap forward in DNA sequencing technology. Rather than only being able to look at tiny but informative parts of ancient genomes, usually concentrated at specific parts of the genome that survive well in archaeological remains, we can now generate vast amounts of information from right across an ancient individual’s genome, giving us an unprecedented insight into their biology and the biology of their ancestors. It is hard to overstate how information-rich studies of ancient DNA have become as a result – analyses of data on high-spec computers can take several days  to complete.

In the early days of ancient DNA, contamination was a major issue. It was very difficult to work out whether aDNA sequence retrieved from ancient remains was genuinely old or was the result of contamination by another human during excavation or handling. The resultant proclamations from ancient geneticists that archaeologists should henceforth don hazmat suits and enforce a sterile environment when excavating human skeletons were met with many lamentations, much wailing and gnashing of teeth. Understandably so – anyone who has worked on a commercial excavation (and archaeological excavation that takes place in advance of development and paid for by the developer) knows that the time pressures involved renders this idea hopelessly impractical in most cases.

However, one of the major benefits of the recent technological advances in ancient DNA is that it allows us us to check for modern contamination. Happily, these checks have shown that with current methods, modern contamination is not a serious problem in most cases, even in remains that were excavated long ago and have been repeatedly handled. Whilst reducing the possibility of modern contamination is still a priority when sampling ancient material for DNA, the days of insisting archaeologists excavate under sterile laboratory conditions are mostly behind us.

As well as many other vaguely notable finds from further back in time (cough, our species breeding with Neanderthals, cough, Denisovans) these innovations have revealed the incredibly messy population history of Europe over the last 30,000 years. This is a bit inconvenient. Most methods that assess natural selection acting on genomes assume that you are looking at the same population through time. However there’s now DNA evidence for several major population changes in European prehistory involving migrations of people from outside of Europe, and that’s only up until around 4000 years ago! We (and many others!) are looking into ways of investigating signs of recent adaptation in British human genomes while taking into account these frequent big population changes.

Drilling Holes in Dead Heads

One of my main roles on the project is to collect and sample ancient human remains for ancient DNA, and that is mostly what I’ve been doing today! I am  in the process of sampling human remains from a variety of dispersed British Early Medieval (c.430-1066 AD) cemeteries held in the anthropology collections of the Natural History Museum.

Another major recent revelation is that the petrous portion of the temporal bone (a bone of the skull located around your ear – the petrous portion is a pyramid shaped outcrop on the internal surface that incorporates your ear canal) is a pretty stupendous source of DNA. This is perhaps unsurprising given that the name ‘petrous’ means ‘stone-like’ as this bone is regarded to be the densest in the human body and most resistant to degradation by microbes and chemicals.  For these reasons, we always try to sample this bone preferentially from ancient human remains. As the petrous portion of the temporal bone is on the inside of the head, it is difficult to sample in complete skulls. Fortunately I’d had a rare bit of foresight before today and purposely chosen examples where the skull bones had broken apart, giving me easy access to the petrous.

One of the temporal bones I sampled today for DNA. The hole in the centre is the outer entrance to the ear canal.

 

A temporal bone showing off the internal surface, The petrous is the sticky-out bit with the eye that looks like a dragon’s head.

Importantly, the petrous is not uniformly good for DNA. There is a ‘sweet spot’ where the DNA preserves particularly well. The size of this sweet spot is dependent on the overall preservation of the bone – in some cases the bone has degraded so far that even DNA preservation in the petrous is low. Identifying and getting at this DNA sweet spot used to involve slicing up temporal bones to ensure it was targetted specifically. This level of destruction is far from ideal and one of the things we have been trying to do at the Natural History Museum is develop a minimally-destructive method of sampling petrous bones for DNA. We’ve now got to the stage where we can sample the productive part of the petrous using a single small hole (roughly 2-5mm wide) in the base. Not only does this method drastically reduce the impact of sampling, it also provides a way of sampling the petrous in complete skulls.

The base of the petrous, where we drill to hit that sweet spot.

To minimise contamination, we sample all bones in the sterile ancient DNA laboratory at the NHM. Sampling ancient remains for DNA is entirely the opposite of what might be expected from archaeology – the key is to stay as clean as possible and maintain this cleanliness at supra-Howard Hughes levels. The first thing I need to do before entering the lab is to suit up! Hazmat suit, face mask, wellies, double-latex-gloves and face guard. I usually feel like a hypochondriac Thunderbird pilot at this point. It sounds like overkill, but theoretically one single stray skin or hair cell during sampling could mess up the entire analysis.

Hypochondriac Thunderbird pilot

The first thing to do when I get into the lab is clean. There is so much cleaning. Cleaning takes up at least 50% of my time when I’m sampling in the lab, not only to avoid modern contamination, but also cross-contamination between samples. I also spend a lot of time changing gloves for the same reason. I get into a mindset where my hands are constantly filthy, essentially a 5-year-old child with adult neuroses levels of self-awareness.  All surfaces are bleached down and special attention given to the drill cabinet where the bone is sampled. The lab itself is fitted with ultraviolet lights that come on every night to make doubley-sure it is free from pesky sources of contamination. The cabinet and surrounding surfaces are thoroughly bleached down between each sample.

I begin by scraping off the outer surface of the bone in the place I want to drill to remove any surface contamination from excavation and handling. I start drilling each temporal bone in more-or-less the same place on the base of the petrous/ One thing I’ve learned doing this job is that no two petrouses (petri?) are alike in terms of preservation and anatomy, therefore my progress from this point is rarely consistent between samples, and relies on me being sensitive to changes in bone consistency.

License to Drill

Sometimes the bone is incredibly hard from the outset and I have to put a lot of pressure on the drill to make a mark. This usually leads to fondly-remembered times where fine white bone powder, pure as driven snow cascades from the drill bit, usually an irresistible sign of high-quality DNA-rich goods. In other cases I barely have to touch the bone or even turn the drill on and the drill bit sinks into the bone like it’s moldy bread, producing a blast of coarse, sandy brown bone powder that looks like it’s been in a fire. I recall these situations less fondly. DNA yield from this type of bone powder tends to be zilch. Nonetheless, even in these cases I continue to drill deeper towards the sweet spot, as samples which seem poor initially can often maintain a well-preserved resilient core. The change when you hit an area of well-preserved bone is palpable, both in terms of how the bone feels, but also in the quality of the bone powder.

Once I’ve collected 50-100 milligrams (a milligram is a thousandth of a gram) I pour the bone powder into a plastic vial, weight it and place it in a cupboard ready for further laboratory work where the DNA is extracted and prepared for sequencing (I make this sound like a doddle, but it involves several more days of intense lab work by individuals cleverer than I!). I then completely clean down the laboratory again. The used drill bits are put in an oven to sterilise. The drill itself is put in the drill cabinet where it is bathed in ultraviolet light.

…like pure driven snow…

Today I managed to drill the four bones quite rapidly, but it still took two hours. In case you’re interested, two were very nicely-preserved indeed, one was like drilling a sand-castle and the last was superficially rotten, but had a nice core. That’s petrous personality.

Base of the petrous after sampling. It’s all about the base.