Fallen but not forgotten: NGS technology identifies Spanish Civil War victims

Isabel Navarro was immersed in her job when the 96-year-old woman sitting beside her began to cry. “She told me that it was the happiest day of her life,” Navarro recalls, “because she knew that she could die and that someone would still be trying to find her father.”

Navarro is head of the forensic laboratory at Citogen in Zaragoza, Spain. That day, she was collecting a DNA sample from the elderly woman as part of a massive historical reconciliation project to identify the remains of victims of the Spanish Civil War.

Five decades after the death of General Francisco Franco, Spain is still grappling with the aftermath of the civil war of 1936 and 35 years of dictatorship. In recent years, the government has passed several measures intended to bring “justice reparation and dignity” to the victims of war.

Among the initiatives is the creation of a national DNA databank to help identify the remains of the tens of thousands of people who were killed either during the war, or afterward for opposing the Franco regime. Their remains still lie in unmarked graves around the country.

In the 1930s, Spain was polarized between left- and right-wing factions. In July 1936, the assassination of right-wing politician José Calvo Sotelo prompted a military coup led by General Francisco Franco, sparking the Spanish Civil War.

Franco's Nationalists clashed with the Republican government, with both sides persecuting perceived enemies through executions, imprisonment, and forced labor. After the Nationalist victory with Franco in power, former adversaries were targeted by confiscating property, revoking qualifications, imprisoning many and executing tens of thousands.

Of the estimated 300,000 to 500,000 lives lost in the war, roughly 150,000 were civilians. 

Identifying war victims

Historians estimate thousands ended up in shallow graves by the sides of the roads where they were killed. In other cases, officers shot their prisoners in cemeteries, where victims fell straight into communal graves (1).

MPS marks a major advance in the ability to identify remains, says Navarro, because it can give more detailed information than traditional methods and do so with less DNA. 

“I am quite excited about MPS,” says Navarro. “It is going to give us clues to get better results and enable the statistical analyses to be able to better identify these people.” 

When Franco died in 1975, Spain embraced democracy. But unlike countries such as Germany and Italy that had long started the process of reckoning with the crimes committed by fascist regimes of their past, Spain chose otherwise. Through a political arrangement known as the “Pact of Forgetting,” the country’s parties agreed to forgo investigation into, or prosecution for, crimes related to the civil war or the dictatorship so that the nation could move forward.

By the early 2000s, however, silence on the country’s brutal past was beginning to give way to a quest for truth and reconciliation. Archaeologists, journalists and families pushed to document and unearth mass graves across the country. The movement was driven in part by advances in forensic technology. With new tools like DNA sequencing and skeletal analysis, forensic specialists could identify remains more easily and match them to living relatives (2).

In 2007, Spain’s socialist government finally officially recognized the atrocities and passed a law on “historical memory” that required the national government to facilitate and provide funding to identify victims of the war. In 2022, a new “Democratic Memory Law” was enacted that included funding for a DNA database and an official list of civil war victims, among other measures.

Identifying remains with MPS

Since the 1980s, labs have used Short Tandem Repeats (STRs) to determine DNA profiles by counting the repeated DNA sequences at specific chromosomal locations. However, CE's limited loci analysis makes identifying distant relatives challenging.

In 2018, Navarro and her team upgraded their lab with MPS. Previously, they used capillary electrophoresis (CE) to identify remains, but CE could only analyze 22 genetic markers, while MPS can handle over 200. By analyzing more loci, MPS offers greater accuracy in identifying distant relatives and degraded DNA.

Navarro’s lab finds MPS especially useful in identifying heavily degraded DNA, which is often the case in old bone samples. MPS also provides more markers. Thus, researchers are more likely to find a match, and even a positive identification, despite the living relatives being only distantly related to the civil war victim.

After receiving a bone or tooth sample, researchers in Navarro’s lab treat it with various reagents to remove exterior contamination. It is then ground into dust using the TissueLyser. Samples are then further processed to extract DNA, which is purified using QIAGEN Investigator kits and quantified using the Investigator Quantiplex Pro Kit.

The kit also quantifies the amount of DNA in the sample, and gives information on the degree of DNA degradation and the possible presence of artifacts that might inhibit the PCR reaction. Researchers then use the ForenSeq MainstAY kit to amplify the DNA and obtain an STR profile.

In addition to autosomal STRs, Navarro’s lab also profiles Y chromosomes, which are passed virtually unchanged through the male lineage. Since the majority of people killed during the civil war were men, the addition of Y chromosome profiling can help positively identify remains with greater certainty.

STR profiles from remains are then compared to a database of profiles obtained from family members who are looking for deceased loved ones. Right now, there is no central database; each region is being planned, Navarro adds, which will make it easier to find matches since any relatives may have moved far away from their family homes.

Finally providing answers

MPS enabled researchers to positively identify five victims from the remains of eight people buried in a shallow grave in the village of Manzanares.

In a poster presented at a forensic science meeting in 2023, the researchers concluded: “Simultaneous amplification from autosomal and Y-STRs in a single reaction allowed by ForenSeq^®MainstAY kit, arises as a very convenient method, especially when working with war victims who are mainly men.”

The MPS workflow that Navarro now uses has made her lab “more efficient,” she says, “it has improved our work in many ways.” Since MPS is more sensitive, “we need less DNA at the outset,” she explains. And her lab has gotten really good results from bone samples on which CE didn’t perform very well, she adds.  

In the village of Manzanares, everyone knew where the unmarked grave containing the remains of family members was located. Even though Spanish history books don’t document the atrocities committed during the civil war because of the “pact of forgetting,” the stories of killings and the violence have been passed down through families.

Despite the fact that the deaths happened 90 years ago, families still feel the trauma, Navarro says. People in the village suspected that family members might be buried in the mass grave, says Navarro, but they had no way to identify them – that is until now.

“In the next few years, we are going to see huge developments in the application of NGS techniques like MPS applied to forensics,” Navarro says. “We still have a lot of work to do in creating standards so that more labs can use these tools, but they will have a huge impact.”

The most “exciting thing” is the potential of the technology, she adds. “I am sure that MPS can provide us with even more insight than what we now know. In the future, it will enable us to do go even further in terms of identifying remains.”