How does ancient DNA technology “find gold in the waves”?

Reconstructing the complete genomes of extinct ancient humans - Neanderthals and Denisovans, mapping the history of global population migration and exchanges, excavating the genetic structure of the oldest modern humans in East Asia, the "pastoral people", revealing the changes in adaptive genes of East Asian populations before and after the Last Glacial Maximum, tracing the formation of the population pattern in northern and southern China, and tracing the origin of the Austronesian people in southern China... In the past decade, researchers have used ancient DNA technology to discover genetic information that has been lost for thousands of years, unraveling it and constantly refreshing human understanding of its own evolutionary process.

On July 21, Cell published a special issue discussing the latest developments in biotechnology. Fu Qiaomie, a researcher at the Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, was invited to lead the ancient DNA frontier field to publish a review paper on the development and future of ancient DNA technology. The article reviewed the development history and breakthroughs of ancient DNA technology, discussed the current technical bottlenecks and solutions, and the future development direction and prospects of ancient DNA technology.

High-throughput sequencing covers the entire ancient DNA field

High-throughput sequencing, also known as second-generation sequencing, is a technology that can quickly determine the sequence of a large number of DNA fragments. Before the popularization of high-throughput sequencing, the ancient DNA field could only rely on PCR technology to determine the sequence of a few specific DNA fragments.

"This method can only obtain very limited DNA information, and it is difficult to distinguish between real ancient DNA and contaminated DNA. High-throughput sequencing can theoretically sequence the information of all DNA molecules in a sample, and the cost is decreasing year by year. Even ancient DNA with extremely low content can be sequenced very effectively," Fu Qiaomie explained. Not only that, through bioinformatics methods, it is also possible to quickly detect whether there is ancient DNA damage in the sample, thereby achieving the purpose of identifying ancient DNA. This method has also become an important standard for ancient DNA detection in the field.

In addition, the researchers have made various adjustments and optimizations to the experimental methods of high-throughput sequencing (DNA library construction) based on the characteristics of ancient DNA.

Fu Qiaomie mentioned that half-UDG treatment and the construction of single-stranded DNA libraries are the two most important technological breakthroughs. Half-UDG technology can both retain some DNA end damage and repair most ancient DNA damage, thereby improving the accuracy of ancient DNA sequencing results while retaining the characteristics of ancient DNA. The single-stranded DNA library directly denatures double-stranded DNA into single-stranded DNA to construct a library, targeting the situation where there are often a large number of single-stranded sticky ends in ancient DNA, so as to more effectively sequence the damaged single-stranded ancient DNA.

DNA capture technology pushes the limits

"Although high-throughput sequencing can sequence ancient DNA relatively effectively, because ancient DNA extracts often contain a large amount of contaminating DNA, most of the sequenced DNA molecules are useless information. Truly useful ancient DNA sequences often account for less than 1% of the sequencing data," said Fu Qiaomie.

In response to this, researchers have developed and applied DNA capture technology in the field of ancient DNA - by designing DNA or RNA probes, they "fish" out the target ancient DNA from the massive amount of contaminating DNA like fishing.

This technology is currently widely used in human ancient genome research, and more than two-thirds of human ancient genome data comes from data captured by a probe group called "1240k".

"DNA capture technology not only greatly improves the efficiency of ancient DNA sequencing, but also can effectively obtain sufficient data from some 'tricky' samples for analysis," said Fu Qiaomie.

A typical example is the genome research of ancient southern populations published by Fu Qiaomie's team in Cell last year. The warm and humid environment and local acidic soil in southern China are not conducive to the preservation of ancient DNA, which has left ancient DNA research in this area blank for a time. At that time, it was by using DNA capture technology that Fu Qiaomie's team successfully obtained genome information of 30 ancient southern populations, revealing the genetic history of the population at the intersection of East Asia and Southeast Asia for more than 10,000 years.

Recently, ancient DNA researchers have further challenged the limits, breaking away from the shackles of fossils and extracting ancient DNA directly from the "soil". This technology has been successfully applied to Denisova Cave and Baishiya Cave, successfully obtaining DNA from extinct ancient humans tens of thousands of years ago. The process of continuous iteration of ancient DNA technology and "gold panning in the waves" is still ongoing.

Difficulties to be overcome in the future

Although the field of ancient DNA has achieved fruitful results, ancient DNA research has always been full of hardships and challenges.

Ancient DNA itself is very susceptible to contamination, and the experiments require extremely sophisticated work. In the past, ancient DNA extraction and library construction almost entirely relied on manual operations. Fu Qiaomie introduced that recently, in a few laboratories around the world, some ancient DNA experimental steps have been successfully integrated into the fully automatic liquid transfer robot platform, which not only greatly saves manpower and material resources, but also reduces the risk of contamination introduced by manual operations.

"Currently, the sample pre-processing steps still have to rely on manual labor. How to integrate this time-consuming and labor-intensive work into the automated system is the next hurdle that ancient DNA experimental technology needs to overcome," Fu Qiaomie pointed out.

In addition, the application of ancient DNA technology goes far beyond human ancient genomes. Fu Qiaomie introduced that tracing ancient epidemics and the evolution of symbiotic microorganisms through ancient microbial DNA information, using ancient epigenetic information to explore the interaction between ancient animals and the environment, and using ancient proteins to explore human evolution on a larger time scale are all important branches of ancient molecular biology. In her opinion, how to obtain this information more effectively and combine the information in multiple dimensions is one of the difficulties in future research.

Ancient DNA is genetic information with a time scale, which records the evolution and adaptation of human beings over tens of thousands of years from a unique perspective. These traces of time not only record the genetic history of human beings, but also continue to affect the physiology and health of people today.

"We speculate that some important functional gene haplotypes come from extinct ancient humans. These genes are involved in innate immunity, lipid metabolism, high altitude adaptability, skin color, susceptibility to severe COVID-19, etc. In addition, there are genotypes related to hair and tooth phenotypes that are unique to our East Asian population, and the frequency also increased after the Last Glacial Maximum. It is speculated that this is related to environmental adaptability." Fu Qiaomie said.

In addition, the functions of many special genotypes discovered by ancient DNA research have yet to be determined. Fu Qiaomie believes that in the future, these findings can be verified by building animal models and combining gene editing technology. "Combining ancient DNA technology with modern cutting-edge molecular biology technology, we can more clearly understand the impact of evolutionary history on human health today."