Violent metaphors

It seems that every week there are exciting new findings from ancient DNA research.  This is wonderful news, because we’re learning incredible things about the relationship between humans and Neandertals, the prehistory of ancient populations, and even previously unknown hominins.  But on the flip side, we’re also seeing news reports of extremely questionable results, and I’ve gotten more than one inquiry recently from people excited or confused by them. I though it would be a good idea to write a bit about how regular people can figure out whether a study is legitimate or not.

The first step in distinguishing good ancient DNA studies from bad ones is the same as distinguishing pseudoscience from legitimate science in general: ask where the results are published. Are they in a peer-reviewed journal? Or does the author present it as “science by press release,” stating something like:

“Peer review will of course be considered, but this information belongs to THE WORLD; not a few academics…”

The next steps require you to know a bit about ancient DNA itself, and how research is conducted. What most casual readers may not understand is how difficult recovering DNA from ancient remains is….and how easily it can become contaminated.

The TL;DR version is that for an ancient DNA study to be considered authentic, at minimum it:

• Must be conducted in the proper facilities
• Must be conducted by personnel practicing sterile techniques
• Must utilize negative controls
• Must have a subset of results reproduced by an outside laboratory
• Must yield phylogenetically reasonable results (or produce extraordinary evidence to support unusual results), that match the characteristics of ancient DNA.
• Must conform to any additional standards necessary, depending on the sample and experimental design

Here’s why:

When an organism dies, its DNA begins to break down into small fragments. This process is affected by several factors, including burial conditions, the age of the sample, and its exposure to chemicals in the soil (or in the laboratory in which it’s housed). The fragmented, damaged DNA is extremely difficult to recover–a success rate of 50% is often considered to be a good outcome! Because the process of extracting DNA from ancient materials (usually) destroys them, it’s important to only conduct research that addresses scientifically interesting questions, that is ethical (done with permissions and support of the relevant descendent communities), and (as best as you can tell) likely to work. You can sometimes determine that last part by doing a test extraction from associated faunal remains, but sometimes there’s simply no way of knowing until you start. But it’s an important consideration, as you don’t want to destroy a sample needlessly if you have reason to believe it won’t work.

Because the DNA in an ancient sample is so fragmented and damaged, the process of copying it into enough quantities to work with will preferentially copy any modern DNA that’s present. So contamination is an enormous concern in the ancient DNA community. Not only do we have to be careful about preventing it from happening, we have to be able to tell the difference between contaminated sequences and legitimate ancient sequences. It’s not as easy as you might think. And so all of us in the ancient DNA community have over the years come to agree on a general list of guidelines for conducting this research. These guidelines have been shown, through experience, to be the best practices for preventing and detecting contamination.

1. The work must be conducted in a proper facility

Ancient DNA laboratories must be physically isolated from laboratories in which modern DNA is handled, and also from spaces in which researchers work with post-amplified DNA.  It is NOT sufficient to work with ancient DNA in a hood in the corner of a regular lab. To get rid of contamination, ancient DNA labs are isolated, positively pressurized, and regularly bleached and irradiated with UV light. The only people allowed into the labs are those who are specially trained in ancient DNA work, and they can only enter the lab if they haven’t been in any other lab earlier in the day. (does this sound crazily stringent? It is absolutely necessary, because it’s easy to bring modern DNA into an isolation lab on your clothes or shoes). Everything that enters the lab–from pipettes to reagents to tubes–is bleached and/or UV irradiated to get rid of surface DNA.

2. Researchers must wear protective clothing and practice sterile technique

Everyone wears protective clothing, but exactly what it constitutes varies somewhat from lab to lab (at minimum: gloves, hairnet, face mask, and suit or coat). Personally, I keep a special set of scrubs in my office that I change into before going to the aDNA lab. Then I don a full bodysuit, hairnet, face mask, hood, gloves, and sleeve guards. THEN I bleach the entire outside of my suit and gloves. Here is a picture of what I wear in the lab (Don’t worry, colleagues, I changed my gloves after shadowboxing):

It may seem excessive, but following this procedure means I very, very rarely find my own DNA in ancient samples I work with.

Sterile technique means following practices such as not exposing samples to potential contamination (such as by leaving tubes open to the air unnecessarily), changing gloves frequently, bleaching surfaces and materials, and a host of other things.

3. Use negative controls

Every single step of the process–from extraction to PCR amplification–introduces another risk of potential contamination. To mitigate that we use special reagents that are guaranteed to be “DNA free”, but even so we need to be able to tell when contamination has occurred. So we use negative controls (“blanks”)–tubes of water with no ancient sample added–that are processed alongside the ancient samples. If one of them turns out to have DNA, we know that the entire group of samples processed alongside it has been contaminated. (We can even sequence that DNA and compare it to sequences of laboratory personnel to find out who the culprit was!). This is CRITICAL, and no ancient DNA can be considered authentic unless negative controls demonstrate that it’s not contaminated.**

4. Reproducibility of results

This takes several forms. First, in order to be considered authentic, targeted ancient DNA studies will generally need to  replicate their sequencing results from two independent extractions (there’s a bit of a different standard applied to complete genome studies, as they’re sequencing many independent fragments). Ideally these extractions take place with at least a month’s time in between them, in order to guarantee that different sets of reagents are used for each extraction. Secondly, a laboratory will send out a subset of their samples for testing to another, independent laboratory. Note that it is NOT sufficient to send already-extracted DNA for processing, but rather the raw material itself (bone, hair, tissue) must be sent in order that the testing be truly independent. If the sequences recovered for the same sample by the two laboratories match, the requirement is met.

5. Reasonableness of results

Here the standard is: the results must make phylogenetic sense, and extraordinary claims must be supported with extraordinary evidence. For example, I work with ancient Native American samples. If one of them suddenly produces a new sequence never before seen in Native American populations, my default assumption will be that it’s contaminated. I will not accept it as a legitimate result until I’ve exhausted all other possibilities. The Denisovan genome is a good example of a highly unusual result–a new hominin species!–that was presented with an extraordinary standard of evidence to back it up.

One way of telling whether a sequence is legitimately ancient DNA (often used in complete genome sequencing) is by looking at the amount of damage it has. Short, damaged fragments are much more likely to be authentic than long, undamaged fragments. In fact, I would be concerned about any study that claimed to get extremely long and undamaged fragments (>250 bases) from their samples. It’s not impossible (given extraordinary preservation conditions), but it is extremely unlikely and would cause me to ask many additional questions about their samples.

There are other steps for authentication, such as cloning the sample, quantitation of the amount of DNA, and testing of additional biomolecules–but they’re a bit technical and I won’t get into them here. You can read much more about ancient DNA quality control here, and here, and here, I’m also happy to point you to other resources, if interested. Finally, I want to stress that this is an ongoing area of discussion in the community: as the field evolves, the criteria evolve as well.

The process of conducting and authenticating ancient DNA research may seem onerous, but each requirement is there for a reason. Even if a researcher doesn’t have access to a proper ancient DNA facility, there are plenty of ancient DNA specialists who are open to the idea of conducting such work on a person’s behalf as collaborators, or to train people in proper techniques and allow them to work in their laboratories. So there is absolutely no reason to circumvent these standards, and no respectable journal should publish a study that doesn’t follow them. Beware of researchers who constantly turn up shocking results that don’t follow these general guidelines, and are only “published” in press releases.

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**There are some additional steps and methodologies utilized in complete genome sequencing, but I won’t get into them here.