A team led by Anqi Chen, Qi Yang, Suhua Zhang, and Chengtao Li at the Institute of Forensic Science of Fudan University in Shanghai systematically compared the two dominant sequencing strategies for reading the full mitochondrial genome. On one side stood Illumina short-read sequencing, the workhorse of modern genomics, which chops DNA into fragments of a few hundred bases and reassembles them computationally. On the other stood nanopore long-read sequencing performed on a QitanTech platform, which threads much longer DNA molecules through protein pores and reads them in real time. The researchers applied both technologies to samples from 177 individuals spanning 39 maternal pedigrees, a design that allowed them to check whether each platform called the same variants in mothers, children, siblings, and cousins who should, by the rules of maternal inheritance, share essentially identical mitochondrial genomes.

The technical heart of the comparison lies in how each platform prepares DNA for sequencing. The short-read workflow relied on multiplex PCR, in which many primer pairs amplify overlapping fragments of the mitochondrial genome in a single reaction. This approach is efficient but notoriously uneven: some regions amplify enthusiastically while others lag behind, producing a patchwork of coverage depth across the genome. The long-read workflow took a fundamentally different route, using a single-amplicon enrichment strategy that amplifies the entire mitochondrial genome in one long piece. The payoff was dramatic. Long-read sequencing achieved a median coverage uniformity of 100 percent, effectively flattening the coverage variability that plagues multiplex PCR approaches and ensuring that every position of the mitochondrial genome is read a comparable number of times.

Coverage uniformity is not merely an aesthetic virtue. In forensic work, uneven coverage can mask genuine variants or inflate the apparent frequency of sequencing errors, complicating the interpretation of mixed or degraded samples. The long-read approach delivered a second, equally important advantage: the ability to see through one of mitochondrial DNA analysis’s most insidious traps. Scattered throughout the human nuclear genome are fragments of ancient mitochondrial DNA that migrated into our chromosomes over evolutionary time. These nuclear mitochondrial DNA segments, known as NUMTs, are molecular fossils that can be mistakenly amplified and sequenced alongside genuine mitochondrial DNA, contaminating forensic profiles with phantom variants. Short reads, typically a few hundred bases long, often cannot distinguish whether a sequence originated from a true mitochondrion or from a nuclear NUMT that happens to match the primer binding sites. Long reads, by contrast, can span the homologous regions entirely, revealing the surrounding nuclear context and allowing analysts to exclude these impostors with confidence.

The short-read data exposed the scale of this problem in vivid detail. The researchers observed pronounced off-target alignment in their Illumina datasets, with spurious reads piling up on particular human chromosomes. Chromosome 17 and chromosome 2 stood out as hotspots, accumulating misaligned sequences because of spurious sequence homology between NUMTs embedded there and the mitochondrial targets being amplified. In a forensic laboratory, such off-target material is more than a nuisance; it consumes sequencing capacity, complicates bioinformatic filtering, and in the worst case can introduce false variants into a profile that might later be used to include or exclude a suspect or identify human remains.

Yet the story is not a simple tale of long reads winning. When the researchers compared the consensus haplotypes produced by the two platforms, the sequences agreed: both technologies called the same set of mitochondrial variants for each individual. The critical divergence emerged in a more delicate measurement, the detection of heteroplasmy. Heteroplasmy refers to the coexistence of two or more mitochondrial DNA variants within a single person, a natural consequence of the way mitochondria are inherited through a genetic bottleneck and copied independently within cells. In forensic science, heteroplasmy can be a double-edged sword. On one hand, a shared rare heteroplasmic variant can provide powerful additional evidence linking relatives or identifying remains. On the other hand, distinguishing a genuine low-level heteroplasmic variant from a random sequencing error requires exquisite measurement fidelity, because true variants may be present at frequencies of just a few percent.

Here the short-read platform revealed its enduring strength. Illumina sequencing exhibited higher accuracy for detecting low-level heteroplasmy, thanks to the sheer depth and low intrinsic error rate of its short reads, which allow genuine minor variants to be separated from background noise with statistical confidence. The long-read workflow, despite its many advantages, carried a heavier background-noise burden. When the team validated their results against the family pedigrees, they found that the current long-read pipeline produced noise signals that were misclassified as heteroplasmy at a 10 percent allele frequency threshold. In other words, sequencing artifacts occurring at low frequencies could masquerade as genuine mixed mitochondrial populations, a potentially serious problem in casework where a false heteroplasmy call could undermine the interpretation of evidence.

Rather than declaring one technology the victor, the researchers proposed something more pragmatic and arguably more useful for the forensic community: a complementary workflow that assigns each platform the task it performs best. Under this integrated strategy, long-read sequencing would be deployed for structural characterization of the mitochondrial genome and for excluding NUMT interference, exploiting its superior coverage uniformity and its ability to span ambiguous regions. Short-read sequencing would then take over for high-fidelity quantification of heteroplasmy, where its precision at low variant frequencies is unmatched. The two technologies, rather than competing, would function as checks on each other, each compensating for the other’s weaknesses.

The implications for forensic casework are substantial. Laboratories handling complex or low-input samples, such as touch DNA from a weapon, hair shafts without roots, or skeletal remains recovered years after death, often face exactly the conditions where these platform trade-offs matter most. A degraded sample may yield too little DNA for confident short-read analysis of NUMT-contaminated regions, while the same sample might benefit enormously from long reads that clarify structural ambiguities. Conversely, a case hinging on a rare heteroplasmic variant shared between a missing person and a maternal relative demands the quantification accuracy that only deep short-read sequencing can provide. The Fudan team’s pedigree-based validation offers laboratories a data-driven rationale for choosing, or combining, their sequencing strategies rather than relying on convention or convenience.

The study also reflects a broader shift in forensic genetics, as nanopore sequencing matures from an experimental curiosity into a validated platform for legal applications. Chinese-made nanopore devices from QitanTech are emerging as alternatives to the long-dominant Oxford Nanopore systems, and their performance in this head-to-head comparison demonstrates that the technology has reached a level of maturity where systematic forensic validation, not just proof-of-concept demonstrations, is possible. With funding from China’s National Key Research and Development Program and the National Natural Science Foundation of China, and computations performed on Fudan University’s CFFF platform, the work signals that the infrastructure for rigorous platform benchmarking is now in place. For a field in which a single base call can help identify a fallen soldier or exonerate the innocent, knowing precisely when to trust long reads and when to demand the precision of short ones is not a technical footnote. It is the difference between evidence that stands up in court and evidence that collapses under scrutiny.

Subject of Research: Comparison of short-read and long-read sequencing platforms for forensic mitochondrial genome analysis

Article Title: Pedigree-Based comparison of short-read and long-read sequencing for forensic mitochondrial genome analysis

Article References: Chen, A., Yang, Q., Cao, Y., Liu, Y., Yang, F., Zhang, S., & Li, C. (2026). Pedigree-Based comparison of short-read and long-read sequencing for forensic mitochondrial genome analysis. International Journal of Legal Medicine. https://doi.org/10.1007/s00414-026-04031-5

Image Credits: AI Generated

DOI: 10.1007/s00414-026-04031-5

Keywords: mitochondrial DNA, forensic genetics, long-read sequencing, short-read sequencing, nanopore sequencing, heteroplasmy, NUMTs, DNA profiling, pedigree analysis, Illumina, QitanTech, human identification