After four decades of development, from DNA fingerprinting to next-generation sequencing (NGS), forensic DNA technology still faces common global challenges in crime laboratories: case backlogs, sample degradation, and database imbalances. Internationally, US state laboratories reported a backlog of up to 3,630 cases (source: https://www.wispolitics.com/2022/state-crime-labs-processed-more-dna-cases-in-2021-but-average-turnaround-time-lagged-2020/). The root causes lie not only in infrastructure and manpower shortages but also in the unpredictability of sample analysis – the success rate for touch DNA varies from 0% to 58.82% depending on the substrate[12]. Furthermore, DNA extraction and quantification steps can lead to sample loss and risk contamination with exogenous DNA[13]. The success rate for typing challenging degraded samples like aged bones, teeth, and decomposed corpses is even lower. Simultaneously, global DNA database coverage shows significant disparities: only 89 out of 194 INTERPOL member states have established forensic DNA databases. The lack of population-specific data further diminishes individual identification effectiveness, highlighting the urgency of the "Faster, Higher, Stronger – Growing Together (Communiter)" technological concept proposed by Butler[14]. Technical bottlenecks are also undeniable. For instance, Rapid DNA systems (e.g., ANDE™ 6C) can process buccal swabs in just 94 minutes (97% success rate)[15], but their detection rate for muscle or rib samples plummets to 0-11%[16]. While single-cell separation technology greatly improves the success rate of mixed sample analysis, it still cannot avoid the possibility of missing alleles.

In contrast, China's forensic DNA field faces unique pressures related to scale expansion and technological demands. By 2018[17], there were 755 DNA laboratories in public security agencies nationwide, handling 800,000 cases annually. The national DNA database expanded at an average annual rate of 8 million profiles, reaching 44 million profiles, the largest globally[18]. This rapid growth has simultaneously generated three closely related technological needs[19]: First, gang rape cases and complex paternity testing (e.g., father-daughter, sister relationships) require obtaining more genetic markers (e.g., X-chromosome markers) in a single test. Second, the expansion of database size and the occurrence of STR mutations in anti-human-trafficking operations necessitate introducing more loci to enhance matching accuracy. Finally, DNA fragmentation and loss of large amplicons in biological evidence collected at crime scenes, affected by temperature, humidity, microbes, etc., is becoming increasingly prominent. This challenge is particularly acute against the backdrop of changing crime patterns: with the advancement of a society governed by law, the number of severe violent crimes like intentional homicide and assault has decreased, while theft cases have risen significantly. The biological evidence extracted from these scenes is predominantly touch DNA[20], characterized by low DNA quantity, susceptibility to degradation, and often mixed profiles, leading to a substantial increase in both the volume and difficulty of casework. However, existing STR kits struggle to meet these demands due to locus limitations. Even the domestically produced GA118-16A platform and its supporting kits developed during China's 11th Five-Year Plan period enhanced technological autonomy, their detection throughput remains insufficient.

The shared domestic and international need for locus expansion directly drives technological iteration. As core STR loci increased from 13 to 20, commercial kits like VeriFiler now integrate over 23 loci, with non-CODIS loci and SNP markers also accelerating in application. Against this backdrop, the high-throughput bottleneck of traditional 6-dye fluorescence systems becomes glaringly apparent. Upgrading to systems with higher channel counts, particularly 8-dye systems, has become a key technological pathway. This choice stems not only from the continuity offered by the mature and widely deployed capillary electrophoresis technology platform, ensuring compatibility with existing and new equipment, but also from its ability to accommodate more loci in a single reaction to meet the growing demand for precise matching. Simultaneously, by integrating more small-sized miniSTR loci and optimizing channel allocation, 8-dye systems significantly improve the detection rate for Low Copy Number (LCN) DNA and degraded DNA samples like touch evidence. Specifically, more fluorescent channels tangibly improve degraded sample detection; for example, touch DNA detection rates in RapidHIT systems are only 32-78%[21], and adding miniSTR loci can effectively mitigate amplicon loss. On the other hand, it supports high-resolution analysis of mixed DNA, avoiding incomplete profiles in single-cell analysis. Especially when NGS data validation relies on capillary electrophoresis consistency, expanding fluorescent channels essentially forms the necessary foundation for high-multiplex detection systems, providing technical support to address global common challenges like degraded sample analysis and database matching accuracy.

Upgrade of Optical Systems

In capillary electrophoresis STR analysis, the optical system's laser and CCD work together to excite and capture fluorescent signals. The laser excites the fluorescent dyes labeling DNA fragments, the emitted spectrum is dispersed by a grating and imaged by the CCD, and overlapping signals are resolved through a color separation matrix to achieve synchronous genotyping of multiple loci[22]. The development of Multidye fluorescence systems places higher demands on optical systems. First, higher-power lasers are required to excite long-wavelength FRET dyes (e.g., ET5 emitting at 670 nm). Second, CCDs with larger imaging areas and higher sensitivity are needed.

In response to these needs, the domestic GA118-24B genetic analyzer implemented significant innovations. First, it employs a high-power 505 nm solid-state laser (50 mW, 12,000-hour lifespan), reducing power consumption by 90% compared to its predecessor[23]. Second, it features a wide-field CCD (512×512 pixels), with an area twice that of imported instruments, offering higher sensitivity and a broader spectral acquisition range. It also possesses the capability for further fluorescent channel expansion, enabling 8-dye and even 9-dye signal acquisition. Additionally, it supports pixel binning mode for targeted collection of weak fluorescent signals[24]. In terms of optical path structure, it pioneered fiber-optic beam splitting technology, replacing traditional mirror/half-mirror assemblies. The laser beam is split by a fiber optic splitter for balanced 1:1 energy transmission to the upper and lower optical paths. Mechanical shutters are eliminated in favor of electronic shutters to control the laser beam. Fiber optic transmission avoids optical path attenuation caused by dust and condensation on lenses. The failure rate of electronic shutters is 90% lower than mechanical shutters, enhancing long-term operational stability, particularly suitable for high-vibration environments like mobile vehicle laboratories.

Development of Novel Fluorescent Dyes

In capillary electrophoresis (CE) technology, fluorescent dye design must strictly adhere to the fundamental principle of spectral separation: the dye's excitation wavelength must match the instrument's laser source (e.g., 488/505 nm), the emission wavelength should have a sufficiently large Stokes shift to avoid excitation light interference, and the emission peak spacing between adjacent dyes needs to be greater than 20 nm to minimize spectral overlap[19]. With the increasing demand for Multidye detection, traditional organic dyes, limited by their emission spectrum range (520-660 nm), struggle to accommodate more independent channels within the finite wavelength window. To address the weakening of long-wavelength signals, Fluorescence Resonance Energy Transfer (FRET) technology was introduced: a donor dye absorbs laser energy and transfers it non-radiatively to an acceptor dye, which then emits long-wavelength fluorescence (>600 nm), significantly increasing detection channel capacity[25]. For example, Bai Xue's team, in a 9-dye system, utilized FRET-modified ET1-ET5 dyes combined with FAM/TET/HEX/NED, successfully expanding the number of channels to nine. The latter five dyes all rely on FRET to achieve stable emission in the 620-670 nm range[26].

Establishment of Multiplex Amplification Systems

In capillary electrophoresis technology, STR multiplex amplification relies on the synergy of multiplex PCR reactions and fluorescent label detection. The fundamental principle involves designing specific primers to simultaneously amplify multiple STR loci in a single reaction system. The amplification products are separated by capillary electrophoresis, and genotyping is achieved based on the fragment length differences labeled with different fluorescent dyes. Key requirements for successful multiplex amplification include[27]: (1) No cross-reactivity between primers to avoid non-specific amplification; (2) Balanced amplification efficiency across all loci to prevent peak height imbalance; (3) High spectral separation of fluorescent dyes to ensure signal resolution accuracy. Currently, in Multidye detection systems, the core of multiplex amplification design lies in maximizing locus throughput and optimizing spectral allocation. The 8-dye STR multiplex amplification system developed by Jiang, B. et al.[28] included 18 autosomal loci and gender markers, all short fragments less than 330 bp. Liu, Y. et al.[29] focused on the Y chromosome, incorporating 59 Y-STRs and 3 Indels. Hao, Y. et al.[30] historically achieved simultaneous detection of 70 loci (29 autosomal STRs + 40 Y-STRs) using 9 dye channels, 29 of which were miniSTRs <300 bp, significantly enhancing adaptability to degraded samples.

It is noteworthy that multiplex amplification systems require careful balancing between the number of loci and amplification performance. Excessively increasing the number of loci can exacerbate primer interactions, affecting amplification uniformity and compatibility with low-quality templates. Capillary electrophoresis remains irreplaceable for inhibited, trace, or degraded samples.

Collaborative Software Optimization

In the field of forensic DNA analysis, capillary electrophoresis combined with Multidye fluorescent labeling is the core method for high-throughput STR fragment detection. Data collection software, acting as the bridge between hardware control and data analysis, relies on spectral calibration to resolve Multidye fluorescence crosstalk. As described by Li Bin[31], before collection, an excitation-emission spectral distribution matrix must be established for each dye. After normalization, overlapping spectra are deconvoluted, effectively utilizing the energy at each wavelength and improving the signal-to-noise ratio. This matrix analysis method is particularly important for multi-channel detection like 8-dye systems, overcoming broad spectral band overlap issues that traditional optical splitting cannot resolve, thereby reducing the stringent requirements on the pre-detection optical splitting system. Currently, the data collection software for the domestic GA118 series sequencers[32]—Genetic Analyzer Data Collection Software—can collect 8-dye and 9-dye data. The GA118 collection software employs a multi-threaded architecture (main thread scheduling, spectral data thread, control command thread), communicates with hardware based on TCP/IP protocol, and is compatible with 4-24 capillary arrays. Its features include dual-buffer data processing to ensure real-time performance and configuration files to adapt spectral collection parameters for different instrument models.

Data analysis software is the critical tool for in-depth processing of collected data. The GAMarker system developed by Guo Tianli et al., built on JAVA and MYSQL, offers significant advantages[33]. The system's core functions include: 1) A five-level quality control system (internal standard, allelic ladder, locus, sample quality, and analysis element checks); 2) Support for 8-dye fluorescence, addressing multi-channel profile overlay issues through expanded chromatogram display and scrolling interfaces; 3) Manual review mechanisms, allowing editing of artifacts and allele labels while logging operation history; 4) Security and traceability, ensuring data integrity through user permissions and system logs. Compared to GeneMapper, GAMarker not only achieves consistent genotyping results (e.g., locus comparison for D3S1358) but also fills the gap for domestic equipment in the 8-dye analysis field.

Regarding instrument advances, genetic analyzers with 8-dye and higher detection capabilities are progressively developing (Table 1). Instruments currently supporting 8-dye STR detection include domestic brands GA118-24B and Honor 1816/1824, as well as imported brands Spectrum CE System and Spectrum Compact CE System. Among these, the Seqstudio Flex Genetic Analyzer requires the manufacturer to provide a specific 8-dye fluorescent spectral calibration dye set definition file to support 8-dye detection. Notably, domestic equipment continues to make breakthroughs in the Multidye domain; literature has confirmed the GA118-24B's potential for 9-dye detection. Suyuan Gene showcased its constructed 10-dye Matrix system at the 12th China International Police Expo in 2025 and announced that a supporting 10-dye STR kit will soon be launched. In terms of loading efficiency, the Spectrum CE System and Seqstudio Flex significantly enhance convenience with designs accommodating continuous runs of four 96-well plates. Regarding software support, apart from GeneMapper™ ID-X v1.7 currently used with SeqStudio Flex, which does not yet support 8-dye data analysis, other brands have dedicated DNA analysis software for processing 8-dye data.

In the field of forensic DNA identification, the differentiated application of first-generation sequencing (Sanger) and second-generation sequencing (NGS) technologies is driving the field towards higher levels of development. First-generation sequencing technology, centered on Short Tandem Repeat (STR) analysis, remains the mainstream technology for case investigation, paternity testing, and database construction due to its high sensitivity, standardized protocols, and compatibility with existing databases. Second-generation sequencing (NGS) technology breaks through the limitations of STR analysis by enabling high-throughput detection of Single Nucleotide Polymorphisms (SNPs), mitochondrial DNA (mtDNA), and whole-genome information, demonstrating unique advantages, especially in degraded evidence, mixed samples, and complex kinship identification. Despite challenges in rapid analysis and alignment with first-generation sequencing results, its application prospects in forensics remain broad.

The current Multidye STR fluorescence detection technology, still underpinned by first-generation sequencing principles, brings new development opportunities to the forensic DNA field. By increasing the number of fluorescent labels and scientifically arranging a larger number of loci, this technology ensures powerful data compatibility, including more Mini-STR loci, with the expectation of improving the success rate of obtaining complete or valid DNA profiles when processing challenging samples. This not only provides stronger support for individual identification and complex mixed sample analysis but also enables the acquisition of more comprehensive genetic information in a single test, enhancing the accuracy and reliability of identification. Furthermore, Multidye STR fluorescence detection technology can be combined with second-generation sequencing technology to further expand the application scope of forensic DNA detection, such as simultaneously detecting various genetic markers like STRs, SNPs, and mtDNA, providing richer data support for forensic genetics research and case investigation.