Being professional geneticists, we have jointly worked on a few projects on ancient samples in the past. All those projects were exciting and fruitful. However, it was only the recent work that gave us countless goosebumps and tears in our eyes.

This is a significant project where, 165 years later, molecular and isotope data provided solid scientific proof to confirm the now-forgotten, brutal Ajnala massacre.

Based on the historic details documented in a book written by the colonial Deputy Commissioner of Amritsar Frederick Henry Cooper, a local historian Surinder Kochhar, identified several disposed skeletons in an abandoned well, in 2014.

Frederick Henry Cooper was the person who himself had ordered the mass killing of Indian freedom fighters in 1857.

This discovery has divided people into two groups. Some believed that these were the remains of the 282 Indian sepoys who mutinied during the 1857 Indian uprising, while others suggested that they were victims of Partition in 1947.

Although this critical discovery was published in several papers, it again went out of memory, and nothing substantial was done to preserve this important event and site related to the Indian Uprising.

Later, in 2018, Forensic Anthropologist, Dr Jagminder Singh Sehrawat from Panjab University brought some skeletons to the CSIR-CCMB ancient DNA lab. He wanted to do their DNA analysis to understand who they were.

During that time, one of us (Dr Niraj Rai) was in-charge of the ancient DNA lab. Though the archaeological and historical data confirmed that the remains were likely to be from the sepoys of 1857, the next challenge was the direct confirmation of their identity.

During the meeting, Dr Sehrawat explained the questions in detail with their alternative hypotheses. He also explained that they found unique coins and medals, which had been made in the year 1856.

The anthropological analyses done by his team revealed that they were all adult males with good dental hygiene, indicative of their military affiliations. Moreover, the radiocarbon dates measured from some teeth collagen samples supported the hypothesis that the remains belong to the mid-19th century.

The scale of horror

We had read about the Jallianwala Bagh massacre. However, this brutal event, the Ajnala massacre, was not in our knowledge. This terrible event, narrated by Dr Sehrawat, distressed us so much that we could not sleep for several days.

It took us almost two years to isolate an adequate amount of DNA, as the first excavation did not use scientific techniques to preserve the specimens.

The DNA quality was poor, so we decided to focus on mitochondrial DNA analysis. Our body is made up of nearly 3.2 billion cells and each cell contains more than one copy of mitochondria, therefore, it is easier to decode the mitochondrial DNA from ancient samples.

We inherit our mitochondrial DNA from our mother and studying mitochondrial DNA is crucial to understanding the genetic history of an individual.

On the other hand, in parallel to the DNA analyses, some good teeth samples had also been sent to the Birbal Sahni Institute of Palaeosciences, Lucknow, for independent analysis of isotopes. Both the groups have worked independently without informing any result to each other.

For the genetic analysis, we designed a panel of 115 markers. This panel was designed in such a way that it captures the overall maternal diversity of the Indian subcontinent. This panel has the power to differentiate any unknown population of Indian origin at the state level.

Now, we considered all these 50 samples as a single (unknown) population and scored the mutations. Based on these mutations, these samples were classified into haplogroups.

A haplogroup is a group of several individuals containing a string of mutations. People in one haplogroup can trace back their maternal ancestry to a single great grandmother. Analysis of these 50 individuals has divided the samples into 13 haplogroups. The most predominant was haplogroup M39 followed by haplogroup R32.

Both of these haplogroups are indigenous to India and are distributed sporadically.

The statistical analysis placed this (unknown) population in close vicinity of Bihar, Uttar Pradesh, West Bengal and Jharkhand states, suggesting a closer genetic affinity of this unknown population with the Gangetic plain populations.

We have also clubbed these haplogroups based on their geographic origin. The geographic ancestry spectrum of the Ajnala samples was very similar to the Ganga plain populations. Thus the genetic analysis was consistent with the historic data.

The independent isotopic analysis by the experts in the Birbal Sahni Institute on the tooth enamel of 85 samples overwhelmingly suggested the mixture C3-C4 diet pattern.

The C3 pattern is found among people consuming wheat, rice, and legumes; whereas the C4 diet is present among people consuming millets.

The archaeological data also supported their homeland in the Ganga plain.

Thus, analyses of unknown samples by using two independent techniques added significant study novelty.

Our analysis is consistent with the historical evidence, stating that the 26th Native Bengal Infantry battalion comprised of soldiers from Bengal, Odisha, Bihar, and eastern parts of Uttar Pradesh.

This is a matter of social and historic debate as to how this issue of national importance went out of memory of the people and the government. Perhaps, After the Jallianwala Bagh massacre, the British Raj did not want to take any other sin on its head. They were indeed successful in hiding this massacre.

The confirmation of results not only testify to the cruel and violent history of the British Raj in India but also suggest that we reconsider our national heroes.

We request that the Government of India obtain their details from the UK so that the appropriate last rituals are performed for these freedom fighters.

Niraj Rai and Gyaneshwer Chaubey

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