
HORSEPOWER
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Interactions between China, Mongolia and the steppe 2000-0 BCE
Five steps to unlocking ancient DNA
A fundamental feature of the Horsepower project is investigating the DNA of ancient horses. This will reveal details of the animals themselves and tell us a lot about the cultures in which they lived. Gaetan Tressieres describes how this work is carried out in the lab run by Ludovic Orlando in Toulouse.
Step One: Hygiene
To limit contamination of the samples, the laboratory has to be isolated by keeping the air pressure higher inside than out. Everyone has to wear lab-suits, masks, hair nets, oversleeves, shoe covers and gloves. And all equipment has to be manipulated in a specific area called “modern” to protect against other sources of DNA content.

Figure 1: Ancient lab in CAGT. The technician, all equipped, is preparing material for bone sampling under hoods. UV light presents in hood on the right helps to decontaminate the area before.
Step Two: Samples
Small pieces are cut from a bone or a tooth and pulverised to produce about 150-200mg of powder which is stored in sterilised tubes.

Figure 2: Sampling preparation with dremel helping to cut a small piece of bone.
Step Three: Extraction
The powder is treated with a bleach solution that’s later washed away by pure water. It’s then put through a series of processes. Immersed in a solvent, the horse DNA sticks to silica which allows everything that’s unwanted to be removed by washing and then drying in a centrifuge. Eventually the result is a tiny but precious 60µL of purified horse DNA that ends up in a test tube stored at -20C.

Figure 3: Digestion step during extraction: tube containing bone powder and digestion buffer are incubated in oven with rotation.
Step Four: Preparation
DNA extracted from ancient samples can contain molecular changes, caused by time, such as fragmentation and ‘cytosine deamination’ which can lead to errors in sequencing. So a treatment known as USER, which takes three hours, recognises and cuts out these mutations. Next, because the DNA may be very degraded, a ‘library’ of repaired samples has to be built up and short sections of known sequences are added. After this ‘library building’, our modified DNA is then ready for Polymerase Chain Reaction which allows sequences to be copied. This replication is repeated several times to increase the amount of DNA available.

Figure 4: Modern lab: used for PCR amplification and DNA purification before sequencing approach.

Figure 5: Short resume of all steps leading to the sequencing of ancient DNA.
Step Five: Sequencing
When the new amplified libraries have been purified and have passed quality criteria, the samples go through a Miniseq sequencer. This can generate more than 25 billion reads. The data are then mapped to a reference genome (for horses, this is known as equCab3) to estimate the proportion of reads referring to the target.

Figure 5: Sequencing approach in CAGT lab with Miniseq monitor.
It’s a lot of painstaking work at the cutting-edge of genetic research but the results are illuminating in ways that earlier generations could only have dreamed of. The colour of the horses, their characteristics and their patterns of kinship can all be determined, even in animals 3,000 years old. The first results should emerge soon and are set to be enthralling.